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1.
PLoS Genet ; 18(7): e1010046, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35857787

RESUMO

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.


Assuntos
Proteínas de Ciclo Celular , Espermatócitos , Complexo Sinaptonêmico , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Microscopia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinases/genética , Recombinases/metabolismo , Espermatócitos/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo
2.
Genome Res ; 26(9): 1202-10, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27510564

RESUMO

The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution.


Assuntos
Cromossomos Sexuais/genética , Processos de Determinação Sexual/genética , Cromossomo X/genética , Cromossomo Y/genética , Animais , Arvicolinae/genética , Cromossomos de Mamíferos/genética , Feminino , Cariotipagem , Masculino , Mamíferos/genética , Camundongos
3.
PLoS Genet ; 9(6): e1003538, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23754961

RESUMO

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.


Assuntos
Pareamento Cromossômico/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Meiose/genética , Inativação do Cromossomo X/genética , Animais , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Feminino , Masculino , Camundongos , Oogênese/genética , Espermatócitos/citologia , Espermatócitos/metabolismo , Cromossomo X/genética , Cromossomo Y/genética
4.
J Cell Sci ; 124(Pt 16): 2837-50, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21807948

RESUMO

RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Meiose , Testículo/metabolismo , Animais , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Histonas/genética , Histonas/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/genética , Testículo/patologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Inativação do Cromossomo X/genética
5.
Cells ; 12(1)2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36611846

RESUMO

Human Perrault syndrome (PRLTS) is autosomal, recessively inherited, and characterized by ovarian insufficiency with hearing loss. Among the genetic causes are mutations of matrix peptidase CLPP, which trigger additional azoospermia. Here, we analyzed the impact of CLPP deficiency on male mouse meiosis stages. Histology, immunocytology, different OMICS and biochemical approaches, and RT-qPCR were employed in CLPP-null mouse testis. Meiotic chromosome pairing and synapsis proceeded normally. However, the foci number of the crossover marker MLH1 was slightly reduced, and foci persisted in diplotene, most likely due to premature desynapsis, associated with an accumulation of the DNA damage marker γH2AX. No meiotic M-phase cells were detected. Proteome profiles identified strong deficits of proteins involved in male meiotic prophase (HSPA2, SHCBP1L, DMRT7, and HSF5), versus an accumulation of AURKAIP1. Histone H3 cleavage, mtDNA extrusion, and cGAMP increase suggested innate immunity activation. However, the deletion of downstream STING/IFNAR failed to alleviate pathology. As markers of underlying mitochondrial pathology, we observed an accumulation of PRLTS proteins ERAL1, PEO1, and HARS2. We propose that the loss of CLPP leads to the extrusion of mitochondrial nucleotide-binding proteins to cytosol and nucleus, affecting late meiotic prophase progression, and causing cell death prior to M-phase entry. This phenotype is more severe than in mito-mice or mutator-mice.


Assuntos
Aminoacil-tRNA Sintetases , Meiose , Masculino , Humanos , Animais , Camundongos , Testículo , Prófase Meiótica I , Mutação , Proteínas Mitocondriais/genética , Mitocôndrias , Aminoacil-tRNA Sintetases/genética , Endopeptidase Clp/genética
6.
Nat Commun ; 12(1): 4605, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326328

RESUMO

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Espermatogênese/fisiologia , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cristalografia por Raios X/métodos , Feminino , Recombinação Homóloga , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Meiose , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência
7.
BMC Genomics ; 11: 367, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20537150

RESUMO

BACKGROUND: The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids. RESULTS: HR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in Hr6b knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in Hr6b knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wild type spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes. CONCLUSIONS: Spermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation.


Assuntos
Espermátides/metabolismo , Espermatócitos/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/genética , Centrômero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Ligados ao Cromossomo X/genética , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Especificidade de Órgãos , Fosfoproteínas/genética , Testículo/metabolismo , Transcrição Gênica , Ativação Transcricional , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Regulação para Cima , Cromossomo X/metabolismo , Cromossomo Y/genética
8.
Gastroenterology ; 136(7): 2195-2203.e1-7, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19272384

RESUMO

BACKGROUND & AIMS: The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. METHODS: We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. RESULTS: Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. CONCLUSIONS: Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse.


Assuntos
Proliferação de Células , Colo/patologia , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/patologia , Transdução de Sinais/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Colo/citologia , Colo/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/citologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Modelos Animais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
9.
Sci Rep ; 9(1): 6068, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988473

RESUMO

X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , RNA Longo não Codificante/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Inativação do Cromossomo X/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Masculino , Modelos Animais , Cultura Primária de Células , Ratos
10.
Fertil Steril ; 112(6): 1059-1070.e3, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31767154

RESUMO

OBJECTIVE: To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects. DESIGN: Retrospective observational study. SETTING: University medical center research laboratory and andrology clinic. PATIENT(S): Forty-eight patients with confirmed spermatogenic impairment (Johnsen scores 3-6) and 15 controls (Johnsen score 10). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative assessment of immunofluorescent analyses of specific markers to determine meiotic entry, chromosome pairing, progression of DNA double-strand break repair, crossover formation, formation of meiotic metaphases, metaphase arrest, and spermatid formation, resulting in a novel classification of human meiotic arrest types. RESULT(S): Complete metaphase arrest was observed most frequently (27%), and the patients with the highest frequency of apoptotic metaphases also displayed a reduction in crossover number. Incomplete metaphase arrest was observed in 17% of the patients. Only four patients (8%) displayed a failure to complete meiotic chromosome pairing leading to pachytene arrest. Two new types of meiotic arrest were defined: premetaphase and postmetaphase arrest (15% and 13%, respectively). CONCLUSION(S): Meiotic arrest in men occurs most frequently at meiotic metaphase. This arrest can be incomplete, resulting in low numbers of spermatids, and often occurs in association with reduced crossover frequency. The phenotyping approach described here provides mechanistic insights to help identify candidate infertility genes and to assess genotype-phenotype correlations in individual cases.


Assuntos
Azoospermia/congênito , Metáfase , Espermatogênese , Espermatozoides/patologia , Testículo/patologia , Apoptose , Azoospermia/patologia , Azoospermia/fisiopatologia , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Humanos , Masculino , Estágio Paquíteno , Estudos Retrospectivos , Testículo/fisiopatologia
11.
Mol Cell Biol ; 25(3): 1041-53, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15657431

RESUMO

During meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination and chromatin silencing in more detail. First, we found that ubiquitinated H2A also marks the silenced X chromosome of the Barr body in female somatic cells. Next, we studied a possible relationship between H2A ubiquitination, chromatin silencing, and unpaired chromatin in meiotic prophase. The mouse models used carry an unpaired autosomal region in male meiosis or unpaired X and Y chromosomes in female meiosis. We show that ubiquitinated histone H2A is associated with transcriptional silencing of large chromatin regions. This silencing in mammalian meiotic prophase cells concerns unpaired chromatin regions and resembles a phenomenon described for the fungus Neurospora crassa and named meiotic silencing by unpaired DNA.


Assuntos
Inativação Gênica/fisiologia , Histonas/metabolismo , Meiose/genética , Cromatina Sexual/metabolismo , Aberrações dos Cromossomos Sexuais , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Meiose/fisiologia , Camundongos , Oócitos/metabolismo , Prófase/fisiologia , Ratos , Espermatócitos/metabolismo
12.
DNA Repair (Amst) ; 63: 25-38, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29414051

RESUMO

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Animais , Proteínas de Ciclo Celular/genética , DNA/metabolismo , DNA/efeitos da radiação , Endodesoxirribonucleases/genética , Feminino , Masculino , Meiose/efeitos da radiação , Camundongos , Camundongos Mutantes , Radiação Ionizante
13.
Epigenetics Chromatin ; 10: 11, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28293300

RESUMO

BACKGROUND: In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols. RESULTS: We observed that pericentric heterochromatin marks, such as H3K9me3, H4K20me3, and HP1 isoforms, were retained on pericentric heterochromatin throughout PGC development. However, the observed immunostaining patterns varied, depending on the fixation method, explaining previous findings of a general loss of pericentric heterochromatic features in PGCs. Also, in contrast to the general clustering of multiple pericentric regions and associated centromeres in DAPI-dense regions in somatic cells, the pericentric regions of PGCs were more frequently organized as individual entities. We also observed a transient enrichment of the chromatin remodeler ATRX in pericentric regions in embryonic day 11.5 (E11.5) PGCs. At this stage, a similar and low level of major satellite repeat RNA transcription was detected in both PGCs and somatic cells. CONCLUSIONS: These results indicate that in pericentric heterochromatin of mouse PGCs, only minor reductions in levels of some chromatin-associated proteins occur, in association with a transient increase in ATRX, between E11.5 and E13.5. These pericentric heterochromatin regions more frequently contain only a single centromere in PGCs compared to the surrounding soma, indicating a difference in overall organization, but there is no de-repression of major satellite transcription.


Assuntos
Células Germinativas/metabolismo , Heterocromatina/metabolismo , Animais , Centrômero/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Inativação Gênica , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Histonas/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
14.
Nucleic Acids Res ; 32(21): 6425-36, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15585666

RESUMO

In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.


Assuntos
Calnexina/genética , Regiões Promotoras Genéticas , Espermatogênese , Testículo/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Proteínas de Ligação ao Cálcio , Sequências Hélice-Alça-Hélice , Masculino , Camundongos , Chaperonas Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Espermatócitos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
15.
PLoS One ; 6(8): e23155, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21858012

RESUMO

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromatina/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação , Raios Ultravioleta , Dedos de Zinco/genética
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