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1.
Genes Dev ; 35(5-6): 354-366, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33602871

RESUMO

Polycomb repressive complex 1 (PRC1) and PRC2 are critical epigenetic developmental regulators. PRC1 and PRC2 largely overlap in their genomic binding and cooperate to establish repressive chromatin domains demarcated by H2AK119ub and H3K27me3. However, the functional contribution of each complex to gene repression has been a subject of debate, and understanding of its physiological significance requires further studies. Here, using the developing murine epidermis as a paradigm, we uncovered a previously unappreciated functional redundancy between Polycomb complexes. Coablation of PRC1 and PRC2 in embryonic epidermal progenitors resulted in severe defects in epidermal stratification, a phenotype not observed in the single PRC1-null or PRC2-null epidermis. Molecular dissection indicated a loss of epidermal identity that was coupled to a strong derepression of nonlineage transcription factors, otherwise repressed by either PRC1 or PRC2 in the absence of its counterpart. Ectopic expression of subsets of PRC1/2-repressed nonepidermal transcription factors in wild-type epidermal stem cells was sufficient to suppress epidermal identity genes, highlighting the importance of functional redundancy between PRC1 and PRC2. Altogether, our studies show how PRC1 and PRC2 function as two independent counterparts, thereby providing a repressive safety net that protects and preserves lineage identity.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células Epidérmicas/citologia , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Bioessays ; 42(5): e1900192, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32196702

RESUMO

Polycomb group proteins are evolutionary conserved chromatin-modifying complexes, essential for the regulation of developmental and cell-identity genes. Polycomb-mediated transcriptional regulation is provided by two multi-protein complexes known as Polycomb repressive complex 1 (PRC1) and 2 (PRC2). Recent studies positioned PRC1 as a foremost executer of Polycomb-mediated transcriptional control. Mammalian PRC1 complexes can form multiple sub-complexes that vary in their core and accessory subunit composition, leading to fascinating and diverse transcriptional regulatory mechanisms employed by PRC1 complexes. These mechanisms include PRC1-catalytic activity toward monoubiquitination of histone H2AK119, a well-established hallmark of PRC1 complexes, whose importance has been long debated. In this review, the central roles that PRC1-catalytic activity plays in transcriptional repression are emphasized and the recent evidence supporting a role for PRC1 complexes in gene activation is discussed.


Assuntos
Histonas , Complexo Repressor Polycomb 1 , Animais , Proteínas de Ciclo Celular , Cromatina , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ubiquitinação
3.
PLoS Genet ; 12(7): e1006151, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27414999

RESUMO

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.


Assuntos
Proteínas Hedgehog/fisiologia , Células de Merkel/citologia , Complexo Repressor Polycomb 2/fisiologia , Transdução de Sinais , Pele/embriologia , Animais , Linhagem da Célula , Proliferação de Células , Epiderme/embriologia , Epiderme/metabolismo , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Folículo Piloso/embriologia , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Pele/metabolismo , Células-Tronco/citologia , Transcrição Gênica
4.
Nucleic Acids Res ; 43(3): 1637-45, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25589543

RESUMO

DNA-damage tolerance (DDT) via translesion DNA synthesis (TLS) or homology-dependent repair (HDR) functions to bypass DNA lesions encountered during replication, and is critical for maintaining genome stability. Here, we present piggyBlock, a new chromosomal assay that, using piggyBac transposition of DNA containing a known lesion, measures the division of labor between the two DDT pathways. We show that in the absence of DNA damage response, tolerance of the most common sunlight-induced DNA lesion, TT-CPD, is achieved by TLS in mouse embryo fibroblasts. Meanwhile, BP-G, a major smoke-induced DNA lesion, is bypassed primarily by HDR, providing the first evidence for this mechanism being the main tolerance pathway for a biologically important lesion in a mammalian genome. We also show that, far from being a last-resort strategy as it is sometimes portrayed, TLS operates alongside nucleotide excision repair, handling 40% of TT-CPDs in repair-proficient cells. Finally, DDT acts in mouse embryonic stem cells, exhibiting the same pattern­mutagenic TLS included­despite the risk of propagating mutations along all cell lineages. The new method highlights the importance of HDR, and provides an effective tool for studying DDT in mammalian cells.


Assuntos
Cromossomos , Dano ao DNA , Animais , Sequência de Bases , Células Cultivadas , Camundongos , Sondas de Oligonucleotídeos
5.
Dev Cell ; 56(18): 2547-2561.e8, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34473941

RESUMO

Ultraviolet (UV) radiation is a prime environmental stressor that our epidermis is exposed to on a daily basis. To avert UV-induced damage, epidermal stem cells (EpSCs) become pigmented via a process of heterotypic interaction between melanocytes and EpSCs; however, the molecular mechanisms of this interaction are not well understood. In this study, we show that the function of a key chromatin regulator, the Polycomb complex, was reduced upon UV exposure in human and mouse epidermis. Genetic ablation of key Polycomb subunits in murine EpSCs, mimicking depletion upon UV exposure, results in an increased number of epidermal melanocytes and subsequent epidermal pigmentation. Genome-wide transcriptional and chromatin studies show that Polycomb regulates the expression of UV-responsive genes and identifies type II collagen (COL2A1) as a critical secreted regulator of melanogenesis and epidermal pigmentation. Together, our findings show how UV exposure induces Polycomb-mediated changes in EpSCs to affect melanocyte behavior and promote epidermal pigmentation.


Assuntos
Células Epidérmicas/citologia , Epiderme/metabolismo , Melanócitos/metabolismo , Células-Tronco/citologia , Animais , Células Cultivadas , Epiderme/patologia , Queratinócitos/metabolismo , Camundongos Transgênicos , Pigmentação/fisiologia , Pigmentação da Pele/fisiologia , Raios Ultravioleta/efeitos adversos
6.
Methods Mol Biol ; 2154: 197-215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32314219

RESUMO

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method designed to detect interactions between chromatin and the proteins bound to it. This method has been widely used for characterizing epigenetic landscapes in many cell types; however, a limiting factor has been the requirement of a high number of cells. Here, we describe a protocol for ChIP in epidermal cells from a newborn mouse, purified by fluorescence-activated cell sorting (FACS). This protocol has been optimized specifically for prefixed, low cell numbers, resulting in enough immunoprecipitated DNA suitable for genome-wide analysis.


Assuntos
Imunoprecipitação da Cromatina , Células Epidérmicas/metabolismo , Citometria de Fluxo , Animais , Animais Recém-Nascidos , Western Blotting , Imunoprecipitação da Cromatina/métodos , Citometria de Fluxo/métodos , Imuno-Histoquímica , Camundongos , Pele
7.
Cell Rep ; 28(1): 257-266.e5, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269445

RESUMO

How tissue patterns are formed and maintained are fundamental questions. The murine tongue epithelium, a paradigm for tissue patterning, consists of an array of specialized fungiform papillae structures that harbor taste cells. The formation of fungiform papillae is preceded by pronounced spatial changes in gene expression, in which taste cell genes such as Shh, initially diffused in lingual epithelial progenitors, become restricted to taste cells when their specification progresses. However, the requirement of spatial restriction of taste cell gene expression for patterning and formation of fungiform papillae is unknown. Here, we show that a chromatin regulator, Polycomb repressive complex (PRC) 1, is required for proper maintenance of fungiform papillae by repressing Shh and preventing ectopic SHH signaling in non-taste cells. Ablation of SHH signaling in PRC1-null non-taste cells rescues the maintenance of taste cells. Altogether, our studies exemplify how epigenetic regulation establishes spatial gene expression patterns necessary for specialized niche structures.


Assuntos
Proteínas Hedgehog/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Papilas Gustativas/metabolismo , Língua/metabolismo , Animais , Padronização Corporal/genética , Ciclo Celular/genética , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Epigênese Genética , Epitélio/metabolismo , Epitélio/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/genética , Ontologia Genética , Proteínas Hedgehog/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA-Seq , Transdução de Sinais/genética , Papilas Gustativas/crescimento & desenvolvimento , Papilas Gustativas/ultraestrutura , Língua/crescimento & desenvolvimento , Língua/fisiologia
8.
Cell Stem Cell ; 22(5): 726-739.e7, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29727681

RESUMO

Polycomb repressive complexes (PRCs) 1 and 2 are essential chromatin regulators of cell identity. PRC1, a dominant executer of Polycomb-mediated control, functions as multiple sub-complexes that possess catalytic-dependent H2AK119 mono-ubiquitination (H2AK119ub) and catalytic-independent activities. Here, we show that, despite its well-established repressor functions, PRC1 binds to both silent and active genes. Through in vivo loss-of-function studies, we show that global PRC1 function is essential for skin development and stem cell (SC) specification, whereas PRC1 catalytic activity is dispensable. Further dissection demonstrated that both canonical and non-canonical PRC1 complexes bind to repressed genes, marked by H2AK119ub and PRC2-mediated H3K27me3. Interestingly, loss of canonical PRC1, PRC1 catalytic activity, or PRC2 leads to expansion of mechanosensitive Merkel cells in neonatal skin. Non-canonical PRC1 complexes, however, also bind to and promote expression of genes critical for skin development and SC formation. Together, our findings highlight PRC1's diverse roles in executing a precise developmental program.


Assuntos
Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Pele/metabolismo , Células-Tronco/metabolismo , Animais , Biocatálise , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Complexo Repressor Polycomb 1/deficiência , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Pele/citologia
9.
Nat Commun ; 9(1): 2333, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899403

RESUMO

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Folículo Piloso/embriologia , Folículo Piloso/metabolismo , Células de Merkel/citologia , Células de Merkel/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOX9/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula , Feminino , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folículo Piloso/citologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Gravidez , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOX9/antagonistas & inibidores , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 9(1): 2854, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018293

RESUMO

The originally published version of this Article contained an error in Figure 2. In panel e, the blue bar was incorrectly labelled 'KRT8(+)/TOMATO(-)'. Furthermore, during the process of preparing a correction, the publication date of the Article was inadvertently changed to June 20th 2018. Both of these errors have been corrected in the PDF and HTML versions of the Article.

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