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1.
Nature ; 600(7890): 737-742, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34880491

RESUMO

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.


Assuntos
Montagem e Desmontagem da Cromatina , Células Germinativas , Animais , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética , Feminino , Células Germinativas/metabolismo , Masculino , Camundongos , Proteínas do Grupo Polycomb/metabolismo
2.
Nucleic Acids Res ; 47(22): 11649-11666, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31701127

RESUMO

CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.


Assuntos
Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster/embriologia , Epigênese Genética/genética , Perfilação da Expressão Gênica , Histona Desacetilases/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Isoformas de Proteínas/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética
3.
Eur J Cell Biol ; 98(2-4): 103-115, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30679029

RESUMO

Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.


Assuntos
Proteínas de Drosophila/genética , Infertilidade Masculina/genética , Proteoma/genética , Testículo/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Masculino , Proteoma/metabolismo , Espermatogênese/genética , Testículo/crescimento & desenvolvimento
4.
Methods Mol Biol ; 1471: 289-303, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28349404

RESUMO

In the course of spermatogenesis, germ cells undergo dramatic morphological changes that affect almost all cellular components. Therefore, it is impossible to study the process of spermatogenesis in its entirety without detailed morphological analyses. Here, we describe a method to visualize chromatin dynamics in differentiating Drosophila male germ cells using immunofluorescence staining. In addition, we demonstrate how to treat Drosophila sperm before immunofluorescence staining to help reveal epitopes in the highly condensed sperm chromatin that otherwise may be inaccessible to antibodies.


Assuntos
Cromatina/metabolismo , Drosophila/fisiologia , Espermatogênese/genética , Animais , Cromatina/imunologia , Drosophila/genética , Epitopos , Feminino , Imunofluorescência/instrumentação , Imunofluorescência/métodos , Masculino , Espermatozoides/citologia , Espermatozoides/fisiologia
5.
Biol Open ; 6(4): 439-448, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-28235844

RESUMO

Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during post-meiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex, and testis-specific TFIID (tTFIID) complex. How the action of these protein complexes is coordinated and which other factors are involved in the regulation of transcription in spermatocytes is not well understood. Here, we show that the bromodomain proteins tBRD-1 and tBRD-2 regulate gene expression in primary spermatocytes and share a subset of target genes. The function of tBRD-1 was essential for the sub-cellular localization of endogenous tBRD-2 but dispensable for its protein stability. Our comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depends on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22, and the tTAF Sa.

6.
Cell Rep ; 13(11): 2327-2335, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26673329

RESUMO

The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. Here, we identify Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Protaminas/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Infertilidade Masculina/patologia , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Testículo/metabolismo
7.
Eur J Cell Biol ; 94(1): 46-59, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25464903

RESUMO

Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the transition-protein-like protein Tpl94D contains an HMG-box domain and is expressed during chromatin reorganization. Here, we searched for additional HMG-box-containing proteins with a similar expression pattern. We identified two proteins specifically expressed in the testis, tHMG-1 and tHMG-2, whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. We demonstrated that Tpl94D, tHMG-1 and tHMG-2 localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. Our results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. We hypothesize that the mechanism of chromatin reorganization is a process highly secured by redundancies.


Assuntos
Drosophila melanogaster/metabolismo , Proteínas HMGB/metabolismo , Histonas/metabolismo , Protaminas/metabolismo , Espermatogênese/fisiologia , Animais , Animais Geneticamente Modificados , Cromatina/metabolismo , Drosophila melanogaster/genética , Masculino , Testículo/metabolismo
8.
PLoS One ; 9(9): e108267, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25251222

RESUMO

Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. Here, we performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. Our data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. We also characterized two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Mapas de Interação de Proteínas , Testículo/metabolismo , Animais , Células Cultivadas , Proteínas de Drosophila/análise , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Masculino , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogênese , Fatores Associados à Proteína de Ligação a TATA , Testículo/citologia
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