72 results on '"David J. Tremethick"'
Search Results
2. The Role of the Histone Variant H2A.Z in Metazoan Development
- Author
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Yasmin Dijkwel and David J. Tremethick
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chromatin ,chromatin remodeling ,development ,differentiation ,epigenetics ,H2A.Z ,Biology (General) ,QH301-705.5 - Abstract
During the emergence and radiation of complex multicellular eukaryotes from unicellular ancestors, transcriptional systems evolved by becoming more complex to provide the basis for this morphological diversity. The way eukaryotic genomes are packaged into a highly complex structure, known as chromatin, underpins this evolution of transcriptional regulation. Chromatin structure is controlled by a variety of different epigenetic mechanisms, including the major mechanism for altering the biochemical makeup of the nucleosome by replacing core histones with their variant forms. The histone H2A variant H2A.Z is particularly important in early metazoan development because, without it, embryos cease to develop and die. However, H2A.Z is also required for many differentiation steps beyond the stage that H2A.Z-knockout embryos die. H2A.Z can facilitate the activation and repression of genes that are important for pluripotency and differentiation, and acts through a variety of different molecular mechanisms that depend upon its modification status, its interaction with histone and nonhistone partners, and where it is deposited within the genome. In this review, we discuss the current knowledge about the different mechanisms by which H2A.Z regulates chromatin function at various developmental stages and the chromatin remodeling complexes that determine when and where H2A.Z is deposited.
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- 2022
- Full Text
- View/download PDF
3. Gene editing of the multi-copy H2A.B gene and its importance for fertility
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Nur Diana Anuar, Sebastian Kurscheid, Matt Field, Lei Zhang, Edward Rebar, Philip Gregory, Thierry Buchou, Josephine Bowles, Peter Koopman, David J. Tremethick, and Tatiana A. Soboleva
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Chromatin ,Histone variants ,H2A.B ,RNA polymerase II ,Pre-mRNA splicing ,Splicing speckles ,Biology (General) ,QH301-705.5 ,Genetics ,QH426-470 - Abstract
Abstract Background Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. Results Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed. Conclusion We have produced the first H2A.B knockout mouse using the TALEN approach.
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- 2019
- Full Text
- View/download PDF
4. The Histone Variant H2A.Z Is a Master Regulator of the Epithelial-Mesenchymal Transition
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Renae Domaschenz, Sebastian Kurscheid, Maxim Nekrasov, Shuyi Han, and David J. Tremethick
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Biology (General) ,QH301-705.5 - Abstract
Summary: Epithelial-mesenchymal transition (EMT) is a profound example of cell plasticity that is crucial for embryonic development and cancer. Although it has long been suspected that chromatin-based mechanisms play a role in this process, no master regulator that can specifically regulate EMT has been identified to date. Here, we show that H2A.Z can coordinate EMT by serving as either an activator or repressor of epithelial or mesenchymal gene expression, respectively. Following induction of EMT by TGF-β, we observed an unexpected loss of H2A.Z across both downregulated epithelial and upregulated mesenchymal promoters. Strikingly, the repression of epithelial gene expression was associated with reduction of H2A.Z upstream of the transcription start site (TSS), while the activation of mesenchymal gene expression was dependent on removal of H2A.Z downstream of the TSS. Therefore, the ability of H2A.Z to regulate EMT is dependent on its position, either upstream or downstream of the TSS. : EMT is one of the most intensely studied differentiation-dedifferentiation processes. Domaschenz et al. now demonstrate that H2A.Z has the unique ability to simultaneously serve as either an activator or a repressor of epithelial or mesenchymal gene expression, respectively. Keywords: EMT, H2A.Z, nucleosomes, TGF-β, gene regulation, promoter organization, cell plasticity, differentiation, chromatin
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- 2017
- Full Text
- View/download PDF
5. Short Histone H2A Variants: Small in Stature but not in Function
- Author
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Xuanzhao Jiang, Tatiana A. Soboleva, and David J. Tremethick
- Subjects
chromatin ,nucleosomes ,histone variants ,H2A.B ,H2A.L ,acidic patch ,Cytology ,QH573-671 - Abstract
The dynamic packaging of DNA into chromatin regulates all aspects of genome function by altering the accessibility of DNA and by providing docking pads to proteins that copy, repair and express the genome. Different epigenetic-based mechanisms have been described that alter the way DNA is organised into chromatin, but one fundamental mechanism alters the biochemical composition of a nucleosome by substituting one or more of the core histones with their variant forms. Of the core histones, the largest number of histone variants belong to the H2A class. The most divergent class is the designated “short H2A variants” (H2A.B, H2A.L, H2A.P and H2A.Q), so termed because they lack a H2A C-terminal tail. These histone variants appeared late in evolution in eutherian mammals and are lineage-specific, being expressed in the testis (and, in the case of H2A.B, also in the brain). To date, most information about the function of these peculiar histone variants has come from studies on the H2A.B and H2A.L family in mice. In this review, we describe their unique protein characteristics, their impact on chromatin structure, and their known functions plus other possible, even non-chromatin, roles in an attempt to understand why these peculiar histone variants evolved in the first place.
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- 2020
- Full Text
- View/download PDF
6. Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells
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David J. Tremethick, Renae Domaschenz, Maxim Nekrasov, Sebastian Kurscheid, Daniel L. Vera, Lauren Cole, and Jonathan H. Dennis
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Epigenomics ,0301 basic medicine ,Nucleosome organization ,animal structures ,Science ,Gene Expression ,General Physics and Astronomy ,RNA polymerase II ,Article ,General Biochemistry, Genetics and Molecular Biology ,Histones ,03 medical and health sciences ,Chromatin analysis ,0302 clinical medicine ,Cell Line, Tumor ,Gene expression ,Humans ,Micrococcal Nuclease ,Nucleosome ,Promoter Regions, Genetic ,Histone variants ,Binding Sites ,Multidisciplinary ,biology ,Chemistry ,Promoter ,General Chemistry ,Chromatin ,Nucleosomes ,Cell biology ,DNA binding site ,030104 developmental biology ,Histone ,embryonic structures ,biology.protein ,RNA Polymerase II ,Transcription ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter., Histone variant H2A.Z has been suggested to contribute to the regulation of promoter accessibility. Here, the authors present high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for human Pol II promoters and provide evidence that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.
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- 2021
7. Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes
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Maxim, Nekrasov and David J, Tremethick
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Histones ,Chromatin Immunoprecipitation ,Binding Sites ,Chromatin Assembly and Disassembly ,Chromatin ,Nucleosomes ,Protein Binding - Abstract
Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.
- Published
- 2021
8. H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma
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Jiayu Wen, Elizabeth Paver, Tatiana A. Soboleva, Amanda Bullman, Xuanzhao Jiang, David J. Tremethick, Yu-Huan Wu, Gege Sun, and Jane E. Dahlstrom
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Male ,biology ,Nucleolus ,RNA ,Ribosome biogenesis ,RNA polymerase II ,Articles ,Hodgkin Disease ,Biochemistry ,Chromatin ,Cell biology ,Histones ,Transcription (biology) ,Testis ,Gene expression ,Genetics ,biology.protein ,RNA polymerase I ,Humans ,Ribosomes ,Molecular Biology - Abstract
Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.
- Published
- 2021
9. Long-range interactions between topologically associating domains shape the four-dimensional genome during differentiation
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Maxim Nekrasov, Jonas Paulsen, Erwan Delbarre, David J. Tremethick, Tharvesh Moideen Liyakat Ali, Marie-Odile Baudement, Sebastian Kurscheid, and Philippe Collas
- Subjects
Heterochromatin ,Somatic cell ,Neurogenesis ,Gene Expression ,Computational biology ,Biology ,Genome ,Chromosome conformation capture ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Neural Stem Cells ,Genetics ,Animals ,Humans ,Gene silencing ,Cell Lineage ,030304 developmental biology ,0303 health sciences ,Adipogenesis ,Nuclear Lamina ,Models, Genetic ,Genome, Human ,Stem Cells ,Cell Differentiation ,Mouse Embryonic Stem Cells ,Chromatin Assembly and Disassembly ,Chromatin ,Nuclear lamina ,Reprogramming ,030217 neurology & neurosurgery - Abstract
Genomic information is selectively used to direct spatial and temporal gene expression during differentiation. Interactions between topologically associating domains (TADs) and between chromatin and the nuclear lamina organize and position chromosomes in the nucleus. However, how these genomic organizers together shape genome architecture is unclear. Here, using a dual-lineage differentiation system, we report long-range TAD-TAD interactions that form constitutive and variable TAD cliques. A differentiation-coupled relationship between TAD cliques and lamina-associated domains suggests that TAD cliques stabilize heterochromatin at the nuclear periphery. We also provide evidence of dynamic TAD cliques during mouse embryonic stem-cell differentiation and somatic cell reprogramming and of inter-TAD associations in single-cell high-resolution chromosome conformation capture (Hi-C) data. TAD cliques represent a level of four-dimensional genome conformation that reinforces the silencing of repressed developmental genes.
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- 2019
10. H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma
- Author
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David J. Tremethick, Elizabeth Paver, Tatiana A. Soboleva, Amanda Bullman, Wu Yu-Huan, Jiayu Wen, Jane E. Dahlstrom, Xuanzhao Jiang, and Gege Sun
- Subjects
biology ,Nucleolus ,Transcription (biology) ,Gene expression ,biology.protein ,RNA polymerase I ,RNA ,Ribosome biogenesis ,RNA polymerase II ,Chromatin ,Cell biology - Abstract
Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin Lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL-derived cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both the nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.
- Published
- 2021
11. Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes
- Author
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Maxim Nekrasov and David J. Tremethick
- Subjects
Affinity purified antibody ,education.field_of_study ,animal structures ,biology ,Chemistry ,Immunoprecipitation ,Population ,Chromatin ,Cell biology ,Histone ,embryonic structures ,biology.protein ,Nucleosome ,education ,Chromatin immunoprecipitation ,Micrococcal nuclease - Abstract
Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.
- Published
- 2021
12. Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications
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Anne Conibear, Christian F. W. Becker, K. Johan Rosengren, David J. Tremethick, Debra Urwin, and Gerhard Niederacher
- Abstract
Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.
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- 2020
13. Short Histone H2A Variants: Small in Stature but not in Function
- Author
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David J. Tremethick, Tatiana A. Soboleva, and Xuanzhao Jiang
- Subjects
animal structures ,chromatoid bodies ,Piwi-interacting RNA ,Review ,piRNA ,Biology ,Genome ,acidic patch ,Histones ,chemistry.chemical_compound ,splicing ,Mice ,Sequence Analysis, Protein ,Histone H2A ,Nucleosome ,Animals ,Humans ,Epigenetics ,histone variants ,lcsh:QH301-705.5 ,Genetics ,Genetic Variation ,General Medicine ,histone-protamine exchange ,Chromatin ,H2A.L ,Histone ,chemistry ,lcsh:Biology (General) ,nucleosomes ,embryonic structures ,biology.protein ,chromatin ,DNA ,H2A.B - Abstract
The dynamic packaging of DNA into chromatin regulates all aspects of genome function by altering the accessibility of DNA and by providing docking pads to proteins that copy, repair and express the genome. Different epigenetic-based mechanisms have been described that alter the way DNA is organised into chromatin, but one fundamental mechanism alters the biochemical composition of a nucleosome by substituting one or more of the core histones with their variant forms. Of the core histones, the largest number of histone variants belong to the H2A class. The most divergent class is the designated “short H2A variants” (H2A.B, H2A.L, H2A.P and H2A.Q), so termed because they lack a H2A C-terminal tail. These histone variants appeared late in evolution in eutherian mammals and are lineage-specific, being expressed in the testis (and, in the case of H2A.B, also in the brain). To date, most information about the function of these peculiar histone variants has come from studies on the H2A.B and H2A.L family in mice. In this review, we describe their unique protein characteristics, their impact on chromatin structure, and their known functions plus other possible, even non-chromatin, roles in an attempt to understand why these peculiar histone variants evolved in the first place.
- Published
- 2020
14. The interplay between H2A.Z and H3K9 methylation in regulating HP1α binding to linker histone-containing chromatin
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David J. Tremethick and Daniel P. Ryan
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0301 basic medicine ,Chromosomal Proteins, Non-Histone ,Methylation ,Histones ,03 medical and health sciences ,Heterochromatin ,Genetics ,Humans ,Nucleosome ,Nucleosome binding ,030102 biochemistry & molecular biology ,biology ,Chromatin binding ,Gene regulation, Chromatin and Epigenetics ,Histone-Lysine N-Methyltransferase ,Linker DNA ,Chromatin ,Nucleosomes ,Cell biology ,030104 developmental biology ,Histone ,Chromobox Protein Homolog 5 ,Histone Methyltransferases ,biology.protein ,Heterochromatin protein 1 ,Protein Processing, Post-Translational ,Linker ,Protein Binding - Abstract
One of the most intensively studied chromatin binding factors is HP1α. HP1α is associated with silenced, heterochromatic regions of the genome and binds to H3K9me3. While H3K9me3 is necessary for HP1α recruitment to heterochromatin, it is becoming apparent that it is not sufficient suggesting that additional factors are involved. One candidate proposed as a potential regulator of HP1α recruitment is the linker histone H1.4. Changes to the underlying make-up of chromatin, such as the incorporation of the histone variant H2A.Z, has also been linked with regulating HP1 binding to chromatin. Here, we rigorously dissected the effects of H1.4, H2A.Z and H3K9me3 on the nucleosome binding activity of HP1α in vitro employing arrays, mononucleosomes and nucleosome core particles. Unexpectedly, histone H1.4 impedes the binding of HP1α but strikingly, this inhibition is partially relieved by the incorporation of both H2A.Z and H3K9me3 but only in the context of arrays or nucleosome core particles. Our data suggests that there are two modes of interaction of HP1α with nucleosomes. The first primary mode is through interactions with linker DNA. However, when linker DNA is missing or occluded by linker histones, HP1α directly interacts with the nucleosome core and this interaction is enhanced by H2A.Z with H3K9me3.
- Published
- 2018
15. Gene editing of the multi-copy H2A.B gene and its importance for fertility
- Author
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Peter Koopman, David J. Tremethick, Tatiana A. Soboleva, Lei Zhang, Josephine Bowles, Matthew A. Field, Sebastian Kurscheid, Philip D. Gregory, Thierry Buchou, Nur Diana Anuar, and Edward J. Rebar
- Subjects
Male ,lcsh:QH426-470 ,Chromosomal Proteins, Non-Histone ,Gene Expression ,RNA polymerase II ,Histones ,03 medical and health sciences ,Pre-mRNA splicing ,0302 clinical medicine ,Transcription Activator-Like Effector Nucleases ,Gene expression ,Animals ,Gene family ,lcsh:QH301-705.5 ,Gene ,Infertility, Male ,030304 developmental biology ,Gene Editing ,Mice, Knockout ,Histone variants ,Genetics ,0303 health sciences ,Base Sequence ,biology ,Research ,Splicing speckles ,Spermatozoa ,Chromatin ,lcsh:Genetics ,TALENs ,Fertility ,Histone ,lcsh:Biology (General) ,Mutation ,RNA splicing ,Knockout mouse ,biology.protein ,Female ,H2A.B ,030217 neurology & neurosurgery ,Genome editing - Abstract
Background Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. Results Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed. Conclusion We have produced the first H2A.B knockout mouse using the TALEN approach. Electronic supplementary material The online version of this article (10.1186/s13059-019-1633-3) contains supplementary material, which is available to authorized users.
- Published
- 2019
16. RChIP-Seq: Chromatin-Associated RNA Sequencing in Developmentally Staged Mouse Testes
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David J. Tremethick and Tatiana A. Soboleva
- Subjects
0301 basic medicine ,biology ,RNA ,RNA-binding protein ,Mouse Testis ,DNA sequencing ,Chromatin ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Histone ,biology.protein ,Function (biology) ,Macromolecule - Abstract
Chromatin is a dynamic macromolecular structure comprised of histones and a wealth of non-histone proteins. Recently, it has become clear that RNA is also an integral component of chromatin playing an important role in regulating its structure and function. Central to the understanding of RNA function is the ability to identify and genomically map interactions between chromatin components and RNA.Here, we describe a new method, RChIP-seq (RNA-associated-Chromatin-Immuno Precipitation followed by next-generation sequencing) that allows the identification of RNA species that are directly bound to specific components of chromatin in the mouse testis.
- Published
- 2018
17. Gene editing of the multi-copy H2A.B gene family by a single pair of TALENS
- Author
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Peter Koopman, Edward J. Rebar, Matthew A. Field, Tatiana A. Soboleva, David J. Tremethick, Josephine Bowles, Lei Zhang, Philip A. Gregory, Sebastian Kurscheid, and Nur Diana Anuar
- Subjects
0303 health sciences ,03 medical and health sciences ,Transcription activator-like effector nuclease ,0302 clinical medicine ,Genome editing ,Single pair ,Gene family ,Computational biology ,Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
In view of the controversy related to the generation of off-target mutations by gene editing approaches, we tested the specificity of TALENs by disrupting a multi-copy gene family using only one pair of TALENS. We show here that TALENS do display a high level of specificity by simultaneously knocking out the function of the three genes that encode for H2A.B.3. This represents the first described knockout of this histone variant.
- Published
- 2017
18. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B
- Author
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Ying Jin Tay, Brian J. Parker, David J. Tremethick, Wei Quan Tng, Marc R. Wilkins, Gene Hart-Smith, Tatiana A. Soboleva, Daniel P. Ryan, and Maxim Nekrasov
- Subjects
0301 basic medicine ,Male ,Cancer Research ,Transcription, Genetic ,RNA splicing ,Molecular biology ,Fluorescent Antibody Technique ,Gene Expression ,RNA polymerase II ,Biochemistry ,Mass Spectrometry ,Histones ,Exon ,0302 clinical medicine ,Sequencing techniques ,Animal Cells ,Testis ,RNA Precursors ,Histone code ,Genetics (clinical) ,Mice, Inbred BALB C ,biology ,Chromosome Biology ,Brain ,RNA-Binding Proteins ,RNA sequencing ,Exons ,Spermatids ,Chromatin ,Nucleosomes ,Nucleic acids ,embryonic structures ,Epigenetics ,RNA Polymerase II ,Transcription Initiation Site ,Cellular Types ,Protein Binding ,Research Article ,animal structures ,lcsh:QH426-470 ,Blotting, Western ,DNA transcription ,03 medical and health sciences ,Histone H1 ,Histone H2A ,DNA-binding proteins ,Genetics ,Animals ,Ecology, Evolution, Behavior and Systematics ,Biology and life sciences ,RNA ,Genetic Variation ,Proteins ,Cell Biology ,Introns ,Sperm ,Research and analysis methods ,lcsh:Genetics ,030104 developmental biology ,Molecular biology techniques ,Germ Cells ,RNA processing ,biology.protein ,RNA Splice Sites ,030217 neurology & neurosurgery - Abstract
The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron—exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z., Author summary The substitution of core histones with their non-allelic variant forms plays a particular important role in regulating chromatin function because they can directly alter the structure of chromatin, and provide new protein interaction interfaces for the recruitment of proteins involved in gene expression. Despite being discovered over a decade ago, the function of H2A.B, a variant of the H2A class, in its proper physiological context (being expressed in the testis and the brain) is unknown. We provide strong evidence that H2A.B has a role in the processing of RNA. It is found in the gene body of an active gene, directly interacts with RNA polymerase II and splicing factors and is located in the nucleus at distinct regions enriched with RNA processing factors (splicing speckles). Most significantly, we show that H2A.B can directly bind to RNA both in vitro and in germ cells. Therefore, H2A.B has the novel ability to bind to both RNA and DNA (as well as proteins) thus directly linking chromatin structure with the function of RNA. Taken together, this suggests that a special mechanism of splicing may operate in the testis and brain.
- Published
- 2016
19. Histone variant selectivity at the transcription start site: H2A.Z or H2A.Lap1
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Cameron Jack, Maxim Nekrasov, David J. Tremethick, and Tatiana A. Soboleva
- Subjects
Male ,RNA polymerase II ,H2A.Bbd ,Crystallography, X-Ray ,Protein Structure, Secondary ,Histones ,Mice ,Transcription (biology) ,Y Chromosome ,Histone methylation ,Promoter Regions, Genetic ,Oligonucleotide Array Sequence Analysis ,Regulation of gene expression ,Genetics ,Mice, Inbred BALB C ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Cycle ,H2A.Z ,Spermatids ,Chromatin ,Trophoblasts ,Nucleosomes ,ChIP-seq ,Meiosis ,Histone ,microarray gene expression analysis ,embryonic structures ,Transcription Initiation Site ,nucleosome positioning ,Protein Binding ,animal structures ,H2A.Lap1 ,X Chromosome ,Blotting, Western ,Molecular Sequence Data ,Models, Biological ,stem cells ,Histone H2A ,Nucleosome ,Animals ,Amino Acid Sequence ,histone variants ,Spermatogenesis ,Gene ,active chromatin ,the transcriptional start site ,Sequence Homology, Amino Acid ,Extra View ,Gene Expression Profiling ,Genetic Variation ,Cell Biology ,Gene Expression Regulation ,biology.protein - Abstract
Considerable attention has been given to the understanding of how nucleosomes are altered or removed from the transcription start site of RNA polymerase II genes to enable transcription to proceed. This has led to the view that for transcriptional activation to occur, the transcription start site (TSS) must become depleted of nucleosomes. However, we have shown that this is not the case with different unstable histone H2A variant-containing nucleosomes occupying the TSS under different physiological settings. For example, during mouse spermatogenesis we found that the mouse homolog of human H2A.Bbd, H2A.Lap1, is targeted to the TSS of active genes expressed during specific stages of spermatogenesis. On the other hand, we observed in trophoblast stem cells, a H2A.Z-containing nucleosome occupying the TSS of genes active in the G 1 phase of the cell cycle. Notably, this H2A.Z-containing nucleosome was different compared with other promoter specific H2A.Z nucleosomes by being heterotypic rather than being homotypic. In other words, it did not contain the expected two copies of H2A.Z per nucleosome but only one (i.e., H2A.Z/H2A rather than H2A.Z/H2A.Z). Given these observations, we wondered whether the histone variant composition of a nucleosome at an active TSS could in fact vary in the same cell type. To investigate this possibility, we performed H2A.Z ChIP-H2A reChIP assays in the mouse testis and compared this data with our testis H2A.Lap1 ChIP-seq data. Indeed, we find that different promoters involved in the expression of genes involved in distinct biological processes can contain either H2A.Z/H2A or H2A.Lap1. This argues that specific mechanisms exist, which can determine whether H2A.Z or H2A.Lap1 is targeted to the TSS of an active gene.
- Published
- 2013
20. Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics
- Author
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Gavin A. Huttley, Tatiana A. Soboleva, Jane Amrichova, David J. Tremethick, Brian J. Parker, Cameron Jack, Maxim Nekrasov, and Rohan B. H. Williams
- Subjects
animal structures ,biology ,Molecular biology ,Histone ,Histone H1 ,Structural Biology ,Histone methyltransferase ,embryonic structures ,Histone H2A ,Histone methylation ,biology.protein ,Histone code ,Histone octamer ,Molecular Biology ,Chromatin immunoprecipitation - Abstract
Although it has been clearly established that well-positioned histone H2A.Z-containing nucleosomes flank the nucleosome-depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in mouse trophoblast stem cells, the amount of histone H2A.Z at promoters decreased during S phase, coinciding with homotypic (H2A.Z-H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z-H2A). To our surprise these nucleosomes remained heterotypic at M phase. At the TSS, we identified an unstable heterotypic histone H2A.Z-containing nucleosome in G1 phase that was lost after DNA replication. These dynamic changes at the TSS mirror a global expansion of the NDR at S and M phases, which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of histone H2A.Z at promoters, histone H2A.Z is targeted to the centromere when mitosis begins.
- Published
- 2012
21. SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation
- Author
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Charlotte, Moretti, Maria-Elisabetta, Serrentino, Côme, Ialy-Radio, Marion, Delessard, Tatiana A, Soboleva, Frederic, Tores, Marjorie, Leduc, Patrick, Nitschké, Joel R, Drevet, David J, Tremethick, Daniel, Vaiman, Ayhan, Kocer, and Julie, Cocquet
- Subjects
Male ,Original Paper ,Sex Chromosomes ,Nuclear Proteins ,Receptors, Cytoplasmic and Nuclear ,DNA ,Chromatin Assembly and Disassembly ,behavioral disciplines and activities ,Chromosomes, Mammalian ,Spermatozoa ,Repressor Proteins ,Adaptor Proteins, Vesicular Transport ,Mice ,Gene Expression Regulation ,Animals ,Promoter Regions, Genetic ,Spermatogenesis ,Adaptor Proteins, Signal Transducing - Abstract
Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.
- Published
- 2016
22. The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression
- Author
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David J. Tremethick, Jiansheng Zhou, Jun Y. Fan, and Danny Rangasamy
- Subjects
Genetics ,biology ,RNA polymerase II ,Protein degradation ,Chromatin remodeling ,Chromatin ,Cell biology ,Structural Biology ,biology.protein ,Nucleosome ,Chromatin structure remodeling (RSC) complex ,Molecular Biology ,ChIA-PET ,Chromatin Fiber - Abstract
Although it is believed that the interconversion between permissive and refractory chromatin structures is important in regulating gene transcription, this process is poorly understood. Central to addressing this issue is to elucidate how a nucleosomal array folds into higher-order chromatin structures. Such findings can then provide new insights into how the folding process is regulated to yield different functional states. Using well-defined in vitro chromatin-assembly and transcription systems, we show that a small acidic region on the surface of the nucleosome is crucial both for the folding of a nucleosomal template into the 30-nm chromatin fiber and for the efficient repression of transcription, thereby providing a mechanistic link between these two essential processes. This structure-function relationship has been exploited by complex eukaryotic cells through the replacement of H2A with the specific variant H2A.Bbd, which naturally lacks an acidic patch.
- Published
- 2007
23. Inhibition of Arginase I Activity by RNA Interference Attenuates IL-13-Induced Airways Hyperresponsiveness
- Author
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Klaus I. Matthaei, Suresh Mahalingam, Ming Yang, Philip J. Thompson, David J. Tremethick, William B. Cowden, Nives Zimmmermann, Simon P. Hogan, Danny Rangasamy, Ailsa J. Frew, Dianne C. Webb, Paul S. Foster, and Marc E. Rothenberg
- Subjects
Immunology ,Inflammation ,Biology ,Arginine ,Gene Expression Regulation, Enzymologic ,Nitric oxide ,Allergic inflammation ,Small hairpin RNA ,Mice ,chemistry.chemical_compound ,Th2 Cells ,medicine ,Animals ,Immunology and Allergy ,Nitric Oxide Donors ,Lung ,Mice, Knockout ,Interleukin-13 ,Arginase ,respiratory system ,Asthma ,respiratory tract diseases ,chemistry ,Interleukin 13 ,RNA Interference ,Bronchoconstriction ,Bronchial Hyperreactivity ,medicine.symptom ,STAT6 Transcription Factor ,Cytokine receptor - Abstract
Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.
- Published
- 2006
24. The X and Y Chromosomes Assemble into H2A.Z, Containing Facultative Heterochromatin, following Meiosis
- Author
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Danny Rangasamy, Ian K. Greaves, David J. Tremethick, Michael Devoy, and Jennifer A. Marshall Graves
- Subjects
Genetics ,Euchromatin ,Spermatid ,Heterochromatin ,Cell Biology ,Biology ,Y chromosome ,Chromatin ,medicine.anatomical_structure ,Histone ,Meiosis ,medicine ,biology.protein ,Molecular Biology ,X chromosome - Abstract
Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1β and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.
- Published
- 2006
25. Chromatin: the dynamic link between structure and function
- Author
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David J. Tremethick
- Subjects
DNA Replication ,Recombination, Genetic ,Genetics ,DNA Repair ,Transcription, Genetic ,Heterochromatin ,DNA ,Link (geometry) ,Biology ,Chromatin ,Nucleosomes ,Structure and function ,Histones ,Biophysics ,Animals ,Humans ,Epigenetics - Published
- 2006
26. The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken
- Author
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Constanze Bonifer, Kimberley Bruce, Fiona A. Myers, David J. Tremethick, Pascal Lefevre, Ian K. Greaves, Alan W. Thorne, Evangelia Mantouvalou, and Colyn Crane-Robinson
- Subjects
animal structures ,Transcription, Genetic ,Heterochromatin ,Chick Embryo ,Article ,Antibodies ,Histones ,03 medical and health sciences ,Genetics ,Animals ,Epigenetics ,Enhancer ,Gene ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Acetylation ,Molecular biology ,Housekeeping gene ,Chromatin ,Cell biology ,Histone ,biology.protein ,Chickens - Abstract
The replacement histone H2A.Z is variously reported\ud as being linked to gene expression and preventing the\ud spread of heterochromatin in yeast, or concentrated\ud at heterochromatin in mammals. To resolve this\ud apparent dichotomy, affinity-purified antibodies\ud against the N-terminal region of H2A.Z, in both a triacetylatedandnon-\ud acetylatedstate, areusedin native\ud chromatin immmuno-precipitation experiments with\ud mononucleosomes from three chicken cell types. The\ud hyperacetylated species concentrates at the 50 end of\ud active genes, both tissue specific and housekeeping\ud but is absent from inactive genes, while the\ud unacetylated form is absent from both active and\ud inactive genes. A concentration of H2A.Z is also\ud found at insulators under circumstances implying a\ud link to barrier activity but not to enhancer blocking.\ud Although acetylated H2A.Z is widespread throughout\ud the interphase genome, at mitosis its acetylation is\ud erased, the unmodified form remaining. Thus,\ud although H2A.Z may operate as an epigenetic marker\ud for active genes, its N-terminal acetylation does not.
- Published
- 2005
27. H2A.Z Alters the Nucleosome Surface to Promote HP1α-Mediated Chromatin Fiber Folding
- Author
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Jun Y. Fan, David J. Tremethick, Karolin Luger, and Danny Rangasamy
- Subjects
Chromatin Immunoprecipitation ,Protein Folding ,Protein Conformation ,Blotting, Western ,Molecular Sequence Data ,Solenoid (DNA) ,Biology ,Models, Biological ,Protein Structure, Secondary ,Chromatin remodeling ,Histones ,Histone H1 ,Heterochromatin ,Animals ,Histone code ,Amino Acid Sequence ,Molecular Biology ,ChIA-PET ,Chromatin Fiber ,Genetics ,Chromatin binding ,Genetic Variation ,Cell Biology ,Chromatin ,Recombinant Proteins ,Nucleosomes ,Protein Structure, Tertiary ,Cell biology ,DNA-Binding Proteins ,Gene Expression Regulation - Abstract
Controlling the degree of higher order chromatin folding is a key element in partitioning the metazoan genome into functionally distinct chromosomal domains. However, the mechanism of this fundamental process is poorly understood. Our recent studies suggested that the essential histone variant H2A.Z and the silencing protein HP1alpha may function together to establish a specialized conformation at constitutive heterochromatic domains. We demonstrate here that HP1alpha is a unique chromatin binding protein. It prefers to bind to condensed higher order chromatin structures and alters the chromatin-folding pathway in a novel way to locally compact individual chromatin fibers without crosslinking them. Strikingly, both of these features are enhanced by an altered nucleosomal surface created by H2A.Z (the acidic patch). This shows that the surface of the nucleosome can regulate the formation of distinct higher order chromatin structures mediated by an architectural chromatin binding protein.
- Published
- 2004
28. Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA
- Author
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David J. Tremethick, Danny Rangasamy, Young-Jun Park, Paul J. Laybourn, Yunhe Bao, Simona Rosu, Karolin Luger, Kasey L. Konesky, and Pamela N. Dyer
- Subjects
Models, Molecular ,Protein Folding ,animal structures ,Transcription, Genetic ,Protein Conformation ,Molecular Sequence Data ,Article ,General Biochemistry, Genetics and Molecular Biology ,Histones ,Histone H4 ,Histone H3 ,Histone H2A ,Histone methylation ,Histone H2B ,Animals ,Humans ,Micrococcal Nuclease ,Histone code ,Nucleosome ,Amino Acid Sequence ,Promoter Regions, Genetic ,skin and connective tissue diseases ,Base Pairing ,Molecular Biology ,Oligonucleotide Array Sequence Analysis ,General Immunology and Microbiology ,biology ,General Neuroscience ,DNA ,Nucleosomes ,Cell biology ,Histone ,Biochemistry ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Sequence Alignment ,Protein Binding - Abstract
H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A-H2B dimer to the (H3-H4)(2) tetramer; in addition, the C-terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd-NCP), and found that Bbd-NCP had a more relaxed structure in which only 118+/-2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd-NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain-swap experiments. Bbd-containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd-NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.
- Published
- 2004
29. RNA interference demonstrates a novel role for H2A.Z in chromosome segregation
- Author
-
David J. Tremethick, Ian K. Greaves, and Danny Rangasamy
- Subjects
Genetics ,Genome ,animal structures ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Heterochromatin ,Molecular Sequence Data ,Biology ,Blotting, Northern ,Chromosome aberration ,Chromatin ,Histones ,Chromosome segregation ,Histone ,Structural Biology ,RNA interference ,embryonic structures ,Centromere ,biology.protein ,RNA Interference ,Molecular Biology ,Gene ,DNA Primers - Abstract
The histone variant H2A.Z plays an essential role in metazoans but its function remains to be determined. Here, we developed a new inducible RNAi strategy to elucidate the role of H2A.Z in mammalian cell lines. We show that in the absence of H2A.Z, the genome becomes highly unstable and that this instability is caused by defects in the chromosome segregation process. Analysis of H2A.Z localization reveals that in these cells it is enriched at heterochromatic foci with HP1alpha on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1alpha-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1alpha. We conclude that H2A.Z is essential for the accurate transmission of chromosomes.
- Published
- 2004
30. Structural Characterization of Histone H2A Variants
- Author
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Srinivas Chakravarthy, David J. Tremethick, Yunhe Bao, Victoria A. Roberts, and Karolin Luger
- Subjects
Models, Molecular ,Macromolecular Substances ,Protein Conformation ,Molecular Sequence Data ,Static Electricity ,Biology ,Biochemistry ,Epigenesis, Genetic ,Evolution, Molecular ,Histones ,Histone H4 ,Mice ,Histone H3 ,Histone H2A ,Genetics ,Histone H2B ,Animals ,Humans ,Nucleosome ,Amino Acid Sequence ,Histone octamer ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,Genetic Variation ,Chromatin ,Nucleosomes ,Cell biology ,Histone ,biology.protein - Abstract
protein to form chromatin, the fundamental unit of which is the nucleosome core particle (NCP). An NCP consists of two copies each of the four core histones H2A, H2B, H3, and H4. This histone octamer binds 147 base pairs of DNA around its outer surface in 1.65 tight superhelical turns (Fig. 1A) (Luger et al. 1997; Richmond and Davey 2003). Linker histones and other nonhistone proteins promote or stabilize the folding of nucleosomal arrays into superstructures of increasing complexity and largely unknown architecture (Hansen 2002). Covalent modification of the core histones and variations in the fundamental biochemical composition of nucleosomes distinguish transcriptionally active from inactive chromatin regions, by either changing the structure of the nucleosomes, altering their ability to interact with other protein factors, or modifying their propensity to fold into varying degrees of higher-order structures (or by any combination of the above). Studying the mechanism for establishing distinct chromatin domains is essential to understanding differential regulation of gene expression and all other DNA-dependent processes. Much progress has been made in this direction in the past few years. Substitution of one or more of the core histones with the corresponding histone variants has the potential to exert considerable influence on the structure and function of chromatin. Histone variants are distinct nonallelic forms of conventional, major-type histones that form the bulk of nucleosomes during replication and whose synthesis is tightly coupled to S phase. Histone variants are characterized by a completely different expression pattern that is not restricted to S phase. They are found in most eukaryotic organisms and are expressed in all tissue types (unlike some H2B isoforms that are found only in specialized tissues such as testes). Compared to their majortype counterparts, histone variants exhibit moderate to significant degrees of sequence homology (Fig. 1B). H2A.X (82%) and H3.3 (~96%) are the least divergent of all histone variants. H2A.Z (~60%), macroH2A (~65%), H2A.Bbd (40%), and CenpA, which has a 93 amino acid domain that is 62% identical to H3 (Palmer et al. 1991; Sullivan et al. 1994), are increasingly divergent in their Structural Characterization of Histone H2A Variants
- Published
- 2004
31. Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development
- Author
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David J. Tremethick, Leise A. Berven, Patricia Ridgway, and Danny Rangasamy
- Subjects
X Chromosome ,Heterochromatin ,Molecular Sequence Data ,Fluorescent Antibody Technique ,General Biochemistry, Genetics and Molecular Biology ,Histones ,Embryonic and Fetal Development ,Mice ,Dosage Compensation, Genetic ,Two-Hybrid System Techniques ,Histone H2A ,medicine ,Animals ,Amino Acid Sequence ,Epigenetics ,Molecular Biology ,Pericentric heterochromatin ,DNA Primers ,Genetics ,Base Sequence ,General Immunology and Microbiology ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,INCENP ,General Neuroscience ,Articles ,Chromatin ,Mice, Inbred C57BL ,Cell nucleus ,Histone ,medicine.anatomical_structure ,biology.protein ,Female ,Protein Binding - Abstract
Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.
- Published
- 2003
32. Histone variants at the transcription start-site
- Author
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David J. Tremethick, Maxim Nekrasov, Tatiana A. Soboleva, and Daniel P. Ryan
- Subjects
Genetics ,Models, Molecular ,genetic structures ,General transcription factor ,Pioneer factor ,Eukaryotic transcription ,Response element ,Molecular Sequence Data ,Promoter ,RNA polymerase II ,Transcription coregulator ,Biology ,Nucleosomes ,Histones ,biology.protein ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Transcription Initiation Site ,Promoter Regions, Genetic ,Cis-regulatory module - Abstract
The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief.
- Published
- 2014
33. Histone variant H2A.Z is required for early mammalian development
- Author
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Renate Faast, Ian Lyons, Thomas C. Schulz, Helen Taylor, Jacquie Beall, Julian R.E. Wells, Peter D. Rathjen, Varaporn Thonglairoam, David J. Tremethick, and Klaus I. Matthaei
- Subjects
Genetics ,Histone-modifying enzymes ,Base Sequence ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Chromatin remodeling ,Cell Line ,Chromatin ,Cell biology ,Histones ,Embryonic and Fetal Development ,Mice ,Histone H1 ,Histone methyltransferase ,Histone H2A ,Histone methylation ,Animals ,Histone code ,General Agricultural and Biological Sciences ,Gene Deletion ,DNA Primers - Abstract
Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1° have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.
- Published
- 2001
- Full Text
- View/download PDF
34. [Untitled]
- Author
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Michael J. Clarkson, Karolin Luger, Robert K. Suto, and David J. Tremethick
- Subjects
animal structures ,Materials science ,biology ,Biochemistry ,Linker DNA ,Molecular biology ,Histone ,Histone H1 ,Structural Biology ,embryonic structures ,Chromatosome ,Histone methylation ,Genetics ,Biophysics ,biology.protein ,Histone code ,Nucleosome ,Histone octamer - Abstract
Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the molecular mechanism by which they function is largely unknown. Here we report the 2.6 A crystal structure of a nucleosome core particle containing the histone variant H2A.Z. The overall structure is similar to that of the previously reported 2.8 A nucleosome structure containing major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)(2) tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the association of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z-H2B dimers.
- Published
- 2000
35. Multiple ISWI ATPase Complexes from Xenopus laevis
- Author
-
Dmitry Guschin, Paul A. Wade, Alan P. Wolffe, David J. Tremethick, Nobuaki Kikyo, and Theresa M. Geiman
- Subjects
Genetics ,biology ,ATPase ,Protein subunit ,ATPase complex ,Xenopus ,Cell Biology ,biology.organism_classification ,Biochemistry ,Chromatin ,Conserved sequence ,Cell biology ,Protein structure ,Histone ,biology.protein ,Molecular Biology - Abstract
The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.
- Published
- 2000
36. The binding of a Fos/Jun heterodimer can completely disrupt the structure of a nucleosome
- Author
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David J. Tremethick, Patricia Ridgway, Donna R. Cohen, and Kean Wooi Ng
- Subjects
Nucleosome assembly ,Proto-Oncogene Proteins c-jun ,Molecular Sequence Data ,Fos-Related Antigen-2 ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Histones ,Mice ,Xenopus laevis ,Animals ,Nucleosome ,Binding site ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Leucine Zippers ,Base Sequence ,General Immunology and Microbiology ,General Neuroscience ,Nuclear Proteins ,Acetylation ,Promoter ,DNA ,Molecular biology ,Sex-Determining Region Y Protein ,Nucleosomes ,Rats ,Chromatin ,Histone displacement ,Cell biology ,DNA-Binding Proteins ,Transcription Factor AP-1 ,Chickens ,Dimerization ,Proto-Oncogene Proteins c-fos ,Research Article ,Protein Binding ,Transcription Factors - Abstract
An important first step in the chromatin remodelling process is the initial binding of a transcriptional activator to a nucleosomal template. We have investigated the ability of Fos/Jun (a transcriptional activator involved in the signal transduction pathway) to interact with its cognate binding site located in the promoter region of the mouse fos-related antigen-2 (fra-2) promoter, when this site was reconstituted into a nucleosome. Two different nucleosome assembly systems were employed to assemble principally non-acetylated or acetylated nucleosomes. The ability of Fos/Jun to interact with an acetylated or an unacetylated nucleosome differed markedly. Fos/Jun bound to an unacetylated nucleosome with only a 4- to 5-fold reduction in DNA binding affinity compared with naked DNA. Strikingly, the binding of Fos/Jun to a single high-affinity site incorporated into an acetylated nucleosome resulted in the complete disruption of nucleosomal structure without histone displacement. Moreover, this disruption was sufficient to facilitate the subsequent binding of a second transcription factor.
- Published
- 1997
37. High Mobility Group Proteins 14 and 17 Can Prevent the Close Packing of Nucleosomes by Increasing the Strength of Protein Contacts in the Linker DNA
- Author
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David J. Tremethick and Luke Hyman
- Subjects
Nucleosome binding ,Binding Sites ,biology ,Base pair ,High Mobility Group Proteins ,DNA ,Cell Biology ,Biochemistry ,Linker DNA ,Nucleosomes ,Chromatin ,Histones ,Xenopus laevis ,High-mobility group ,Histone ,biology.protein ,Biophysics ,Animals ,Humans ,Nucleosome ,Magnesium ,Molecular Biology ,Micrococcal nuclease - Abstract
High mobility group (HMG) proteins 14 and 17 are abundant chromatin-associated proteins found in all higher eukaryotic nuclei. This observation demonstrates that HMGs 14 and 17 must have an important and universal function with regard to the structure and function of chromatin. What this function is, including how they interact with a nucleosomal array in vivo, is not known. Recently, we have demonstrated that HMGs 14 and 17 can organize nucleosomes into a regular array and increase the repeat length from 145 to about 160-165 base pairs in vitro. In addition, they can increase the apparent repeat length of chromatin deficient in histones H2A/H2B from 125 to approximately 145 base pairs. Importantly, this template was transcriptionally active. In this study, we report five new observations that begin to address the mechanism by which HMGs 14 and 17 space nucleosomal particles. First, we demonstrate that both human placenta HMG 14 and HMG 17 can space nucleosomes to produce a chromatin template with a repeat length around 160 base pairs. This result further highlights the similarity between these proteins in terms of protein structure and perhaps function. Second, we show that digestion of HMG containing chromatin with micrococcal nuclease produces DNA fragments that were approximately 10 and 20 base pairs longer than nucleosome core-particle DNA. This suggests that HMG 14 or HMG 17 can protect, directly or indirectly, at least an additional 10 base pairs of linker DNA from micrococcal digestion. However, this HMG-containing particle does not produce a strong kinetic block, and further digestion results in the eventual accumulation of DNA fragments 145 base pairs in length. Third, by comparing the full-length protein with different domains, we demonstrate that the acidic carboxyl-terminal domain is absolutely required for nucleosome spacing, neither the nucleosome binding domain of HMG 14 or HMG 17 nor the amino-terminal domain plus the nucleosome binding domain of HMG 14 could space nucleosomes. Fourth, we demonstrate that extensive micrococcal nuclease digestion of chromatin deficient in histones H2A/H2B led to the accumulation of DNA fragments about 110 base pairs in length, which is presumably the length of DNA associated with a nucleosomal particle deficient in one H2A/H2B dimer. Incorporation of either HMG 14 or HMG 17 into this chromatin results in the disappearance of this band and increase in the accumulation of fragments around 140-150 base pairs in length. Finally, in contrast to spacing of complete nucleosomes, we find that the nucleosome binding domain of HMG 17 (but not the nucleosome binding of HMG 14) is the only domain required for spacing of H2A/H2B-deficient chromatin.
- Published
- 1996
38. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?
- Author
-
Mekonnen Lemma Dechassa, Karolin Luger, and David J. Tremethick
- Subjects
Models, Molecular ,Histone-modifying enzymes ,DNA Repair ,Protein Conformation ,Molecular Conformation ,Biology ,Article ,Protein Structure, Secondary ,Histones ,Histone H2B ,Histone code ,Nucleosome ,Animals ,Humans ,Amino Acids ,Scaffold/matrix attachment region ,Molecular Biology ,Genetics ,Cell Biology ,DNA ,Linker DNA ,Chromatin ,Cell biology ,Nucleosomes ,Protein Structure, Tertiary ,Histone ,biology.protein ,Protein Processing, Post-Translational - Abstract
The compaction of genomic DNA into chromatin has profound implications for the regulation of key processes such as transcription, replication and DNA repair. Nucleosomes, the repeating building blocks of chromatin, vary in the composition of their histone protein components. This is the result of the incorporation of variant histones and post-translational modifications of histone amino acid side chains. The resulting changes in nucleosome structure, stability and dynamics affect the compaction of nucleosomal arrays into higher-order structures. It is becoming clear that chromatin structures are not nearly as uniform and regular as previously assumed. This implies that chromatin structure must also be viewed in the context of specific biological functions.
- Published
- 2012
39. Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer
- Author
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Shalima S. Nair, Mark D. Robinson, David J. Tremethick, Jenny Z. Song, Clare Stirzaker, Aaron L. Statham, Kate I. Patterson, Fatima Valdes-Mora, Dario Strbenac, Susan J. Clark, University of Zurich, and Clark, Susan J
- Subjects
Male ,Transcriptional Activation ,2716 Genetics (clinical) ,animal structures ,medicine.disease_cause ,Models, Biological ,Epigenesis, Genetic ,Histones ,Epigenetics of physical exercise ,1311 Genetics ,Cell Line, Tumor ,Neoplasms ,Genetics ,medicine ,Humans ,Genes, Tumor Suppressor ,Epigenetics ,Cancer epigenetics ,Promoter Regions, Genetic ,Genetics (clinical) ,Regulation of gene expression ,biology ,Research ,Prostatic Neoplasms ,Promoter ,Acetylation ,Oncogenes ,DNA Methylation ,10124 Institute of Molecular Life Sciences ,Nucleosomes ,Gene Expression Regulation, Neoplastic ,Protein Transport ,Histone ,DNA methylation ,embryonic structures ,biology.protein ,570 Life sciences ,Transcription Initiation Site ,Carcinogenesis - Abstract
Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.
- Published
- 2012
40. A unified phylogeny-based nomenclature for histone variants
- Author
-
George A. M. Cross, Jan Postberg, Detlef Doenecke, Geneviève Almouzni, Bing Zhu, Robert Schneider, Stefan Dimitrov, Sarah Holec, David J. Tremethick, Steven E. Jacobsen, Maria-Elena Torres-Padilla, Barbara A. Hamkalo, Harmit S. Malik, John R. Pehrson, Mohan Singh, Saadi Khochbin, Liwang Cui, Kami Ahmad, W. Zacheus Cande, M. Mitchel Smith, Kinga Kamieniarz, Sandra B. Hake, Simon W. L. Chan, William M. Bonner, Martin A. Gorovsky, Benjamin Loppin, Steven Henikoff, Jakob H. Waterborg, Ramesh Yelagandula, Eric M. Thompson, Heike Wollmann, Frédéric Berger, José M. Eirín-López, Prem L. Bhalla, Juan Ausió, Andreas G. Ladurner, Brian P. Chadwick, John A Latham, Bryan M. Turner, Paul B. Talbert, David Landsman, William F. Marzluff, Basic Sciences Division, Fred Hutchinson Cancer Research Center [Seattle] (FHCRC)-Howard Hughes Medical Institute (HHMI), Department of Biological Chemistry and Molecular Pharmacology (DBCMP), Harvard Medical School [Boston] (HMS), Dynamique nucléaire et plasticité du génome (DNPG), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry and Microbiology, University of Victoria [Canada] (UVIC), Temasek Lifesciences Laboratory, National University of Singapore (NUS), Plant Molecular Biology and Biotechnology Group, University of Melbourne-Melbourne School of Land and Environment, Laboratory of Molecular Pharmacology, National Cancer Institute (NCI)-National Institute of Health (NIH)-Centre for Cancer Research (CCR), Department of Molecular & Cell Biology [Berkeley], University of California [Berkeley] (UC Berkeley), University of California (UC)-University of California (UC), Department of Biological Science, Florida State University [Tallahassee] (FSU), Department of Plant Biology, Howard Hughes Medical Institute (HHMI)-University of California [Davis] (UC Davis), University of California (UC)-University of California (UC)-Davis campus-Gordon and Betty Moore Foundation, Laboratory of Molecular Parasitology, Rockefeller University [New York], Department of Entomology, Pennsylvania State University (Penn State), Penn State System-Penn State System, INSERM U823, équipe 4 (Chromatine et Epigénétique), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Biochemistry, Georg-August-University = Georg-August-Universität Göttingen, Department of Cellular and Molecular Biology, University of A Coruña (UDC), Department of Biology, University of Rochester [USA], Center for Integrated Protein Science (CIPSM), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Ludwig-Maximilians-Universität München (LMU)-Helmholtz Zentrum München = German Research Center for Environmental Health, Department of Molecular Biology and Biochemistry, University of California [Irvine] (UC Irvine), Department of Molecular Cellular and Developmental Biology, University of California [Los Angeles] (UCLA), University of California (UC)-University of California (UC)-Howard Hughes Medical Institute (HHMI), Max Planck Institute of Immunobiology and Epigenetics (MPI-IE), Max-Planck-Gesellschaft, INSERM U823, équipe 6 (Epigénétique et Signalisation Cellulaire), Department of Physiological Chemistry, Butenandt Institute and Biomedical Center-Ludwig-Maximilians University [Munich] (LMU), National Center for Biotechnology Information (NCBI), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Program in Molecular Biology and Biotechnology, University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC)-University of North Carolina System (UNC), Department of Animal Biology, University of Pennsylvania, HELIOS Medical Centre Wuppertal, Witten/Herdecke University-Paediatrics Centre, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Department of Biological Sciences [Bergen] (BIO / UiB), University of Bergen (UiB), Genome Biology Department, Australian National University (ANU)-The John Curtin School of Medical Research, College of Medical and Dental Sciences, University of Birmingham [Birmingham], Cell Biology and Biophysics, School of Biological Sciences-University of Missouri [Kansas City] (UMKC), University of Missouri System-University of Missouri System, Chromatin Lab, National Institute of Biological Sciences, We are grateful for support from EMBO, the Howard Hughes Medical Institute, and the Intramural Research Program of the NIH, National Library of Medicine, University of California [Berkeley], University of California-University of California, University of California-University of California-Davis campus-Gordon and Betty Moore Foundation, University of Göttingen - Georg-August-Universität Göttingen, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Helmholtz-Zentrum München (HZM)-Ludwig Maximilian University of Munich [Germany] (LMU München), University of California [Irvine] (UCI), University of California-University of California-Howard Hughes Medical Institute (HHMI), Butenandt Institute and Biomedical Center-Ludwig Maximilians University of Munich, Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, University of Pennsylvania [Philadelphia], University of Virginia [Charlottesville], University of California [Davis] (UC Davis), University of California-University of California-Howard Hughes Medical Institute (HHMI)-Davis campus-Gordon and Betty Moore Foundation, and BMC, Ed.
- Subjects
Genetics ,0303 health sciences ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,lcsh:QH426-470 ,Phylogenetic tree ,030302 biochemistry & molecular biology ,[SDV.GEN] Life Sciences [q-bio]/Genetics ,Review ,Biology ,Histone H4 ,lcsh:Genetics ,03 medical and health sciences ,Histone H3 ,Histone phosphorylation ,Phylogenetics ,Evolutionary biology ,Histone H2A ,Clade ,Nomenclature ,Molecular Biology ,030304 developmental biology - Abstract
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.
- Published
- 2012
41. High mobility group proteins 14 and 17 can space nucleosomal particles deficient in histones H2A and H2B creating a template that is transcriptionally active
- Author
-
David J. Tremethick
- Subjects
Nucleosomal Repeat Length ,biology ,Base pair ,Cell Biology ,Biochemistry ,Molecular biology ,Chromatin ,chemistry.chemical_compound ,Histone ,High-mobility group ,chemistry ,biology.protein ,Biophysics ,Nucleosome ,Molecular Biology ,DNA ,Micrococcal nuclease - Abstract
Recently, using a well defined nucleosomal assembly system, we demonstrated that high mobility group proteins (HMGs) 14 and 17 can organize nucleosomes into a regular array with a nucleosomal repeat length of 160-165 base pairs in vitro. Interestingly, such a short repeat length has been described for lower eukaryotes and for active chromatin. To begin to investigate how these proteins may prevent the close packing of nucleosomes, assembly reactions were carried out in which the relative amounts of HMGs 14 and 17, histones H2A and H2B, and the N1/N2.(H3, H4) complex were varied in assembly reactions. Under conditions in which histones H2A and H2B were limiting and in the absence of HMGs 14 and 17, micrococcal nuclease digestion of the assembled product produced a ladder of DNA fragments that was much less well defined and which included DNA that was associated with subnucleosomal structures. The apparent repeat length for this chromatin template was around 125 base pairs. Most interestingly, when HMGs 14 and 17 were added to this assembly reaction, "nucleosome-like" structures were reassembled as shown by the restoration of a regular, well defined ladder of DNA fragments upon micrococcal nuclease digestion. The apparent repeat length increased from 125 to approximately 145 base pairs. Analysis of the protein composition of chromatin formed in the presence or absence of HMGs 14 and 17 reveals that HMGs 14 and 17 might be able to substitute for a histone H2A-H2B dimer in a H2A/H2B-deficient nucleosome. The ability to form a regularly spaced nucleosomal template is also lost when excess HMGs 14 and 17 are used in assembly reactions. Spacing can be restored by the addition of poly(glutamate, alanine), a chemical polymer of negative charge, which may indicate that carrier proteins (specific or nonspecific) may be required for the proper incorporation of all chromatin assembly components into chromatin in vivo. Finally, although the mechanism of action is not known, HMGs 14 and 17 can partially overcome inhibition of initiation of transcription caused by the formation of nucleosomal particles deficient in histones H2A and H2B.
- Published
- 1994
42. High mobility group proteins 14 and 17 can space nucleosomes in vitro
- Author
-
H.R. Drew and David J. Tremethick
- Subjects
Cell Biology ,Solenoid (DNA) ,Hmg protein ,Biology ,Biochemistry ,Molecular biology ,Chromatin ,Histone H1 ,Histone methylation ,Biophysics ,Histone code ,Nucleosome ,Histone octamer ,Molecular Biology - Abstract
Recently we partially purified from Xenopus laevis ovaries a novel, ATP-dependent, spacing activity that can convert a DNA template consisting of irregularly spaced nucleosomes into a chromatin structure made up of regularly spaced nucleosomes with a repeat length of 160-165 base pairs. In a second independent step, the longer spacing of higher eukaryotic chromatin can be generated by the addition of histone H1. The partially purified spacing fraction contains several proteins that display chromatographic properties and mobilities on polyacrylamide gels similar to high mobility group (HMG) proteins. For that reason, different HMG proteins were tested for their ability to generate chromatin structures with regularly spaced nucleosomes. In this report, using two different nucleosome assembly systems, we show that the addition of phosphorylated HMGs 14 and 17 to the histone octamer results in the formation of chromatin with a repeat length of 160-165 base pairs. The results are similar to those obtained from studies of chromatin structure in simple cells, such as fungi and yeast, and in active genes.
- Published
- 1993
43. Partial purification, from Xenopus laevis oocytes, of an ATP-dependent activity required for nucleosome spacing in vitro
- Author
-
David J. Tremethick and M Frommer
- Subjects
Nucleosomal Repeat Length ,Base pair ,Xenopus ,Cell Biology ,Biology ,biology.organism_classification ,Biochemistry ,Molecular biology ,Chromatin ,Histone H1 ,Biophysics ,Nucleosome ,A-DNA ,Histone octamer ,Molecular Biology - Abstract
A critical feature of chromatin with regard to structure and function is the regular spacing of nucleosomes. In vivo, spacing of nucleosomes occurs in at least two steps, but the mechanism is not understood. In this report, we have mimicked the two-step process in vitro. A novel spacing activity has been partially purified from Xenopus laevis ovaries. When this activity is added, either at the beginning or at the end of a nucleosomal assembly reaction, it can convert a DNA template consisting of irregularly spaced nucleosomes into a chromatin structure made up of regularly spaced nucleosomes with a repeat length of about 165 base pairs. The reaction requires ATP. Histone H1 is able to increase the nucleosomal repeat from 165 to 190 base pairs. This two-step increase in nucleosomal repeat length suggests that both the spacing activity and histone H1 contribute to generating repeat lengths of greater than 165 base pairs and that their contributions may be additive. Alternatively, the critical step in the spacing reaction may not be the formation of the 165-base pair repeat but may be the sliding of nucleosomes or the reorganization of the octamer structure induced by the spacing activity.
- Published
- 1992
44. Interplay between chromatin remodeling and epigenetic changes during lineage-specific commitment to granzyme B expression
- Author
-
Torsten, Juelich, Elissa L, Sutcliffe, Elissa, Sutcliffe, Alice, Denton, Yiqing, He, Peter C, Doherty, Christopher R, Parish, Christopher, Parish, Stephen J, Turner, Steven J, Turner, David J, Tremethick, David, Tremethick, and Sudha, Rao
- Subjects
CD4-Positive T-Lymphocytes ,Transcription, Genetic ,Immunology ,Gene Expression ,Biology ,CD8-Positive T-Lymphocytes ,Lymphocyte Activation ,Chromatin remodeling ,Granzymes ,GZMB ,Epigenesis, Genetic ,Histones ,Histone H3 ,Mice ,Histone H1 ,T-Lymphocyte Subsets ,Histone H2A ,Immunology and Allergy ,Histone code ,Animals ,Cell Lineage ,Promoter Regions, Genetic ,Epigenomics ,Oligonucleotide Array Sequence Analysis ,Microscopy, Confocal ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Differentiation ,Chromatin Assembly and Disassembly ,Flow Cytometry ,Molecular biology ,Gene Expression Regulation ,Histone methyltransferase ,RNA Polymerase II - Abstract
The role of chromatin remodeling and histone posttranslational modifications and how they are integrated to control gene expression during the acquisition of cell-specific functions is poorly understood. We show here that following in vitro activation of CD4+ and CD8+ T lymphocytes, both cell types show rapid histone H3 loss at the granzyme B (gzmB) proximal promoter region. However, despite the gzmB proximal promoter being remodeled in both T cell subsets, only CD8+ T cells express high levels of gzmB and display a distinct pattern of key epigenetic marks, notably differential H3 acetylation and methylation. These data suggest that for high levels of transcription to occur a distinct set of histone modifications needs to be established in addition to histone loss at the proximal promoter of gzmB.
- Published
- 2009
45. Dynamic histone variant exchange accompanies gene induction in T cells
- Author
-
Elissa L Sutcliffe, M. Louise Tierney, Danny Rangasamy, Sudha Rao, David J. Tremethick, Ian A. Parish, Torsten Juelich, Yi Qing He, Christopher R. Parish, and Peter J. Milburn
- Subjects
Antigens, Differentiation, T-Lymphocyte ,Transcriptional Activation ,Transcription, Genetic ,T-Lymphocytes ,Biology ,Hydroxamic Acids ,Chromatin remodeling ,Chromatography, Affinity ,Histones ,Histone H3 ,Jurkat Cells ,Histone H1 ,Antibody Specificity ,Antigens, CD ,Histone H2A ,Histone methylation ,Histone code ,Humans ,Immunoprecipitation ,Lectins, C-Type ,RNA, Messenger ,Promoter Regions, Genetic ,Molecular Biology ,Glucuronidase ,Cell Biology ,Articles ,Chromatin ,Kinetics ,Biochemistry ,Gene Expression Regulation ,Histone methyltransferase ,RNA Polymerase II ,Protein Processing, Post-Translational - Abstract
Changes in chromatin composition are often a prerequisite for gene induction. Nonallelic histone variants have recently emerged as key players in transcriptional control and chromatin modulation. While the changes in chromatin accessibility and histone posttranslational modification (PTM) distribution that accompany gene induction are well documented, the dynamics of histone variant exchange that parallel these events are still poorly defined. In this study, we have examined the changes in histone variant distribution that accompany activation of the inducible CD69 and heparanase genes in T cells. We demonstrate that the chromatin accessibility increases that accompany the induction of both of these genes are not associated with nucleosome loss but instead are paralleled by changes in histone variant distribution. Specifically, induction of these genes was paralleled by depletion of the H2A.Z histone variant and concomitant deposition of H3.3. Furthermore, H3.3 deposition was accompanied by changes in PTM patterns consistent with H3.3 enriching or depleting different PTMs upon incorporation into chromatin. Nevertheless, we present evidence that these H3.3-borne PTMs can be negated by recruited enzymatic activities. From these observations, we propose that H3.3 deposition may both facilitate chromatin accessibility increases by destabilizing nucleosomes and compete with recruited histone modifiers to alter PTM patterns upon gene induction.
- Published
- 2009
46. Specific patterns of histone marks accompany X chromosome inactivation in a marsupial
- Author
-
David J. Tremethick, Julie Chaumeil, Jennifer A. Marshall Graves, Edda Koina, and Ian K. Greaves
- Subjects
Genetics ,Dosage compensation ,X Chromosome ,Barr body ,Biology ,Fibroblasts ,biology.organism_classification ,X-inactivation ,Histones ,Histone ,Marsupialia ,X Chromosome Inactivation ,biology.protein ,Animals ,XIST ,Female ,Epigenetics ,X chromosome ,In Situ Hybridization, Fluorescence ,Marsupial - Abstract
The inactivation of one of the two X chromosomes in female placental mammals represents a remarkable example of epigenetic silencing. X inactivation occurs also in marsupial mammals, but is phenotypically different, being incomplete, tissue-specific and paternal. Paternal X inactivation occurs also in the extraembryonic cells of rodents, suggesting that imprinted X inactivation represents a simpler ancestral mechanism. This evolved into a complex and random process in placental mammals under the control of the XIST gene, involving notably variant and modified histones. Molecular mechanisms of X inactivation in marsupials are poorly known, but occur in the absence of an XIST homologue. We analysed the specific pattern of histone modifications using immunofluorescence on metaphasic chromosomes of a model kangaroo, the tammar wallaby. We found that all active marks are excluded from the inactive X in marsupials, as in placental mammals, so this represents a common feature of X inactivation throughout mammals. However, we were unable to demonstrate the accumulation of inactive histone marks, suggesting some fundamental differences in the molecular mechanism of X inactivation between marsupial and placental mammals. A better understanding of the epigenetic mechanisms underlying X inactivation in marsupials will provide important insights into the evolution of this complex process.
- Published
- 2008
47. Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines
- Author
-
Danny Rangasamy, David J. Tremethick, and Ian K. Greaves
- Subjects
Gene knockdown ,Ecdysone ,Expression vector ,Genetic Vectors ,RNA ,Biology ,Transfection ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Cell Line ,Small hairpin RNA ,Mice ,Gene Expression Regulation ,Genetic Techniques ,RNA interference ,Gene expression ,Gene silencing ,Animals ,Humans ,RNA Interference ,Cloning, Molecular ,RNA, Small Interfering ,Gene - Abstract
RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.
- Published
- 2008
48. Histone-mediated transduction as an efficient means for gene delivery
- Author
-
Kylie M. Wagstaff, David J. Tremethick, Dominic J. Glover, and David A. Jans
- Subjects
Recombinant Fusion Proteins ,Molecular Sequence Data ,Nuclear Localization Signals ,DNA, Recombinant ,Gene delivery ,Karyopherins ,Protein Engineering ,Cell Line ,Histones ,Histone H1 ,Transduction, Genetic ,Histone H2A ,Drug Discovery ,Chlorocebus aethiops ,medicine ,Histone H2B ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Pharmacology ,Cell Nucleus ,biology ,Genetic Therapy ,Molecular biology ,Cell biology ,Rats ,Cell nucleus ,medicine.anatomical_structure ,Histone ,COS Cells ,biology.protein ,Molecular Medicine ,Nuclear transport ,Dimerization ,Nuclear localization sequence ,HeLa Cells ,Protein Binding - Abstract
Gene delivery into the nucleus of eukaryotic cells is inefficient, largely because of the significant barriers within the target cell of the plasma membrane and nuclear envelope. Recently, a group of basic proteins, including the HIV-1 Tat protein and the four core histones, have been shown to enter cells through a novel energy- and receptor-independent manner. Here, we show that engineered histone H2B proteins are able to mediate the efficient delivery of either green fluorescent protein or DNA into HeLa cells through the process of "Histone-Mediated Transduction" (HMT), with further enhancement achieved by utilizing a dimer of histones H2B and H2A. Subsequent nuclear delivery was accelerated approximately two-fold by the addition of an optimized nuclear localization signal to histone H2B, thereby increasing the affinity of interaction with components of the cellular nuclear import machinery, resulting in increased expression of a reporter gene. Further, we demonstrate that the domains responsible for this histone transduction are located in the N-terminal tail and globular regions of histone H2B. HMT represents a new, efficient, and technically non-demanding means to deliver DNA to the nucleus of intact cells, including embryonic stem cells, which has important applications in gene therapy and cancer therapeutics.
- Published
- 2007
49. H2A.Z contributes to the unique 3D structure of the centromere
- Author
-
Danny Rangasamy, David J. Tremethick, Patricia Ridgway, and Ian K. Greaves
- Subjects
Models, Molecular ,animal structures ,Heterochromatin ,Chromosomal Proteins, Non-Histone ,Centromere ,Biology ,DNA, Satellite ,Autoantigens ,Cell Line ,Histones ,Mice ,X Chromosome Inactivation ,Centromere Protein A ,Histone H2A ,Nucleosome ,Animals ,Humans ,Pericentric heterochromatin ,Genetics ,Chromosomes, Human, X ,Multidisciplinary ,Chromosome ,Biological Sciences ,Cell biology ,Chromatin - Abstract
Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure.
- Published
- 2006
50. Quantitative analysis of HP1alpha binding to nucleosomal arrays
- Author
-
David J. Tremethick, Jun Y. Fan, and Jiansheng Zhou
- Subjects
Chromosomal Proteins, Non-Histone ,Titrimetry ,Biology ,Chromatin Assembly and Disassembly ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Chromatin remodeling ,Chromatin ,Cell biology ,Nucleosomes ,Protein Structure, Tertiary ,Histone H3 ,Mice ,Spectrometry, Fluorescence ,Histone H1 ,Models, Chemical ,Nonlinear Dynamics ,Chromobox Protein Homolog 5 ,Histone H2A ,Histone code ,Nucleosome ,Animals ,Molecular Biology ,Chromatin immunoprecipitation ,Protein Binding - Abstract
Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The “histone code” hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1α to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1α (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.
- Published
- 2006
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