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1.
Mol Cell ; 70(3): 408-421.e8, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29628311

RESUMEN

The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.


Asunto(s)
Complejo Represivo Polycomb 2/genética , Proteínas Represoras/genética , Vertebrados/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Células HEK293 , Histonas/genética , Humanos , Metilación , Metiltransferasas/genética , Ratones , Neoplasias/genética , Alineación de Secuencia
2.
Genes Dev ; 29(21): 2231-43, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26494712

RESUMEN

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas del Grupo Polycomb/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proliferación Celular/genética , Células Cultivadas , Cromatina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F/metabolismo , Humanos , Ratones , Proteínas del Grupo Polycomb/genética , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína/genética
3.
Mol Cell Proteomics ; 15(11): 3450-3460, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27634302

RESUMEN

Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12, and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency data set reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, whereas PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.


Asunto(s)
Células Madre de Carcinoma Embrionario/citología , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 2/metabolismo , Proteómica/métodos , Diferenciación Celular , Línea Celular Tumoral , Células Madre de Carcinoma Embrionario/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias , Células Madre Pluripotentes/metabolismo , Mapas de Interacción de Proteínas , Factores de Transcripción
4.
Proteomics ; 15(21): 3603-12, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26223664

RESUMEN

Native gel electrophoresis enables separation of cellular proteins in their non-denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co-migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes.


Asunto(s)
Complejos Multiproteicos/análisis , Electroforesis en Gel de Poliacrilamida Nativa/métodos , Proteómica/métodos , Cromatina/química , Células HEK293 , Humanos , Immunoblotting/métodos , Complejos Multiproteicos/aislamiento & purificación
5.
FEBS J ; 282(18): 3455-73, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26094870

RESUMEN

The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis.


Asunto(s)
Neoplasias de la Mama/genética , Redes Reguladoras de Genes , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Proliferación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Estudios de Cohortes , Femenino , Genes p16 , Humanos , Metástasis Linfática/genética , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Ratones , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Estrógenos/metabolismo , Factores de Riesgo , Análisis de Matrices Tisulares
6.
Nat Struct Mol Biol ; 19(12): 1273-81, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23160351

RESUMEN

Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb repressive complex 2 (PRC2) component PHF19 binds trimethylated histone H3 Lys36 (H3K36me3), a mark of active chromatin, via its Tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and it is required to recruit the PRC2 complex and NO66 to stem cell genes during differentiation, leading to PRC2-mediated trimethylation of histone H3 Lys27 (H3K27), loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during mouse embryonic stem cell differentiation to transiently bind the H3K36me3 mark via its Tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Células Madre Embrionarias/citología , Ratones
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