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Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3–9, HP1a or Su(var)4–20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation., MICIN/AEI 10.13039/501100011033 [BFU2015-65082-P and PGC2018-094538-B-100]; ‘FEDER, una manera de hacer Europa’; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; this work was carried out within the framework of the ‘Centre de Referencia en Biotecnologia’ of ` the Generalitat de Catalunya. Funding for open access charge: MINECO [PGC2018-094538-B-100]. Conflict of interest statement. None declared.

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels., This work was funded by Spanish Ministry of Health Instituto Carlos III-FIS (grant numbers PI17/01822 and PI20/01562) and the Fanconi Research Fund awarded to C.K. and A.R.-E.; the German Research Foundation Clinical Research Unit “Male Germ Cells” (DFG CRU326), grants to F.T. and C.F.; the National Institutes of Health (R01HD078641), awarded to D.F.C., L.N., and K.I.A.; the Netherlands Organisation for Scientific Research (918-15-667) as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V.; and the MICINN (BFU2015-65082-P and PGC2018-094538-B-I00), the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204), and the European Community FEDER program to F.A.

Post-translational modifications (PTMs) of histones are important epigenetic determinants and specific core histones PTMs correlate with functional chromatin states. However, despite linker histone H1s are heavily post-translationally modified, little is known about the genomic distribution of H1s PTMs and their association with epigenetic chromatin states. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase cells, it is also detected at intercalary heterochromatin. ChIPseq experiments show that dH1K27me2 enriched regions cluster at both the assembled and unassembled heterochromatin regions of all four Drosophila chromosomes. More than 98% of the dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. We also show that dH1K27me2 is independent of H3K9 methylation, as it is equally detected in flies carrying a H3K9R mutation. Moreover, dH1K27me2 is not affected by depletion of Su(var)3-9, HP1a and Su(var)4-20. Altogether these results suggest that dH1K27me2 is a novel epigenetic mark of Drosophila heterochromatin that acts upstream of the major Su(var)3-9/HP1a pathway of heterochromatin formation.

Centromere identity is determined by the specific deposition of CENP-A, a histone H3 variant localizing exclusively at centromeres. Increased CENP-A expression, which is a frequent event in cancer, causes mislocalization, ectopic kinetochore assembly and genomic instability. Proteolysis regulates CENP-A expression and prevents its misincorporation across chromatin. How proteolysis restricts CENP-A localization to centromeres is not well understood. Here we report that, in Drosophila, CENP-ACID expression levels are regulated throughout the cell cycle by the combined action of SCFPpa and APC/CCdh1. We show that SCFPpa regulates CENP-ACID expression in G1 and, importantly, in S-phase preventing its promiscuous incorporation across chromatin during replication. In G1, CENP-ACID expression is also regulated by APC/CCdh1. We also show that Cal1, the specific chaperone that deposits CENP-ACID at centromeres, protects CENP-ACID from SCFPpa-mediated degradation but not from APC/CCdh1-mediated degradation. These results suggest that, whereas SCFPpa targets the fraction of CENP-ACID that is not in complex with Cal1, APC/CCdh1 mediates also degradation of the Cal1-CENP-ACID complex and, thus, likely contributes to the regulation of centromeric CENP-ACID deposition., MINECO [BFU2015-65082-P]; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; European Community FEDER program; ‘Centre de Referència en Biotecnologia’ of the Generalitat de Catalunya. Funding for open access charge: MINECO [BFU2015-65082-P]; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; European Community FEDER program.

© 2021 The Authors., Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too., This work was supported by grants from MICINN (BFU2015-65082-P and PGC2018-094538-B-I00), the Generalitat de Catalunya (SGR2009-1023, SGR2014-204) and the European Community FEDER program to F.A., and from National Research, Development and Innovation Office (OTKA-116372) and Ministry for National Economy of Hungary (GINOP-2.3.2-15-2016-00032) to L.H./I.M.B.

© The Author(s) 2020, Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression., This work was supported by grants from MICINN (BFU2015-65082-P and PGC2018-094538-B-I00), the Generalitat de Catalunya (SGR2009-1023, SGR2014-204) and the European Community FEDER program to F.A., and the Ministry for National Economy of Hungary (GINOP-2.3.2-15-2016-00032 and GINOP-2.3.2-15-2016-00001) and the Hungarian Academy of Sciences (LP2017-7/2017) to Z.L. Work in MRP lab is supported by The Wellcome Trust. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the “Generalitat de Catalunya”. S.M.-G. acknowledges receipt of an FPU fellowship from the MINECO. P.E. acknowledges receipt of an FPI fellowship from MINECO.

© The Author(s) 2020., Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription., MINECO [BFU2015-65082-P, PGC2018-094538-B-100]; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; European Community FEDER program (to F.A.); MEC ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; MINECO [SAF2016-75006-P to M.B.]; ‘Centre de Referència en Biotecnologia’ of the Generalitat de Catalunya. Funding for open access charge: MINECO.

In an earlier paper (Pérez-Montero et al., 2013), we reported that the embryonic linker histone of Drosophila dBigH1 was essential for early Drosophila embryogenesis since embryos homozygous for the bigH1100 mutation showed strong defects and did not survive beyond zygotic genome activation (ZGA) at cellularization. Recent results challenge these observations since null bigH1 mutations generated by CRISPR/Cas9 methodology turn out to be homozygous viable, as reported in Li et al. (2019) and here. In this regard, Li et al. described a novel mechanism by which lack of dBigH1 is compensated by the early expression of maternal dH1. Here, we confirm this observation and show that such compensatory mechanism is not activated in bigH1100 embryos.

In Drosophila, the Heterochromatin Protein 1c (HP1c) forms a transcriptional complex with the zinc-finger proteins WOC and ROW, and the extraproteasomal ubiquitin receptor Dsk2. This complex localizes at promoters of active genes and it is required for transcription. The functions played by the different components of the HP1c complex are not fully understood. In this study we show that WOC and ROW are required for chromatin binding of both Dsk2 and HP1c. However, while impairing chromatin binding strongly destabilizes HP1c, it does not affect Dsk2 stability. We also show that WOC, but not ROW, is required for nuclear localization of Dsk2. Moreover, WOC and Dsk2 co-immunoprecitate upon ROW depletion. These results suggest that WOC and Dsk2 interact to form a subcomplex that mediates nuclear translocation of Dsk2. We also show that ROW mediates chromatin binding of the WOC/Dsk2 subcomplex, as well as of HP1c. Altogether these observations favor a model by which the interaction with WOC recruits Dsk2 to the HP1c complex that, in its turn, binds chromatin in a ROW-dependent manner., This work was supported by grants from MICINN (BFU2015-65082-P and PGC2018-094538-B-100), the “Generalitat de Catalunya” (SGR2017-475) and the European Community FEDER program. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the “Generalitat de Catalunya”. GDM acknowledges receipt of a “Severo Ochoa” FPI fellowship from MINECO.

1Abstract1.1BackgroundsIn the last years we have faced an unprecedented growth in the availability of high-throughput epigenomics data related to the genomic distribution of epigenetic marks, namely transcription factors, histone modifications and other DNA binding proteins. This also pointed out the need for efficient tools for integration, visualization and functional analysis of genomics and epigenomics data. On this subject, we previously developed a computational framework, chroGPS, to integrate and visualize the associations between epigenetic factors and their relation to functional genetic elements in low-dimensional maps. We demonstrated the usefulness of our approach with several practical case studies based on well-defined biological hypothesis.1.2ResultsHere we introduce chroGPS version 2, a major update of our previously developed software with new functionalities for differential analysis of epigenomes. Methods are provided for efficient integration and comparison of data from different conditions or biological backgrounds, accounting and adjusting for systematic biases in order to provide an efficient and statistically robust base for differential analysis. We also include new useful functionalities for general data assessment and quality control prior to comparing maps, such as functions to study chromatin domain conservation between epigenomic backgrounds, to detect gross technical outliers and also to help in the selection of candidate marks for de-novo epigenome mapping. Our software, implemented in R as a Bioconductor package, provides detailed reference and user manuals to allow the final user to reproduce the presented case studies and use them as a starting point for generating and exploring epigenetic maps according to their own experimental needs.1.3ConclusionchroGPS2 extends our previously developed software, providing now a complete, intuitive, efficient and statistically robust framework for generation, visualization, characterization and differential analysis of epigenomic maps, in a way which is easy to adapt to different biological and technical backgrounds allowing exploration and hypothesis testing in multiple biological scenarios.

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process., This work was supported by grants from MINECO (BFU2015-65082-P), the Generalitat de Catalunya (SGR2014-204), and the European Community FEDER program. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the Generalitat de Catalunya. EŠ acknowledges receipt of a NEWFELPRO Fellowship of the Croatian Government and Ministry of Science and Education within EU FP7 Programme framework.

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6–12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. Here, we report the essential role of the germline linker histone dBigH1 during early spermatogenesis. Our results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression., This work was supported by grants from the MINECO (BFU2012-30724 and BFU2015-65082P), the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204), and the European Community FEDER program. This work was carried out within the framework of the ‘‘Centre de Refere` ncia en Biotecnologia’’ of the ‘‘Generalitat de Catalunya.’’ S.P.-M. and P.C.-C. acknowledge receipt of FPI fellowships from the MINECO.

© 2015 Elsevier Inc. dKDM5/LID regulates transcription of essential developmental genes and, thus, is required for different developmental processes. Here, we report the essential contribution of dKDM5/LID to hematopoiesis in Drosophila. Our results show that dKDM5/LID is abundant in hemocytes and that its depletion induces over-proliferation and differentiation defects of larval hemocytes and disrupts organization of the actin cytoskeleton. We also show that dKDM5/LID regulates expression of key factors of hematopoietic development. In particular, dKDM5/LID depletion up-regulates expression of several transcription factors involved in hemocytes proliferation and differentiation as well as of several small-GTPases that link signaling effectors to actin cytoskeleton formation and dynamics., This work was supported by grants from MICINN (BFU2012-30724) and the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204). This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the “Generalitat de Catalunya”

Linker histone H1 is an important structural component of chromatin that stabilizes the nucleosome and compacts the nucleofilament into higher-order structures. The biology of histone H1 remains, however, poorly understood. Here we show that Drosophila histone H1 (dH1) prevents genome instability as indicated by the increased γH2Av (H2AvS137P) content and the high incidence of DNA breaks and sister-chromatid exchanges observed in dH1-depleted cells. Increased γH2Av occurs preferentially at heterochromatic elements, which are upregulated upon dH1 depletion, and is due to the abnormal accumulation of DNA:RNA hybrids (R-loops). R-loops accumulation is readily detectable in G1-phase, whereas γH2Av increases mainly during DNA replication. These defects induce JNK-mediated apoptosis and are specific of dH1 depletion since they are not observed when heterochromatin silencing is relieved by HP1a depletion. Altogether, our results suggest that histone H1 prevents R-loops-induced DNA damage in heterochromatin and unveil its essential contribution to maintenance of genome stability., This work was supported by grants from MICINN (BFU2012-30724 and BFU2015-65082-P), the Generalitat de Catalunya (SGR2014-204) and by the European Community FEDER program. A.B.-F. and A.C.-L. acknowledge receipt of FPU (MED) and FPI (MINECO) fellowships, respectively.

The centromere is a specialized chromosomal structure that dictates kinetochore assembly and, thus, is essential for accurate chromosome segregation. Centromere identity is determined epigenetically by the presence of a centromere‐specific histone H3 variant, CENP‐A, that replaces canonical H3 in centromeric chromatin. Here, we discuss recent work by Roulland et al. that identifies structural elements of the nucleosome as essential determinants of centromere function. In particular, CENP‐A nucleosomes have flexible DNA ends due to the short αN helix of CENP‐A. The higher flexibility of the DNA ends of centromeric nucleosomes impairs binding of linker histones H1, while it facilitates binding of other essential centromeric proteins, such as CENP‐C, and is required for mitotic fidelity. This work extends previous observations indicating that the differential structural properties of CENP‐A nucleosomes are on the basis of its contribution to centromere identity and function. Here, we discuss the implications of this work and the questions arising from it

Histone H1 is an essential chromatin component. Metazoans usually contain multiple stage-specific H1s. In particular, specific variants replace somatic H1s during early embryogenesis. In this regard, Drosophila was an exception because a single dH1 was identified that, starting at cellularization, is detected throughout development in somatic cells. Here, we identify the embryonic H1 of Drosophila, dBigH1. dBigH1 is abundant before cellularization occurs, when somatic dH1 is absent and the zygotic genome is inactive. Upon cellularization, when the zygotic genome is progressively activated, dH1 replaces dBigH1 in the soma, but not in the primordial germ cells (PGCs) that have delayed zygotic genome activation (ZGA). In addition, a loss-of-function mutant shows premature ZGA in both the soma andPGCs. Mutant embryos die at cellularization, showing increased levels of active RNApol II and zygotic transcripts, along with DNA damage and mitotic defects. These results show an essential function of dBigH1 in ZGA regulation. © 2013 Elsevier Inc., This work was supported by grants from the Ministerio de Ciencia e Innovación (MICINN) (CSD2006-49, BFU2009-07111, and BFU2012-30724) and the Generalitat de Catalunya (SGR2009-1023). This work was carried out within the framework of the Centre de Referència en Biotecnologia of the Generalitat de Catalunya. S.P.-M. received an FPI fellowship from the MICINN.

It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites., This work was supported by grants from MICINN (BFU2012-30724 and BFU2015-65082-P), the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204), the European Community FEDER program and NIH grant R01GM105831 NIH/NIGMS (to MZ). This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the “Generalitat de Catalunya”. AD was funded by a Marie Curie Reintegration Grant within the 7th European Community Framework ProgramPIRG7-GA-2010-268400.

Centromere identity and function is determined by the specific localization of CenH3 (reviewed in [1-7]). Several mechanisms regulate centromeric CenH3 localization, including proteasome-mediated degradation that, both in budding yeast and Drosophila, regulates CenH3 levels and prevents promiscuous misincorporation throughout chromatin [8, 9]. CenH3CENP-A proteolysis has also been reported in senescent human cells [10] or upon infection with herpes simplex virus 1 [11]. Little is known, however, about the actual mechanisms that regulate CenH3 proteolysis. Recent work in budding yeast identified Psh1 as an E3-ubiquitin ligase that mediates degradation of CenH3Cse4p [12, 13], but E3-ligases regulating CenH3 stability in metazoans are unknown. Here, we report that the F box protein partner of paired (Ppa), which is a variable subunit of the main E3-ligase SCF [14-17], mediates CenH3CID stability in Drosophila. Our results show that Ppa depletion results in increased CenH3CID levels. Ppa physically interacts with CenH3CID through the CATDCID that, in the fly, mediates Ppa-dependent CenH3CID stability. Altogether, these results strongly suggest that, in Drosophila, SCFPpa regulates CenH3CID proteolysis. Interestingly, most known SCF complexes are inactive when, at mitosis, de novo CenH3CID deposition takes place at centromeres, suggesting that, in Drosophila, CenH3CID deposition and proteolysis are synchronized events. © 2011 Elsevier Ltd. All rights reserved., S.M.-G. acknowledges receipt of a Formación del Profesorado Universitario fellowship from the Ministerio de Ciencia e Innovación (MCINN). This work was supported by grants from MCINN (CSD2006-49 and BFU2009-07111), the CSIC (200420E391), and the Generalitat de Catalunya (SGR2009-1023).

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere-specific histone H3 variant CenH3, originally called CENP-A, which replaces canonical histone H3 at centromeres. CenH3-chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3-chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.

The homeotic Abdominal-B (Abd-B) gene expression depends on a modular cis-regulatory region divided into discrete functional domains (iab) that control the expression of the gene in a particular segment of the fly. These domains contain regulatory elements implicated in both initiation and maintenance of homeotic gene expression and elements that separate the different domains. In this paper we have performed an extensive analysis of the iab-6 regulatory region, which regulates Abd-B expression at abdominal segment A6 (PS11), and we have characterized two new polycomb response elements (PREs) within this domain. We report that PREs at Abd-B cis-regulatory domains present a particular chromatin structure which is nuclease accessible all along Drosophila development and both in active and repressed states. We also show that one of these regions contains a dCTCF and CP190 dependent activity in transgenic enhancer-blocking assays, suggesting that it corresponds to the Fab-6 boundary element of the Drosophila bithorax complex. © 2008 The Author(s), The Ministerio de Educación y Ciencia (MEC) (BMC2002-00905, BMC2006-01627 and CSD2006-00049); the Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) (2001SGR00344). Funding for open access charge: MEC, BMC2006-01627

In this article, we characterize histone demethylase activity of the entire family of JmjC+N proteins of Drosophila melanogaster. Our results show that Lid (little imaginal discs), which is structurally homologous to JARID1, demethylates H3K4me3. However, contrary to what would be inferred from its demethylase activity, lid contributes to the establishment of transcriptionally competent chromatin states as: (i) is required for histone H3 acetylation; (ii) contributes to expression of the homoeotic gene Ultrabithorax (Ubx); and (iii) antagonizes heterochromatin-mediated gene silencing (PEV). These results, which are consistent with the identification of lid as a trithorax group (trxG) gene, are discussed in the context of current models for the contribution of H3K4me3 to the regulation of gene expression. Here, we also show that the two Drosophila JMJD2 homologues, dJMJD2(1)/CG15835 and dJMJD2(2)/CG33182, are capable of demethylating both H3K9me3 and H3K36me3. dJMJD2(1)/CG15835 regulates heterochromatin organization, as its over-expression induces spreading of HP1, out of heterochromatin, into euchromatin, without affecting the actual pattern of histone modifications of heterochromatin. dJMJD2(1)/CG15835 is excluded from heterochromatin and localizes to multiple euchromatic sites, where it regulates H3K36 methylation. These results indicate that dJMJD2(1)/CG15835 contributes to delimit hetero- and euchromatic territories through the regulation of H3K36 methylation in euchromatin. On the other hand, dJARID2/CG3654 shows no demethylase activity on H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H4K20me3.

The retrotransposons that maintain telomeres in Drosophila melanogaster have unique features that are shared across all Drosophila species but are not found in other retrotransposons. Comparative analysis of these features provides insight into their importance for telomere maintenance in Drosophila . Gag proteins encoded by HeT-A mel and TART mel are efficiently and cooperatively targeted to telomeres in interphase nuclei, a behavior that may facilitate telomere-specific transposition. Drosophila virilis , separated from D. melanogaster by 60 MY, has telomeres maintained by HeT-A vir and TART vir . The Gag proteins from HeT-A mel and HeT-A vir have only 16% amino acid identity, yet several of their functional features are conserved. Using transient transfection of cultured cells from both species, we show that the telomere association of HeT-A vir Gag is indistinguishable from that of HeT-A mel Gag. Deletion derivatives show that organization of localization signals within the two proteins is strikingly similar. Gag proteins of TART mel and TART vir are only 13% identical. In contrast to HeT-A , surprisingly, TART vir Gag does not localize to the nucleus, although TART vir is a major component of D. virilis telomeres, and localization signals in the protein have much the same organization as in TART mel Gag. Thus, the mechanism of telomere targeting of TART vir differs, at least in a minor way, from that of TART mel . Our findings suggest that, despite dramatic rates of protein evolution, protein and cellular determinants that correctly localize these Gag proteins have been conserved throughout the 60 MY separating these species.

Eukaryotic genomes are structured in the formof chromatin with the help of a set of five small basic proteins, the histones. Four of them are highly conserved through evolution, form the basic unit of the chromatin, the nucleosome, and have been intensively studied and are well characterized. The fifth histone, histone H1, adds to this basic structure through its interaction at the entry/exit site of DNA in the nucleosomeandmakes an essential contribution to the higher order folding of the chromatin fiber. Histone H1 is the less conserved histone and the less known of them. Though for long time considered as a general repressor of gene expression, recent studies in Drosophila have rejected this view and have contributed to uncover important functions on genome stability and development. Here we present some of the most recent data obtained in the Drosophila model system and discuss how the lessons learnt in these studies compare and could be applied to all other eukaryotes. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan., Work in the authors' laboratory is supported by grants from MINECO (BFU2012-30724) and the Generalitat de Catalunya (SGR2014-204)

The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties., Work in the authors’ laboratory is supported by grants from MINECO (BFU2012-30724) and the Generalitat de Catalunya (SGR2014-204)

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports. © 2005 Elsevier Ltd. All rights reserved., This work was financed by grants from the Ministerio de Ciencia y Tecnología (BMC2003-243; BMC2003-616; BIO2002-2301) and the CIRIT (2001SGR00344; 2001SGR00047). X.A. acknowledges receipt of a doctoral fellowship from the CIRIT. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the Generalitat de Catalunya

Background: The Drosophila melanogaster DDP1 protein is a highly evolutionarily conserved protein that is characterised by the presence of 15 tandemly organized KH domains, known for mediating high-affinity binding to single-stranded nucleic acids (RNA and ssDNA). Consistent with its molecular organization, DDP1 binds single-stranded nucleic acids with high affinity, in vitro. It was shown earlier that, in polytene chromosomes, DDP1 is found in association with chromocenter heterochromatin, suggesting a contribution to heterochromatin formation and/or maintenance. Results: In this paper, the actual contribution of DDP1 to the structural and functional properties of heterochromatin was determined through the analysis of the phenotypes associated with the hypomorphic ddp115.1 mutation that was generated through the mobilization of a P element inserted in the second intron of ddp1. ddp1 15.1 behaves as a dominant suppressor of PEV in the variegated rearrangement In(1)wm4 as well as in several transgenic lines showing variegated expression of a hsp70-white+ transgene. In polytene chromosomes from homozygous ddp115.1 larvae, histone H3-K9 methylation and HP1 deposition at chromocentre heterochromatin are strongly reduced. Our results also show that, when the maternal contribution of DDP1 is reduced, chromosome condensation and segregation are compromised. Moreover, in a ddp115.1 mutant background, transmission of the nonessential Dp1187 minichromosome is reduced. Conclusions: We conclude that DDP1 contributes to the structural and functional properties of heterochromatin. These results are discussed in the context of current models for the formation and maintenance of heterochromatin; in these models, HP1 deposition depends on H3-K9 methylation that, in turn, requires the contribution of the RNAi pathway., This work was financed by grants from the Ministerio de Ciencia y Tecnologı́a (BMC2000-878; BMC2003-243) and the Comissió Interdepertamental d'Investigació i Innovació Tecnològica (2001SGR00344). D.H. acknowledges receipt of a Postdoctoral Contract from the Ministerio de Ciencia y Tecnologı́a. A.C. received a doctoral fellowship from the Comissió Interdepertamental d'Investigació i Innovació Tecnològica. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the Generalitat de Catalunya

9 pages, 7 figures.-- PMID: 12737631 [PubMed].-- PMCID: PMC1223549., In this paper, the human phosphodiesterase 7A1 (h PDE7A1 ) promoter region was identified and functionally characterized. Transient transfection experiments indicated that a 2.9 kb fragment of the h PDE7A1 5'-flanking region, to position -2907, has strong promoter activity in Jurkat T-cells. Deletion analysis showed that the proximal region, up to position -988, contains major cis -regulatory elements of the h PDE7A1 promoter. This minimal promoter region contains a regulatory CpG island which is essential for promoter activity. The CpG island contains three potential cAMP-response-element-binding protein (CREB)-binding sites that, as judged by in vivo dimethyl sulphate (DMS) footprinting, are occupied in Jurkat T-cells. Moreover, over-expression of CREB results in increased promoter activity, but, on the other hand, promoter activity decreases when a dominant-negative form of CREB (KCREB) is over-expressed. In vivo DMS footprinting strongly indicates that other transcription factors, such Ets-2, nuclear factor of activated T-cells 1 (NFAT-1) and nuclear factor kappaB (NF-kappaB), might also contribute to the regulation of h PDE7A1 promoter. Finally, h PDE7A1 promoter was found to be induced by treatment with PMA, but not by treatment with dibutyryl cAMP or forskolin. These results provide insights into the factors and mechanisms that regulate expression of the h PDE7A gene., This study was supported by a grant (PB-98-0468) from the Ministerio de Ciencia y Tecnología.

© 2015 Macmillan Publishers Limited. All rights reserved. dDsk2 is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. Here we report that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1, a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications., This work was supported by grants from MICINN (CSD2006-49, BFU2009-07111 and BFU2012-30724) and the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204). This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. R.K. acknowledges receipt of a ‘La Caixa’ PhD fellowship

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.

The present work describes the genetic modification of a hybridoma cell line with the aim to change its metabolic behaviour, particularly reducing the amounts of ammonia and lactate produced by the cells. The cellular excretion of ammonia was eliminated by transfection of a cloned glutamine synthetase gene. The metabolic characterisation of the transformed cell line includes the analysis of the changes introduced in its intracellular metabolic fluxes by means of a stoichiometric model. Furthermore, the reduction of lactate accumulation was attempted through an antisense mRNA approach, aiming to generate a rate limiting step in the glycolytic pathway, thus lowering the glucose consumption rate. The physiological results obtained with the transformed cells are discussed. A maximum reduction of about 47% in the glucose consumption rate was obtained for one of the transformations. However a main drawback was the lack of stability of the transformed cells

In the presence of specific metal-ions (namely zinc but also cadmium, cobalt and manganese), d(GA x TC)n DNA sequences can form non-B-DNA conformations. At low metal-ion concentration they form [GA(GA x TC)] intramolecular triplexes but, upon increasing the metal concentration, the formation of (GA x GA) intramolecular hairpins is detected. In this paper we address the question of the specific effects of zinc on the structure of the d(GA x TC)n sequences. In the presence of zinc, the DMS-reactivity of the (GA x GA) hairpins is strongly reduced suggesting a direct interaction of the metal-ion with the N7-group of the guanines. This effect is specific for antiparallel-stranded d(GA)n homoduplexes. No such strong decrease in DMS-reactivity is observed in B-DNA duplexes or in d(GGA)n and d(GGGA)n homoduplexes. In addition, the thermal stability of antiparallel-stranded d(GA)n homoduplexes increases in the presence of zinc. On the contrary, the melting temperature of similar B-DNA molecules decreases upon increasing the zinc concentration. Altogether, these result indicate that zinc plays an specific role on the stabilization of the (GA x GA) intramolecular hairpins.

The centromeric dodeca-satellite of Drosophila forms unusual DNA structures in which its purine-rich strand (GTACGGGACCGA) n folds into very stable intramolecular hairpins. These intramolecular hairpins contain groups of tandem 5′-GA:GA-3′ mismatches that, as judged by gel electrophoresis analysis and UV-melting studies, have a determinant contribution to their stability. Duplexes of the dodeca-satellite purine-rich strand, carrying tandem 5′-GA:GA-3′ mismatches, are as stable as equivalent fully Watson-Crick duplexes containing tandem 5′-TA:TA-3′ Watson-Crick pairs in place of the non-Watson-Crick G·A pairs. On the other hand, duplexes carrying any of the other three possible tandem combinations of purine·purine mismatches, including G·A pairs on the opposite orientation 5′-AG:AG-3′, are very unstable. The high stability of the dodeca-satellite hairplus suggests that the tandem G·A pairs are on the sheared configuration although they are found within the less favourable 5′-G-(G-A)-C-3′ sequence context. Other centromeres DNA sequences, including the AAGAG satellite of Drosophila and the mammalian CENP-B box sequence, have the potential of forming intramolecular hairpins stabilised by similar purine·purine interactions.

Development of tools to jointly visualize the genome and the epigenome remains a challenge. chroGPS is a computational approach that addresses this question. chroGPS uses multidimensional scaling techniques to represent similarity between epigenetic factors, or between genetic elements on the basis of their epigenetic state, in 2D/3D reference maps. We emphasize biological interpretability, statistical robustness, integration of genetic and epigenetic data from heterogeneous sources, and computational feasibility. Although chroGPS is a general methodology to create reference maps and study the epigenetic state of any class of genetic element or genomic region, we focus on two specific kinds of maps: chroGPSfactors, which visualizes functional similarities between epigenetic factors, and chroGPSgenes, which describes the epigenetic state of genes and integrates gene expression and other functional data. We use data from the modENCODE project on the genomic distribution of a large collection of epigenetic factors in Drosophila, a model system extensively used to study genome organization and function. Our results show that the maps allow straightforward visualization of relationships between factors and elements, capturing relevant information about their functional properties that helps to interpret epigenetic information in a functional context and derive testable hypotheses. © 2013 The Author(s). Published by Oxford University Press, The Spanish ‘Ministerio de Economia y Competitividad’ [CSD2006-49, BFU2009-07111 and BFU2012-30724] and the ‘Generalitat de Catalunya’ [SGR2009-1023]; This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. Funding for open access charge: Spanish ‘Ministerio de Economia y Competitividad’ [BFU2012-30724]

Simple repeating d(GA·TC)(n) DNA sequences are frequently found at eukaryotic promoters, and in some cases they have been shown to be nucleosome free in vivo. These sequences show a high degree of structural polymorphism and are capable of adopting several types of non-B-DNA conformations. Here we show that the structural versatility of these sequences affects their ability to be packed into nucleosomes. Nucleosome assembly onto short double-stranded DNA fragments carrying d(GA·TC)(n) sequences of different length (n = 10 and n = 22) is very efficient. However, when the simple repeating sequence is forming a [CT(GA·TC)] triplex, nucleosome assembly is either prevented, as in the case of the d(GA·TC)22 sequence, or results in the destabilization of the triple-stranded conformation, as in the case of the d(GA·TC)10 sequence. Similarly, formation of triple-stranded DNA is hindered when the sequence is organized as nucleosomes. Efficient formation of triplex DNA occurs only at relatively high ionic strength (0.6 M NaCl), when the nucleosome is partially destabilized, and results in the disruption of the nucleosomal particle. These results indicate that nucleosome assembly and triplex formation are competing processes, This work was supported in part by Grants PB93-102 from the Spanish Dirección General de Investigación Cientifica y Técnica and CHRX-CT94-0047 from Commission of the European Communities and by fellowships from the Comissió Interdepertamental de Recerce i Tecnologia of the Generalitat de Catalunya (to M. L. E. and E. J.-G.). This work was carried out in the framework of the Center de Referència en Biotecnologia of the Generalitat de Catalunya

9 pages, 6 figures.-- PMID: 8760879 [PubMed].-- PMCID: PMC146023., The binding of TBP (TFIID) to the TATA box has been considered to direct promoter recognition and pre-initiation complex formation because it is the first event leading to basal transcription by RNA polymerase II. Here, we analyse the binding of yeast TBP to a consensus TATAAA box and two point mutations, TAAAAA (inactive) and TATATA (active). Despite the fact that the TAAAAA sequence does not support transcription in vitro, yeast TBP binds the three sequences showing, in this sense, only a limited sequence specificity. However, the TBP-TAAAAA complex cannot be recognised by other basal transcription factors, in particular by TFIIB. DNase I footprinting patterns of the TBP-TAAAAA complex are different from those observed in functional TBP-TATA box complexes, indicating that, most likely, it is a different spatial arrangement of the TBP-DNA complex that prevents formation of the TFIIB-TBP-TAAAAA complex, also seriously impairing entry of TFIIA to the complex. DNA deformability of the A/T-rich sequences appears to be an important determinant in the formation of a productive TBP-TATA complex. These results indicate that the transcriptional competence of A/T-rich sequences is determined not only by TBP binding, but also by the ability of other basal transcription factors to recognise the preformed TBP-DNA complexes., The financial support of grants PB93-102 from the Spanish DGICYT and PL-932217 from the CEC is gratefully acknowledged. This work was carried out within the framework of the ‘Centre de Referéncia en Biotecnologia’ of the Generalitat de Catalunya.

DNA sequences containing homopurine d(G1-3A)n tracts are known to be capable of adopting non-B-DNA conformations. The structural polymorphism of these sequences is a direct consequence of the structural properties of the homopurine d(G1-3A)n tracts. Depending on the conditions, d(GA)n DNA sequences can form antiparallel- and parallel-stranded homoduplexes, multistranded complexes, and ordered single-stranded conformations. On the other hand, much less is known about the structural properties of d(GGA)n and d(GGGA)n sequences. In this paper, we show that d(GGA)n and d(GGGA)n repeats form antiparallel-stranded, intramolecular hairpins. Under physiological salt and pH conditions, the thermal stability of these hairpin forms is high, showing, at 50 mM NaCl, melting temperatures in the range of 40−50 °C. The base-pairing interactions involved in the formation of the d(GGA)n and d(GGGA)n hairpins are different. G·A pairs importantly contribute to the stability of the d(GGA)n hairpins. On the other hand, the d(G...

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License., H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol llo ser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription. © 2012 The Author(s)., Seed Funding for Applied Research (Project No. 201007160001 to M.S.Y.H.); URC-PDF Scheme, Centre for Cancer Research HKU [to S.M.H.S. and S.S.D. (in part)]; Faculty Development Fund. Funding for open access charge: Seed Funding for Applied Research, University of Hong Kong (Project No. 201007160001 to M.S.Y.H.).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, Histone H1 is an intrinsic component of chromatin, whose important contribution to chromatin structure is well-established in vitro. Little is known, however, about its functional roles in vivo. Here, we have addressed this question in Drosophila, a model system offering many advantages since it contains a single dH1 variant. For this purpose, RNAi was used to efficiently deplete dH1 in flies. Expression-profiling showsthatdH1depletion affects expression of a relatively small number of genes in a regional manner. Furthermore, depletion up-regulates inactive genes, preferentially those located in heterochromatin, while active euchromatic genes are down-regulated, suggesting that the contribution of dH1 to transcription regulation is mainly structural, organizing chromatin for proper gene-expression regulation. Up-regulated genes are remarkably enriched in transposons. In particular, R1/R2 retrotransposons, which specifically integrate in the rDNA locus, are strongly up-regulated. Actually, depletion increases expression of transposon-inserted rDNA copies, resulting in synthesis of aberrant rRNAs and enlarged nucleolus. Concomitantly, dH1-depleted cells accumulate extra-chromosomal rDNA, show increased γH2Av content, stop proliferation and activate apoptosis, indicating that depletion causes genome instability and affects proliferation. Finally, the contributions to maintenance of genome integrity and cell proliferation appear conserved in human hH1s, as their expression rescues proliferation of dH1-depleted cells. © The Author(s) 2012. Published by Oxford University Press., MICINN (CSD2006-49 and BFU2009-07111); CSIC (200420E583 and 201120E001); Generalitat de Catalunya (SGR2009-1023); IRB fellowship (to O.V.). This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. Funding for open access charge: MICINN.

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date. © 2012 Elsevier B.V., This work was supported by grants from MICINN (CSD2006-49, BIO2008-00799 and BFU2009-07111), the CSIC (200420E391) and the “Generalitat de Catalunya” (SGR2009-1023 and SGR2009-1005). This work was carried out within the framework of the “Xarxa de Referència en Biotecnologia” of the “Generalitat de Catalunya”.

Telomeric DNA sequences are known to adopt unusual DNA structures upon protonation when contained into negatively supercoiled DNA. In this paper, the structural properties of (T(2)AG(3))(n) telomeric sequences of different length is analyzed in detail. Transition to the protonated form is observed at very low pH for (T(2)AG(3))(n8) sequences. Formation of the protonated form is facilitated by negative supercoiling. The patterns of chemical modification obtained with different chemical reagents indicate that protonation induces denaturation of the (T(2)AG(3))(n) telomeric sequences. Upon denaturation, the "C-rich" strand becomes structured forming, most likely, hairpin-like conformations stabilized by the formation of C(+)·C pairs and, probably, of A(+)·A pairs. The "G-rich" strand of the (T(2)AG(3))(8) sequence shows also signs of becoming structured giving rise to various structural conformers which might include triple- and tetra-stranded conformations. However, in the case of shorter sequences, the "G-rich" strand remains basically single-stranded.

AT-rich DNA sequences other than AnTm tracts (n + m > or = 4) are known to be intrinsically curved. The AATAT-element constitutes one known example of these sequences. In this paper, the elucidation of the structural basis of the curvature induced by this sequence element was addressed. As judged by the patterns of cleavage by the hydroxyl radical and DNase I, the AATAT sequence shows a narrow minor groove. Furthermore, the 5' adenine residue of the AA dinucleotide contained within the sequence is hyperreactive to diethylpyrocarbonate. Similar structural properties are shown by several sequences inducing intrinsic DNA curvature, such as an A5-tract or the closely related ATAAT, AATATA and TAATAT sequences, which are also shown here to induce curvature. On the other hand, other related sequences, such as TATAA and ATATA, that do not induce curvature, show different structural characteristics.

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in S. cerevisiae, Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null ddp1Δ mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic ddp115.1 mutation. Similar results were obtained in the fission yeast Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly. © 2010 Elsevier B.V., Este trabajo fue financiado por las subvenciones del Ministerio de Ciencia e Innovación, España (BFU2006-1627, CSD2006-49 y BFU2009-07111) y la Generalitat de Catalunya (SGR2005-678 y SGR2009-1023)