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1.
Development ; 149(22)2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36355065

RESUMO

Female mouse embryonic stem cells (mESCs) present differently from male mESCs in several fundamental ways; however, complications with their in vitro culture have resulted in an under-representation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female and male mESCs apart. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state that is near indistinguishable from male mESCs. If female pluripotency is to be studied effectively in this system, it is crucial that high-quality cultures of XX mESCs are available. Here, we report a method for better maintaining XX female mESCs in culture that also stabilises the male karyotype and makes study of female-specific pluripotency more feasible.


Assuntos
Células-Tronco Embrionárias Murinas , Inativação do Cromossomo X , Masculino , Animais , Feminino , Camundongos , Diferenciação Celular/fisiologia , Inativação do Cromossomo X/genética , Cariótipo
2.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34121118

RESUMO

Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.


Assuntos
Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Pulmão/embriologia , Fatores de Transcrição SOX9/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Organogênese , Fatores de Transcrição SOX9/genética
3.
Hum Mol Genet ; 27(4): 716-731, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29281018

RESUMO

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Haploinsuficiência/fisiologia , Animais , Western Blotting , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Fibroblastos/metabolismo , Citometria de Fluxo , Haploinsuficiência/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele , Timócitos
4.
Proc Natl Acad Sci U S A ; 112(27): E3535-44, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26091879

RESUMO

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Genoma , Animais , Sítios de Ligação/genética , Western Blotting , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Fator de Ligação a CCCTC , Células Cultivadas , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Histonas/metabolismo , Masculino , Metilação , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genética
5.
Blood ; 125(12): 1890-900, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25645357

RESUMO

Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function.


Assuntos
Regulação Neoplásica da Expressão Gênica , Complexo Repressor Polycomb 2/metabolismo , Animais , Antígenos CD34/metabolismo , Linhagem da Célula , Perfilação da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/citologia , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia
6.
Proc Natl Acad Sci U S A ; 109(8): 2766-71, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-21768359

RESUMO

Overexpression of the prosurvival protein BCL-2 is common in breast cancer. Here we have explored its role as a potential therapeutic target in this disease. BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the proapoptotic BH3-only ligand BIM were found to be coexpressed at relatively high levels in a substantial proportion of heterogeneous breast tumors, including clinically aggressive basal-like cancers. To determine whether the BH3 mimetic ABT-737 that neutralizes BCL-2, BCL-XL, and BCL-W had potential efficacy in targeting BCL-2-expressing basal-like triple-negative tumors, we generated a panel of primary breast tumor xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel, or a combination. Tumor response and overall survival were significantly improved by combination therapy, but only for tumor xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumor cells to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2. Notably, BH3 mimetics also appeared effective in BCL-2-expressing xenograft lines that harbored p53 mutations. Our findings provide in vivo evidence that BH3 mimetics can be used to sensitize primary breast tumors to chemotherapy and further suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer.


Assuntos
Compostos de Bifenilo/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Nitrofenóis/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Docetaxel , Feminino , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Indução de Remissão , Sulfonamidas/farmacologia , Taxoides/farmacologia , Taxoides/uso terapêutico , Proteína bcl-X/metabolismo
7.
Nat Commun ; 14(1): 5466, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37749075

RESUMO

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.


Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Distrofia Muscular Facioescapuloumeral , Animais , Camundongos , Cromatina/genética , Epigenômica , Inativação Gênica , Genes Homeobox , Distrofia Muscular Facioescapuloumeral/genética , Proteínas Cromossômicas não Histona/genética
8.
Nat Commun ; 13(1): 4295, 2022 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-35879318

RESUMO

Parents transmit genetic and epigenetic information to their offspring. Maternal effect genes regulate the offspring epigenome to ensure normal development. Here we report that the epigenetic regulator SMCHD1 has a maternal effect on Hox gene expression and skeletal patterning. Maternal SMCHD1, present in the oocyte and preimplantation embryo, prevents precocious activation of Hox genes post-implantation. Without maternal SMCHD1, highly penetrant posterior homeotic transformations occur in the embryo. Hox genes are decorated with Polycomb marks H2AK119ub and H3K27me3 from the oocyte throughout early embryonic development; however, loss of maternal SMCHD1 does not deplete these marks. Therefore, we propose maternal SMCHD1 acts downstream of Polycomb marks to establish a chromatin state necessary for persistent epigenetic silencing and appropriate Hox gene expression later in the developing embryo. This is a striking role for maternal SMCHD1 in long-lived epigenetic effects impacting offspring phenotype.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Animais , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Expressão Gênica , Camundongos , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Gravidez
9.
iScience ; 25(7): 104684, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35856023

RESUMO

SMCHD1 (structural maintenance of chromosomes hinge domain containing 1) is a noncanonical SMC protein that mediates long-range repressive chromatin structures. SMCHD1 is required for X chromosome inactivation in female cells and repression of imprinted and clustered autosomal genes, with SMCHD1 mutations linked to human diseases facioscapulohumeral muscular dystrophy (FSHD) and bosma arhinia and micropthalmia syndrome (BAMS). We used a conditional mouse model to investigate SMCHD1 in hematopoiesis. Smchd1-deleted mice maintained steady-state hematopoiesis despite showing an impaired reconstitution capacity in competitive bone marrow transplantations and age-related hematopoietic stem cell (HSC) loss. This phenotype was more pronounced in Smchd1-deleted females, which showed a loss of quiescent HSCs and fewer B cells. Gene expression profiling of Smchd1-deficient HSCs and B cells revealed known and cell-type-specific SMCHD1-sensitive genes and significant disruption to X-linked gene expression in female cells. These data show SMCHD1 is a regulator of HSCs whose effects are more profound in females.

10.
Epigenetics Chromatin ; 15(1): 26, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35843975

RESUMO

Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1's role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.


Assuntos
Proteínas Cromossômicas não Histona , RNA Longo não Codificante , Inativação do Cromossomo X , Animais , Blastocisto/fisiologia , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Desenvolvimento Embrionário , Impressão Genômica , Camundongos , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X
11.
Nat Commun ; 13(1): 1658, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35351876

RESUMO

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.


Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Animais , Cromatina/genética , Mecanismo Genético de Compensação de Dose , Epigênese Genética , Feminino , Camundongos , RNA Longo não Codificante/genética , Cromossomo X/genética , Inativação do Cromossomo X/genética
12.
NAR Genom Bioinform ; 3(2): lqab028, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33937765

RESUMO

Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs ('sequins') as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.

13.
Elife ; 92020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33186096

RESUMO

Genomic imprinting establishes parental allele-biased expression of a suite of mammalian genes based on parent-of-origin specific epigenetic marks. These marks are under the control of maternal effect proteins supplied in the oocyte. Here we report epigenetic repressor Smchd1 as a novel maternal effect gene that regulates the imprinted expression of ten genes in mice. We also found zygotic SMCHD1 had a dose-dependent effect on the imprinted expression of seven genes. Together, zygotic and maternal SMCHD1 regulate three classic imprinted clusters and eight other genes, including non-canonical imprinted genes. Interestingly, the loss of maternal SMCHD1 does not alter germline DNA methylation imprints pre-implantation or later in gestation. Instead, what appears to unite most imprinted genes sensitive to SMCHD1 is their reliance on polycomb-mediated methylation as germline or secondary imprints, therefore we propose that SMCHD1 acts downstream of polycomb imprints to mediate its function.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Impressão Genômica/genética , Animais , Blastocisto , Proteínas Cromossômicas não Histona/genética , Metilação de DNA , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , Proteínas de Fluorescência Verde , Masculino , Camundongos , Células-Tronco Neurais
14.
Nat Struct Mol Biol ; 25(9): 766-777, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127357

RESUMO

The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Genes Homeobox , Família Multigênica , Cromossomo X , Animais , Proteínas Cromossômicas não Histona/genética , Elementos Facilitadores Genéticos , Deleção de Genes , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
Cell Rep ; 25(7): 1912-1923.e9, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428357

RESUMO

We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Complexo Repressor Polycomb 1/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X/genética , Animais , Sequência de Bases , Diferenciação Celular , Feminino , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oligonucleotídeos/metabolismo , Transporte Proteico
16.
Artigo em Inglês | MEDLINE | ID: mdl-27195021

RESUMO

BACKGROUND: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. RESULTS: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. CONCLUSIONS: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.

17.
Cell Rep ; 3(2): 411-26, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23375371

RESUMO

The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.


Assuntos
Epigênese Genética , Glândulas Mamárias Animais/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Glândulas Mamárias Animais/citologia , Metilação , Camundongos , Fosforilação/efeitos dos fármacos , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/metabolismo , Gravidez , Progesterona/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo
18.
Cancer Cell ; 24(1): 120-9, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23845444

RESUMO

The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Receptores de Estrogênio/análise , Sulfonamidas/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos SCID , Nitrofenóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Piperazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cell Biol ; 31(22): 4609-22, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21930782

RESUMO

The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of "luminal-like" cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Caspase 14/metabolismo , Fator de Transcrição GATA3/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Neoplasias Mamárias Experimentais/metabolismo , Adulto , Idoso , Animais , Antígenos Transformantes de Poliomavirus/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Caspase 14/biossíntese , Diferenciação Celular , Transformação Celular Neoplásica/genética , Feminino , Fator de Transcrição GATA3/biossíntese , Fator de Transcrição GATA3/genética , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Pessoa de Meia-Idade , Células-Tronco
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