Epigenetic mechanisms are crucial for sustaining cell type-specific transcription programs. Among the distinct factors, Polycomb group (PcG) proteins are major negative regulators of gene expression in mammals. These proteins play key roles in regulating the proliferation, self-renewal, and differentiation of stem cells. During hematopoietic differentiation, many PcG proteins are fundamental for proper lineage commitment, as highlighted by the fact that a lack of distinct PcG proteins results in embryonic lethality accompanied by differentiation biases. Correspondingly, proteins of these complexes are frequently dysregulated in hematological diseases. In this review, we present an overview of the role of PcG proteins in normal and malignant hematopoiesis, focusing on the compositional complexity of PcG complexes, and we briefly discuss the ongoing clinical trials for drugs targeting these factors. ; Support to the Di Croce laboratory comes from grants from the Spanish Ministry of Economy, Industry, and Competitiveness (BFU2016-75008-P), the European Regional Development Fund (FEDER), Fundacion Vencer El Cancer, and Agència de Gestió d'Ajuts Universitaris i de Recerca; to V. Di Carlo from an ImPuLSe Marie Curie Postdoctoral Fellowship of the European Union Seventh Framework Program (FP7/2007-2013; grant 608959); and I. Mocavini from an FPI fellowship. We acknowledge support of the Spanish Ministry of Economy, Industry, and Competitiveness through the Instituto de Salud Carlos III and for the European Molecular Biology Laboratory partnership; Centro de Excelencia Severo Ochoa; and the Centres de Recerca de Catalunya Program/Generalitat de Catalunya.
De novo identification of chromatin interactors can reveal unexpected pathways relevant to physiology and human disease. Inspired by the DNA mediated chromatin pull-down (Dm-ChP) technology (also known as iPOND [isolation of proteins on nascent DNA]) for the proteomic characterization of nascent DNA, we have recently reported a new experimental protocol that allows for the identification of proteins on total DNA (iPOTD) for bulk chromatome profiling and de novo identification of chromatin-bound proteins. Here, we detail a step-by-step protocol to survey the cellular chromatin-bound proteome in a simple, robust, and unbiased manner. For complete details on the use and execution of this protocol, please refer to Aranda et al. (2019). ; The Proteomics Unit is supported by EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union. The Di Croce Laboratory is supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2016-75008-P) and by AGAUR.
Profiling the chromatin-bound proteome (chromatome) in a simple, direct, and reliable manner might be key to uncovering the role of yet uncharacterized chromatin factors in physiology and disease. Here, we have designed an experimental strategy to survey the chromatome of proliferating cells by using the DNA-mediated chromatin pull-down (Dm-ChP) technology. Our approach provides a global view of cellular chromatome under normal physiological conditions and enables the identification of chromatin-bound proteins de novo. Integrating Dm-ChP with genomic and functional data, we have discovered an unexpected chromatin function for adenosylhomocysteinase, a major one-carbon pathway metabolic enzyme, in gene activation. Our study reveals a new regulatory axis between the metabolic state of pluripotent cells, ribosomal protein production, and cell division during the early phase of embryo development, in which the metabolic flux of methylation reactions is favored in a local milieu. ; We acknowledge support from the Spanish Ministry of Economy, Industry and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (to S.A. and A.A.-C.). The CRG/UPF Proteomics Unit is a member of the ProteoRed PRB3 consortium that was supported by grant PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII), ERDF, and "Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya" (2017SGR595). The Di Croce Laboratory was supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2016-75008-P), AGAUR, and La Marato TV3
Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but little is known regarding the function of CBX6. Here, we show that CBX6 is essential for ESC identity. Its depletion destabilizes the pluripotency network and triggers differentiation. Mechanistically, we find that CBX6 is physically and functionally associated to both canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. Notably, in contrast to CBX7, CBX6 is recruited to chromatin independently of H3K27me3. Taken together, our findings reveal that CBX6 is an essential component of ESC biology that contributes to the structural and functional complexity of the PRC1 complex. ; This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2016-75008-P), Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208), AGAUR, Fundació "La Marató de TV3," CERCA Programme/Generalitat de Catalunya, and EU FP7 Programs 4DCellFate (277899) to L.D.C. This work aided by collaboration with the National Center for Dynamic Interactome Research and the National Resource for the Mass Spectrometric Analysis of Biological Macromolecules, supported in part by NIH grant P41 GM109824 to Michael P. Rout and grant P41 GM103314 to Brian T. Chait. This paper is subject to the NIH Public Access Policy.
The unicellular ancestor of animals had a complex repertoire of genes linked to multicellular processes. This suggests that changes in the regulatory genome, rather than in gene innovation, were key to the origin of animals. Here, we carry out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression, and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora. Moreover, we demonstrate conservation of animal developmental transcription-factor networks and extensive network interconnection in this premetazoan organism. In contrast, however, Capsaspora lacks animal promoter types, and its regulatory sites are small, proximal, and lack signatures of animal enhancers. Overall, our results indicate that the emergence of animal multicellularity was linked to a major shift in genome cis-regulatory complexity, most notably the appearance of distal enhancer regulation. ; This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, a European Research Council Consolidator Grant (ERC-2012-Co-616960), and a grant from Ministerio de Economía y Competitividad (MINECO) (BFU-2011-23434) (to I.R.-T.). We also acknowledge financial support from Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (project 2014 SGR 619). The work in L.D.'s laboratory was supported by grants from the Spanish "Ministerio de Educación y Ciencia" (SAF2013-48926-P), AGAUR, and the European Commission's 7th Framework Program 4DCellFate (277899). A.S.-P. is supported by an EMBO Long-Term Fellowship (ALTF 841-2014). J.L.G.-S. was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). J.J.T. has a postdoctoral grant from the University Pablo de Olavide. The CRG/UPF Proteomics Unit is part of the "Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)" supported by a grant from Instituto de Salud Carlos III (ISCIII) (PT13/0001). We thank Guadalupe Espadas for her support with the histone derivatization protocol and Núria Ros and Meritxell Antó for technical support. Finally, we thank the CRG Genomics Unit for helping with ChIP-seq and RNA-seq sequencing.
The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis. ; The S.A.B. laboratory research is supported by the European Research Council (ERC), the Worldwide Cancer Research Foundation, the Foundation La Marató de TV3, the Spanish Ministry of Economy and Development, the Foundation Vencer el Cancer (''Beat Cancer''), the Government of Cataluña (SGR and Mario Salvia' grants), the Foundation Fundación Botín, and the Institute for Research in Biomedicine (IRB-Barcelona). L.R. is a La Caixa Foundation Ph.D. fellow. G.S. was supported by an AXA postdoctoral fellowship. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). L.D.C. was supported by grants from the Spanish Ministerio de Educación y Ciencia (SAF2013-48926-P) and the European Commission's 7th Framework Program 4DCellFate grant number 277899. We are grateful to the Common Fund's Epigenomic Program from the NIH (USA) for providing the bisulphite whole genome sequencing data of human EpSCs
Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis. ; The work in the Di Croce laboratory is supported by grants from the Spanish of Economy, Industry, and Competitiveness (MEIC) (BFU2016-75008-P), "Fundación Vencer El Cancer" (VEC), the European Regional Development Fund (FEDER), Fundació "La Marató de TV3," and from AGAUR. The laboratory of A.B. is supported by SAF2016-75613-R from the Ministerio de Ciencia, Innovación y Universidades. H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII) and received funding from the European Union's Horizon 2020 research and innovation programme (MSCA-ITN-2015-675752) and the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89109-P; AEI/FEDER, UE). P.V. was supported by "Fundación Científica de la Asociación Española Contra el Cáncer."
Changes in the epigenetic regulation of gene expression have a central role in evolution. Here, we extensively profiled a panel of human, chimpanzee, gorilla, orangutan, and macaque lymphoblastoid cell lines (LCLs), using ChIP-seq for five histone marks, ATAC-seq and RNA-seq, further complemented with whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS). We annotated regulatory elements (RE) and integrated chromatin contact maps to define gene regulatory architectures, creating the largest catalog of RE in primates to date. We report that epigenetic conservation and its correlation with sequence conservation in primates depends on the activity state of the regulatory element. Our gene regulatory architectures reveal the coordination of different types of components and highlight the role of promoters and intragenic enhancers (gE) in the regulation of gene expression. We observe that most regulatory changes occur in weakly active gE. Remarkably, novel human-specific gE with weak activities are enriched in human-specific nucleotide changes. These elements appear in genes with signals of positive selection and human acceleration, tissue-specific expression, and particular functional enrichments, suggesting that the regulatory evolution of these genes may have contributed to human adaptation. ; R.G.-P. was supported by a fellowship from MICINN (FPU13/01823). P.E.-C. was supported by a Formació de Personal Investigador fellowship from Generalitat de Catalunya (FI_B00122). M.K. was supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship (KU 3467/1-1) and the Postdoctoral Junior Leader Fellowship Program from "la Caixa" Banking Foundation (LCF/BQ/PR19/11700002). D.J. was supported by a Juan de la Cierva fellowship (FJCI2016-29558) from MICINN. T.M-B. is supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement EC-H2020-ERC-CoG-ApeGenomeDiversity-864203), BFU2017-86471-P (AEI/FEDER, UE), "Unidad de Excelencia María de Maeztu", funded by the AEI (CEX2018-000792-M), Howard Hughes International Early Career, NIH 1R01HG010898-01A1, Obra Social "La Caixa" and Secretaria d'Universitats i Recerca and CERCA Program del Departament d'Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). G.M., V.D.C., and L.D.C. were supported by grants from the Spanish of Economy, Industry, and Competitiveness (MEIC) (BFU2016-75008-P) and G.M. was also supported by the "Convocatoria de Ayudas Fundación BBVA a Investigadores, Innovadores y Creadores Culturales". J.L.G.-S. was supported by the Spanish government (grants BFU2016-74961-P), an institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687) and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 740041). A.N. was supported by Fondo Europeo de Desarrollo Regional (FEDER) with project grants BFU2016-77961-P and PGC2018- 101927-B-I00 and by the Spanish National Institute of Bioinformatics (PT17/0009/0020).
Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1–induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors. ; S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC).
Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs ; This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) FIS/FEDER (PI15/00396; CPII14/0006), Ministerio de Economía y Competitividad (MINECO) (SAF2013-40922-R1; FPU14/0407; BFU2016-75008-P), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional-FEDER (SAF2016-76461-R), Generalitat de Catalunya (2014 SGR 32), Fundació FERO, Fundació La Marató TV3, and La Caixa Foundation. We also thank the Advanced Light Microscopy Unit at the CRG for their assistance and the Cellex Foundation for providing research facilities and equipment. M.A.M.-R. acknowledges funding from the European Research Council under the 7th Framework Program (FP7/2010-2015, ERC grant agreement 609989), the European Union's Horizon 2020 research and innovation program (agreement 676556), the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P), and the Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208) to the CRG
Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1-induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors. ; S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC).
INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor alpha (ERalpha) are among the most effective systemic treatments for ERalpha-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERalpha transcriptional regulatory plasticity. Here, we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERalpha was evaluated by molecular docking analyses, an agonist fluoligand assay, and short-hairpin (sh) RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot of signaling and proliferation markers and shRNA-mediated protein depletion in viability and clonogenic assays were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine the association with therapy response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase) but instead a result of binding to ERalpha. VAV3 was selectively reduced upon exposure to YC-1 or ERalpha depletion and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 x 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and rationale signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer. ; We wish to thank all study participants and their clinicians for their valuable contributions. This work was supported by grants from the Eugenio Rodriguez Pascual Foundation (2012, to MAP), the Government of Catalonia (2009-SGR283, to AV and MAP), the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R01 DK015556, to JAK), the Red Cooperative Research Thematic Network on Cancer (RTICC) (12/0036/0002 to XRB and 12/0036/0008 to XRB and MAP) and the Spanish Ministry of Health, Fund for Health Research-Institute of Health Carlos III (11/00951 to AU and 12/01528 to MAP).
