ABSTRACT
Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.
Subject(s)
Adenosine Triphosphatases , Chromatin , Chromosome Pairing , DNA-Binding Proteins , Drosophila Proteins , Animals , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Binding Sites , Chromatin/metabolism , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Osmotic Pressure , Protein Binding , Zinc FingersABSTRACT
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.
Subject(s)
Azoospermia , Infertility, Male , Oligospermia , Azoospermia/genetics , Humans , Infertility, Male/genetics , Male , Spermatogenesis/genetics , X ChromosomeABSTRACT
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.
Subject(s)
Drosophila Proteins , Histones , Animals , Histones/genetics , Histones/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Drosophila/genetics , Methylation , Lysine/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , DNA Transposable Elements , Epigenesis, Genetic , Protein Processing, Post-Translational , Chromatin/metabolismABSTRACT
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.
Subject(s)
Chromatin/metabolism , Drosophila Proteins/metabolism , Gene Silencing , Histones/metabolism , Animals , Cell Line , Down-Regulation , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/chemistry , Histone Code , Histones/chemistry , Protein Domains , RNA Polymerase II/metabolismABSTRACT
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.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/metabolism , Cdh1 Proteins/metabolism , Cell Cycle , Centromere Protein A/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Centromere/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , G1 Phase , S PhaseABSTRACT
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.
Subject(s)
Centromere/metabolism , Nucleosomes/metabolism , Animals , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Histones/metabolism , Humans , Yeasts/genetics , Yeasts/metabolismABSTRACT
Eukaryotic genomes are structured in the form of 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 nucleosome and makes 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.
Subject(s)
Histones/physiology , Amino Acid Sequence , Animals , Drosophila , Genomic Instability , Heterochromatin/chemistry , Histones/chemistry , Molecular Sequence DataABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , RNA Polymerase II/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Transcription, Genetic , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cell Nucleolus/ultrastructure , Centromere/metabolism , Centromere/ultrastructure , Centromere Protein B/genetics , Centromere Protein B/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , DNA-Binding Proteins/deficiency , Heterochromatin/metabolism , Heterochromatin/ultrastructure , RNA Polymerase II/metabolism , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retroelements , Schizosaccharomyces/metabolism , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
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 â¼30 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.
Subject(s)
Eukaryota/metabolism , Germ Cells/metabolism , Histones/metabolism , Nucleosomes , Animals , Female , Histones/physiology , Humans , Male , Protein IsoformsABSTRACT
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.
Subject(s)
Drosophila Proteins/physiology , Drosophila/enzymology , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Histone Demethylases/physiology , Actins/metabolism , Animals , Animals, Genetically Modified , Cell Differentiation , Cell Movement , Cell Proliferation , Cytoskeleton/metabolism , Drosophila/embryology , Female , Hemocytes/cytology , Hemocytes/metabolism , Immunohistochemistry , Larva/enzymology , Male , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
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: chroGPS(factors), which visualizes functional similarities between epigenetic factors, and chroGPS(genes), 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.
Subject(s)
Chromatin/metabolism , Epigenesis, Genetic , Epigenomics/methods , Software , Animals , Cell Line , Computer Graphics , Drosophila/genetics , Gene Expression , Genes, Insect , Signal Transduction/geneticsABSTRACT
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 shows that dH1 depletion 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.
Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Gene Silencing , Genomic Instability , Histones/physiology , Retroelements , Animals , Cell Proliferation , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Gene Expression Regulation , Genome, Insect , Heterochromatin/metabolism , Histones/antagonists & inhibitors , Histones/genetics , Humans , RNA InterferenceABSTRACT
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.
Subject(s)
Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Transcription Initiation Site , Transcription, Genetic , Animals , Cell Line , Drosophila/enzymology , Drosophila/genetics , Drosophila/metabolism , Histone Demethylases , Nuclear Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.
Subject(s)
Drosophila Proteins , Heterochromatin , Histones , Animals , Drosophila/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Heterochromatin/metabolism , Histones/metabolism , Homeostasis , Nucleosomes/metabolism , R-Loop Structures , RNA/metabolism , RNA/genetics , RNA Polymerase II/metabolism , Male , FemaleABSTRACT
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.
Subject(s)
Autoantigens/genetics , Autoantigens/metabolism , Centromere/genetics , Centromere/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Amino Acid Sequence , Animals , Autoantigens/analysis , Autoantigens/chemistry , Centromere/chemistry , Centromere Protein A , Chromatin/chemistry , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/analysis , Chromosomal Proteins, Non-Histone/chemistry , Epigenesis, Genetic , Humans , Molecular Sequence Data , Sequence AlignmentABSTRACT
In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. Here, we address this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. Here, we identify the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. We also report that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, we show that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, our results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.
ABSTRACT
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 ddp1(Δ) 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 ddp1(15.1) 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.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endoplasmic Reticulum, Rough/metabolism , Animals , Animals, Genetically Modified , Blotting, Western , Cells, Cultured , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Endoplasmic Reticulum, Rough/ultrastructure , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/metabolism , Immunohistochemistry , Lysine/metabolism , Male , Methylation , Microscopy, Immunoelectron , Mutation , Protein Binding , RNA Interference , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
In fission yeast, mating-type switching involves replacing genetic information contained at the expressed mat1 locus by that of either the mat2P or mat3M donor loci. Donor selection is nonrandom, as mat1P cells preferentially use mat3M for switching, whereas mat1M cells use mat2P. Switching directionality is determined by the cell-type-specific distribution of the Swi2-Swi5 complex that, in mat1P cells, localises to mat3M and, only in mat1M cells, spreads to mat2P in a heterochromatin-dependent manner. Mechanisms regulating spreading of Swi2-Swi5 across heterochromatin are not fully understood. Here, we show that the fission yeast homologue of CENP-B, Abp1, binds to the silent domain of the mating-type locus and regulates directionality of switching. Deletion of abp1 prevents utilisation of mat2P, as when heterochromatin is disrupted and spreading of Swi2-Swi5 is impaired. Our results show that, indeed, deletion of abp1 abolishes spreading of Swi2-Swi5 to mat2P. However, in abp1Delta cells, heterochromatin organisation at the mating-type locus is preserved, indicating that Abp1 is actually required for efficient spreading of Swi2-Swi5 through heterochromatin. Cbh1 and Cbh2, which are also homologous to CENP-B, have only a minor contribution to the regulation of directionality of switching, which is in contrast with the strong effects observed for Abp1.
Subject(s)
Centromere Protein B/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Mating Type, Fungal , Genes, Switch , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Sequence Homology, Amino Acid , Gene Deletion , Heterochromatin/metabolism , Models, Biological , Mutation/genetics , Protein Transport , Schizosaccharomyces/cytologyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/biosynthesis , Drosophila Proteins/metabolism , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Histones/biosynthesis , Animals , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Histones/geneticsABSTRACT
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.