22 results on '"Maxim Nekrasov"'
Search Results
2. Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans
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Henry J. Sutton, Racheal Aye, Azza H. Idris, Rachel Vistein, Eunice Nduati, Oscar Kai, Jedida Mwacharo, Xi Li, Xin Gao, T. Daniel Andrews, Marios Koutsakos, Thi H.O. Nguyen, Maxim Nekrasov, Peter Milburn, Auda Eltahla, Andrea A. Berry, Natasha KC, Sumana Chakravarty, B. Kim Lee Sim, Adam K. Wheatley, Stephen J. Kent, Stephen L. Hoffman, Kirsten E. Lyke, Philip Bejon, Fabio Luciani, Katherine Kedzierska, Robert A. Seder, Francis M. Ndungu, and Ian A. Cockburn
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B cell memory ,atypical B cells ,alternative B cell lineage ,malaria ,sporozoite ,vaccination ,Biology (General) ,QH301-705.5 - Abstract
Summary: The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.
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- 2021
- Full Text
- View/download PDF
3. 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
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4. POWERDRESS-mediated histone deacetylation is essential for thermomorphogenesis in Arabidopsis thaliana.
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Celine Tasset, Avilash Singh Yadav, Sridevi Sureshkumar, Rupali Singh, Lennard van der Woude, Maxim Nekrasov, David Tremethick, Martijn van Zanten, and Sureshkumar Balasubramanian
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Genetics ,QH426-470 - Abstract
Ambient temperature affects plant growth and even minor changes can substantially impact crop yields. The underlying mechanisms of temperature perception and response are just beginning to emerge. Chromatin remodeling, via the eviction of the histone variant H2A.Z containing nucleosomes, is a critical component of thermal response in plants. However, the role of histone modifications remains unknown. Here, through a forward genetic screen, we identify POWERDRESS (PWR), a SANT-domain containing protein known to interact with HISTONE DEACETYLASE 9 (HDA9), as a novel factor required for thermomorphogenesis in Arabidopsis thaliana. We show that mutations in PWR impede thermomorphogenesis, exemplified by attenuated warm temperature-induced hypocotyl/petiole elongation and early flowering. We show that inhibitors of histone deacetylases diminish temperature-induced hypocotyl elongation, which demonstrates a requirement for histone deacetylation in thermomorphogenesis. We also show that elevated temperature is associated with deacetylation of H3K9 at the +1 nucleosomes of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and YUCCA8 (YUC8), and that PWR is required for this response. There is global misregulation of genes in pwr mutants at elevated temperatures. Meta-analysis revealed that genes that are misregulated in pwr mutants display a significant overlap with genes that are H2A.Z-enriched in their gene bodies, and with genes that are differentially expressed in mutants of the components of the SWR1 complex that deposits H2A.Z. Our findings thus uncover a role for PWR in facilitating thermomorphogenesis and suggest a potential link between histone deacetylation and H2A.Z nucleosome dynamics in plants.
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- 2018
- Full Text
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5. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.
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Tatiana A Soboleva, Brian J Parker, Maxim Nekrasov, Gene Hart-Smith, Ying Jin Tay, Wei-Quan Tng, Marc Wilkins, Daniel Ryan, and David J Tremethick
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Genetics ,QH426-470 - 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.
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- 2017
- Full Text
- View/download PDF
6. CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells
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Yuwei Hao, Bahar Miraghazadeh, Rochna Chand, Ainsley R. Davies, Chelisa Cardinez, Kristy Kwong, Morgan B. Downes, Rebecca A. Sweet, Pablo F. Cañete, Lloyd J. D’Orsogna, David A. Fulcher, Sharon Choo, Desmond Yip, Geoffrey Peters, Sonia Yip, Matthew J. Witney, Maxim Nekrasov, Zhi-Ping Feng, David C. Tscharke, Carola G. Vinuesa, Matthew C. Cook, Vinuesa, Carola G [0000-0001-9799-0298], Cook, Matthew C [0000-0002-3331-9363], and Apollo - University of Cambridge Repository
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Model organisms ,CTLA4 ,CD4-Positive T-Lymphocytes ,Human Biology & Physiology ,B-Lymphocytes ,FOS: Clinical medicine ,Terminal differentiation ,Immunology ,Gene Expression ,Cell Differentiation ,Cell Biology ,Cell exhaustion ,Infectious Diseases ,CD57 Antigens ,Cytotoxic CD4+ T cells ,Immunology and Allergy ,Immunodeficiency ,Humans ,CTLA-4 Antigen ,Genetics & Genomics ,CD57 - Abstract
As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.
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- 2023
7. CTLA4 protects against maladaptive cytotoxicity during differentiation of effector and follicular CD4+ T cells
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Matthew Cook, Yuwei Hao, Bahar Miraghazadeh, Rochna Chand, Ainsley Davies, Chelisa Cardinez, Kristy Kwong, Morgan Downes, Rebecca Sweet, Pablo Cañete, Lloyd D’Orsogna, David Fulcher, Sharon Choo, Desmond Yip, Geoffrey Peters, Sonia Yip, Matthew Witney, Maxim Nekrasov, Zhi-Ping Feng, David Tscharke, and Carola Vinuesa
- Abstract
As chronic antigenic stimulation from infection and autoimmunity are features of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T cell differentiation, and explain how environmental exposures modify clinical phenotypes conferred by single gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while CD57+ CD4+ T cells are normally rare in the circulation, we found that they are increased in patients with PAD, and markedly by CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil. Circulating CD57+ CD4+ T cells (CD4cyt) exhibit a cytotoxic transcriptome similar to CD8+ effectors, can kill B cells, and inhibit B cell responses. CTLA4 restrains the formation of CD4cyt. While CD57 also marks an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset has a precursor of exhaustion transcriptomic signature marked by TCF7, TOX, ID3, and constitutive expression of CTLA4, and are robust to becoming cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T cell phenotypes of cytotoxicity and exhaustion are compartmentalized between blood and germinal centres. CTLA4 is a key modifier of CD4+ T cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated in CTLA4 deficiency by the environmental stimulus of infection.
- Published
- 2023
8. ZEB2 regulates the development of CD11c+ atypical B cells
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Xin Gao, Qian Shen, Jonathan A. Roco, Katie Frith, C. Mee Ling Munier, Maxim Nekrasov, Becan Dalton, Jin-Shu He, Rebecca Jaeger, Matthew C. Cook, John J. Zaunders, and Ian A. Cockburn
- Abstract
CD11c+ atypical B cells (ABC) are an alternative memory B cell lineage identified both in normal immune responses as well as pathogenic responses in autoimmunity. While it is clear that ABCs have a distinct transcriptional program, the factors that direct this program have not been identified. Here, we generated a human tonsil single-cell RNA-seq dataset and identified candidate transcription factors associated with the ABC population. We selected 8 of these transcription factors for further analysis based on their conserved expression in mouse ABC bulk RNA-seq datasets. Using an optimized CRSPR-Cas9 knockdown method we found that only zinc finger E-box binding homeobox 2 (Zeb2) knock-out impaired ABC formation. To assess the role of Zeb2 in ABC formation in vivo we used Zeb2fl/fl mice crossed to a CD23Cre line. Germinal center and plasma cell responses in these mice after Plasmodium sporozoite immunization were largely unaltered but we observed a specific defect in ABC formation. We further determined that ZEB2 haploinsufficient Mowat Wilson syndrome patients also have decreased circulating ABCs in the blood, supporting a role for this transcription factor in humans as well as mice. In sum, we identified Zeb2 as a key TF governing the formation of ABCs.
- Published
- 2022
9. 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.
- Published
- 2021
10. 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.
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- 2021
11. 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
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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.
- Published
- 2019
12. Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes
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Maxim Nekrasov and David J. Tremethick
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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
13. Atypical B cells are a normal component of immune responses to vaccination and infection in humans
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Thi H. O. Nguyen, B. Kim Lee Sim, Rachel Vistein, Francis M. Ndungu, Adam K. Wheatley, Eunice Nduati, Fabio Luciani, Stephen L. Hoffman, Stephen J. Kent, Peter J. Milburn, Philip Bejon, Xin Gao, Maxim Nekrasov, Henry J. Sutton, Sumana Chakravarty, T. Daniel Andrews, Xi Li, Oscar Kai, Jedida Mwacharo, Kirsten E. Lyke, Robert A. Seder, Azza H. Idris, Andrea A. Berry, Natasha Kc, Racheal Aye, Marios Koutsakos, Ian A. Cockburn, Katherine Kedzierska, and Auda Ethala
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education.field_of_study ,Population ,CD11c ,Biology ,medicine.disease_cause ,Autoimmunity ,Transcriptome ,medicine.anatomical_structure ,Immune system ,Antigen ,Immunology ,medicine ,biology.protein ,Antibody ,education ,B cell - Abstract
The full diversity of the circulating human B cell compartment is unknown. Flow cytometry analysis suggests that in addition to naïve and memory B cells, there exists a population of CD11c+, CD27− CD21− “atypical” B cells, that are associated with chronic or recurrent infection and autoimmunity. We used single cell RNA-seq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and individuals naturally-exposed to recurrent malaria infections. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and an atypical-like lineage. Surprisingly, the atypical lineage was common in both malaria exposed individuals and non-exposed healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that atypical lineage cells in healthy individuals lack many atypical B markers and thus represent an undercounted cryptic population. We further determined using antigen specific probes that atypical cells can be induced by primary vaccination in humans and can be recalled upon boosting. Collectively these data suggest that atypical cells are not necessarily pathogenic but can be a normal component of B responses to antigen.
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- 2020
14. Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans
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B. Kim Lee Sim, Henry J. Sutton, Andrea A. Berry, Auda A. Eltahla, Rachel Vistein, Natasha Kc, Kirsten E. Lyke, Francis M. Ndungu, Sumana Chakravarty, Adam K. Wheatley, Racheal Aye, Thi H. O. Nguyen, Oscar Kai, Stephen J. Kent, Azza H. Idris, Peter J. Milburn, Katherine Kedzierska, Marios Koutsakos, Xi Li, Jedida Mwacharo, Ian A. Cockburn, Maxim Nekrasov, T. Daniel Andrews, Eunice Nduati, Fabio Luciani, Stephen L. Hoffman, Philip Bejon, Robert A. Seder, and Xin Gao
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Adult ,Male ,0301 basic medicine ,Plasmodium ,Lineage (genetic) ,atypical B cells ,Population ,Antibodies, Protozoan ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Malaria Vaccines ,medicine ,sporozoite ,Humans ,Compartment (development) ,RNA-Seq ,Child ,education ,lcsh:QH301-705.5 ,B cell ,B-Lymphocytes ,education.field_of_study ,Vaccination ,Plasmodium falciparum ,biology.organism_classification ,Malaria ,030104 developmental biology ,medicine.anatomical_structure ,lcsh:Biology (General) ,Immunization ,alternative B cell lineage ,B cell memory ,Child, Preschool ,Immunology ,Female ,030217 neurology & neurosurgery - Abstract
Summary: The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.
- Published
- 2021
15. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B
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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
16. 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
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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
17. Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics
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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
18. Decoding of Methylated Histone H3 Tail by the Pygo-BCL9 Wnt Signaling Complex
- Author
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Jürg Müller, Marc Fiedler, Juliusz Mieszczanek, Mariann Bienz, Maxim Nekrasov, María José Sánchez-Barrena, Phil Evans, and Vladimir Rybin
- Subjects
Models, Molecular ,Protein Conformation ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Crystallography, X-Ray ,Methylation ,Article ,Histones ,Histone H3 ,Histone H2A ,Animals ,Drosophila Proteins ,Humans ,Histone code ,Amino Acid Sequence ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Histone binding ,Regulation of gene expression ,Binding Sites ,biology ,Lysine ,Intracellular Signaling Peptides and Proteins ,DNA ,Cell Biology ,Molecular biology ,Neoplasm Proteins ,Cell biology ,Wnt Proteins ,Drosophila melanogaster ,Histone ,SIGNALING ,PHD finger ,Multiprotein Complexes ,Histone methyltransferase ,biology.protein ,Peptides ,Sequence Alignment ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
Summary Pygo and BCL9/Legless transduce the Wnt signal by promoting the transcriptional activity of β-catenin/Armadillo in normal and malignant cells. We show that human and Drosophila Pygo PHD fingers associate with their cognate HD1 domains from BCL9/Legless to bind specifically to the histone H3 tail methylated at lysine 4 (H3K4me). The crystal structures of ternary complexes between PHD, HD1, and two different H3K4me peptides reveal a unique mode of histone tail recognition: efficient histone binding requires HD1 association, and the PHD-HD1 complex binds preferentially to H3K4me2 while displaying insensitivity to methylation of H3R2. Therefore, this is a prime example of histone tail binding by a PHD finger (of Pygo) being modulated by a cofactor (BCL9/Legless). Rescue experiments in Drosophila indicate that Wnt signaling outputs depend on histone decoding. The specificity of this process provided by the Pygo-BCL9/Legless complex suggests that this complex facilitates an early step in the transition from gene silence to Wnt-induced transcription.
- Published
- 2008
- Full Text
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19. 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
20. Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes
- Author
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Hendrik G. Stunnenberg, Matthias Wilm, Adrian Cohen, Bernadett Papp, Sven Fraterman, Tetyana Klymenko, Katarzyna Oktaba, Maxim Nekrasov, Jürg Müller, and Thomas Köcher
- Subjects
Cell Extracts ,Embryo, Nonmammalian ,Polycomb-Group Proteins ,Repressor ,Genes, Insect ,macromolecular substances ,Methylation ,Article ,General Biochemistry, Genetics and Molecular Biology ,PcG ,Substrate Specificity ,Histones ,gene silencing ,Histone methylation ,Polycomb-group proteins ,Animals ,Drosophila Proteins ,histone methylation ,Protein Methyltransferases ,Molecular Biology ,trxG ,Genetics ,General Immunology and Microbiology ,biology ,Lysine ,General Neuroscience ,fungi ,Histone-Lysine N-Methyltransferase ,musculoskeletal system ,Chromatin ,Repressor Proteins ,Drosophila melanogaster ,Histone ,Histone methyltransferase ,Histone Methyltransferases ,biology.protein ,Drosophila ,lipids (amino acids, peptides, and proteins) ,PRC1 ,PRC2 - Abstract
PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state.
- Published
- 2007
21. Nucleosome binding and histone methyltransferase activity of Drosophila PRC2
- Author
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Brigitte Wild, Maxim Nekrasov, and Jürg Müller
- Subjects
Histone methyltransferase activity ,Chromosomal Proteins, Non-Histone ,Xenopus ,Scientific Report ,Electrophoretic Mobility Shift Assay ,macromolecular substances ,Biochemistry ,Methylation ,Histone H4 ,Histone H3 ,Histone methylation ,Genetics ,Nucleosome ,Animals ,Drosophila Proteins ,Gene Silencing ,Protein Methyltransferases ,Molecular Biology ,Polycomb Repressive Complex 1 ,Nucleosome binding ,biology ,Polycomb Repressive Complex 2 ,Nuclear Proteins ,Histone-Lysine N-Methyltransferase ,Molecular biology ,Chromatin ,Cell biology ,Nucleosomes ,Repressor Proteins ,Protein Subunits ,Histone ,Histone methyltransferase ,biology.protein ,Histone Methyltransferases ,Drosophila ,Retinoblastoma-Binding Protein 4 ,Baculoviridae ,Molecular Chaperones ,Plasmids - Abstract
The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55. Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. Here, we investigate the function of the PRC2 subunits. We show that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, we show that Su(z)12–Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. We find that nucleosome binding of PRC2 is not sufficient for histone methylation and that only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity.
- Published
- 2005
22. The mRNA-binding protein YB-1 (p50) prevents association of the eukaryotic initiation factor eIF4G with mRNA and inhibits protein synthesis at the initiation stage
- Author
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Elizaveta A. Kovrigina, Konstantin G. Chernov, John W.B. Hershey, Lev P. Ovchinnikov, Maxim Nekrasov, Valentina Evdokimova, Maria Ivshina, and Adri A. M. Thomas
- Subjects
Cytoplasm ,Sucrose ,Reticulocytes ,Time Factors ,Blotting, Western ,Biology ,Biochemistry ,Models, Biological ,chemistry.chemical_compound ,Polysome ,Eukaryotic initiation factor ,Translational regulation ,P-bodies ,Centrifugation, Density Gradient ,Initiation factor ,Animals ,RNA, Messenger ,Molecular Biology ,Cell-Free System ,Dose-Response Relationship, Drug ,EIF4G ,EIF4E ,Nuclear Proteins ,Cell Biology ,Blotting, Northern ,Molecular biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Internal ribosome entry site ,NFI Transcription Factors ,chemistry ,Protein Biosynthesis ,CCAAT-Enhancer-Binding Proteins ,RNA ,Rabbits ,Y-Box-Binding Protein 1 ,Eukaryotic Initiation Factor-4G ,Ribosomes ,Protein Binding ,Transcription Factors - Abstract
The cytoplasmic messenger ribonucleoprotein particles of mammalian somatic cells contain the protein YB-1, also called p50, as a major core component. YB-1 is multifunctional and involved in regulation of mRNA transcription and translation. Our previous studies demonstrated that YB-1 stimulates initiation of translation in vitro at a low YB-1/mRNA ratio, whereas an increase of YB-1 bound to mRNA resulted in inhibition of protein synthesis in vitro and in vivo. Here we show that YB-1-mediated translation inhibition in a rabbit reticulocyte cell-free system is followed by a decay of polysomes, which is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it affects neither elongation nor termination of polypeptide chains and only occurs at the stage of initiation. YB-1 induces accumulation of mRNA in the form of free messenger ribonucleoprotein particles, i.e. it blocks mRNA association with the small ribosomal subunit. The accumulation is accompanied by eukaryotic initiation factor eIF4G dissociation from mRNA. The C-terminal domain of YB-1 is responsible for inhibition of translation as well as the disruption of mRNA interaction with eIF4G.
- Published
- 2003
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