Publications by authors named "Marian Martínez-Balbás"

27 Publications

  • Page 1 of 1

PHF2 histone demethylase prevents DNA damage and genome instability by controlling cell cycle progression of neural progenitors.

Proc Natl Acad Sci U S A 2019 09 5;116(39):19464-19473. Epub 2019 Sep 5.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Spain;

Histone H3 lysine 9 methylation (H3K9me) is essential for cellular homeostasis; however, its contribution to development is not well established. Here, we demonstrate that the H3K9me2 demethylase PHF2 is essential for neural progenitor proliferation in vitro and for early neurogenesis in the chicken spinal cord. Using genome-wide analyses and biochemical assays we show that PHF2 controls the expression of critical cell cycle progression genes, particularly those related to DNA replication, by keeping low levels of H3K9me3 at promoters. Accordingly, PHF2 depletion induces R-loop accumulation that leads to extensive DNA damage and cell cycle arrest. These data reveal a role of PHF2 as a guarantor of genome stability that allows proper expansion of neural progenitors during development.
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http://dx.doi.org/10.1073/pnas.1903188116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765295PMC
September 2019

E proteins sharpen neurogenesis by modulating proneural bHLH transcription factors' activity in an E-box-dependent manner.

Elife 2018 08 10;7. Epub 2018 Aug 10.

Department of Developmental Biology, Instituto de Biología Molecular de Barcelona, Barcelona, Spain.

Class II HLH proteins heterodimerize with class I HLH/E proteins to regulate transcription. Here, we show that E proteins sharpen neurogenesis by adjusting the neurogenic strength of the distinct proneural proteins. We find that inhibiting BMP signaling or its target ID2 in the chick embryo spinal cord, impairs the neuronal production from progenitors expressing ATOH1/ASCL1, but less severely that from progenitors expressing NEUROG1/2/PTF1a. We show this context-dependent response to result from the differential modulation of proneural proteins' activity by E proteins. E proteins synergize with proneural proteins when acting on CAGSTG motifs, thereby facilitating the activity of ASCL1/ATOH1 which preferentially bind to such motifs. Conversely, E proteins restrict the neurogenic strength of NEUROG1/2 by directly inhibiting their preferential binding to CADATG motifs. Since we find this mechanism to be conserved in corticogenesis, we propose this differential co-operation of E proteins with proneural proteins as a novel though general feature of their mechanism of action.
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http://dx.doi.org/10.7554/eLife.37267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126921PMC
August 2018

Lineage specific transcription factors and epigenetic regulators mediate TGFβ-dependent enhancer activation.

Nucleic Acids Res 2018 04;46(7):3351-3365

Department of Molecular Genomics. Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona 08028, Spain.

During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFβ signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR-Cas9 technology to demonstrate that the TGFβ-responsive Neurog2 enhancer is essential for proper neuronal polarization.
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http://dx.doi.org/10.1093/nar/gky093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909450PMC
April 2018

MicroRNA-200, associated with metastatic breast cancer, promotes traits of mammary luminal progenitor cells.

Oncotarget 2017 Oct 7;8(48):83384-83406. Epub 2017 Sep 7.

Institute of Molecular Biology, Spanish National Research Council (IBMB-CSIC), Barcelona, Spain.

MicroRNAs are critical regulators of gene networks in normal and abnormal biological processes. Focusing on invasive ductal breast cancer (IDC), we have found dysregulated expression in tumor samples of several microRNAs, including the miR-200 family, along progression from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b and miR-7 in IDC patients with regional or distant metastases relative to patients with primary node-negative tumors. Forced expression of miR-200s in MCF10CA1h mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization . MiR-200s also induced the constitutive activation of the PI3K-Akt signaling through downregulation of PTEN, and the enhanced mammosphere growth and ALDH activity induced in MCF10CA1h cells by miR-200s required the activation of this signaling pathway. Interestingly, the morphology of tumors formed by cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC). Indeed, the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. We propose that microRNAs of the miR-200 family promote traits of highly proliferative breast luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.
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http://dx.doi.org/10.18632/oncotarget.20698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663523PMC
October 2017

The histone demethylase PHF8 is a molecular safeguard of the IFNγ response.

Nucleic Acids Res 2017 04;45(7):3800-3811

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona 08028, Spain.

A precise immune response is essential for cellular homeostasis and animal survival. The paramount importance of its control is reflected by the fact that its non-specific activation leads to inflammatory events that ultimately contribute to the appearance of many chronic diseases. However, the molecular mechanisms preventing non-specific activation and allowing a quick response upon signal activation are not yet fully understood. In this paper we uncover a new function of PHF8 blocking signal independent activation of immune gene promoters. Affinity purifications coupled with mass spectrometry analysis identified SIN3A and HDAC1 corepressors as new PHF8 interacting partners. Further molecular analysis demonstrated that prior to interferon gamma (IFNγ) stimulation, PHF8 is bound to a subset of IFNγ-responsive promoters. Through the association with HDAC1 and SIN3A, PHF8 keeps the promoters in a silent state, maintaining low levels of H4K20me1. Upon IFNγ treatment, PHF8 is phosphorylated by ERK2 and evicted from the promoters, correlating with an increase in H4K20me1 and transcriptional activation. Our data strongly indicate that in addition to its well-characterized function as a coactivator, PHF8 safeguards transcription to allow an accurate immune response.
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http://dx.doi.org/10.1093/nar/gkw1346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397186PMC
April 2017

EZH2 orchestrates apicobasal polarity and neuroepithelial cell renewal.

Neurogenesis (Austin) 2016 17;3(1):e1250034. Epub 2016 Nov 17.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC) , Barcelona, Spain.

During early stages of neural development, neuroepithelial cells translocate their nuclei along the apicobasal axis in a harmonized manner with the cell cycle. How cell cycle progression and neuroepithelium polarity are coordinated remains unclear. It has been proposed that developmental cues, epigenetic mechanisms and cell cycle regulators must be linked in order to orchestrate these processes. We have recently discovered that a master epigenetic factor, EZH2 is essential to coordinate these events. EZH2 directly represses the cell cycle regulator in the chicken spinal cord. By doing so, EZH2 controls neural progenitor cell renewal and fine-tunes Rho signaling pathway, which is essential to maintain neuroepithelial structure. Our findings point to a new role of EZH2 during development that could have potential implication in other areas as cancer.
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http://dx.doi.org/10.1080/23262133.2016.1250034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129905PMC
November 2016

EZH2 regulates neuroepithelium structure and neuroblast proliferation by repressing p21.

Open Biol 2016 Apr 20;6(4):150227. Epub 2016 Apr 20.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona 08028, Spain

The function of EZH2 as a transcription repressor is well characterized. However, its role during vertebrate development is still poorly understood, particularly in neurogenesis. Here, we uncover the role of EZH2 in controlling the integrity of the neural tube and allowing proper progenitor proliferation. We demonstrate that knocking down the EZH2 in chick embryo neural tubes unexpectedly disrupts the neuroepithelium (NE) structure, correlating with alteration of the Rho pathway, and reduces neural progenitor proliferation. Moreover, we use transcriptional profiling and functional assays to show that EZH2-mediated repression of p21(WAF1/CIP1) contributes to both processes. Accordingly, overexpression of cytoplasmic p21(WAF1/CIP1) induces NE structural alterations and p21(WAF1/CIP1) suppression rescues proliferation defects and partially compensates for the structural alterations and the Rho activity. Overall, our findings describe a new role of EZH2 in controlling the NE integrity in the neural tube to allow proper progenitor proliferation.
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http://dx.doi.org/10.1098/rsob.150227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852452PMC
April 2016

Jumonji family histone demethylases in neural development.

Cell Tissue Res 2015 Jan 21;359(1):87-98. Epub 2014 Jun 21.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Parc Científic de Barcelona (PCB), Barcelona, 08028, Spain.

Central nervous system (CNS) development is driven by coordinated actions of developmental signals and chromatin regulators that precisely regulate gene expression patterns. Histone methylation is a regulatory mechanism that controls transcriptional programs. In the last 10 years, several histone demethylases (HDM) have been identified as important players in neural development, and their implication in cell fate decisions is beginning to be recognized. Identification of the physiological roles of these enzymes and their molecular mechanisms of action will be necessary for completely understanding the process that ultimately generates different neural cells in the CNS. In this review, we provide an overview of the Jumonji family of HDMs involved in neurodevelopment, and we discuss their roles during neural fate establishment and neuronal differentiation.
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http://dx.doi.org/10.1007/s00441-014-1924-7DOI Listing
January 2015

Regulation of CBP and Tip60 coordinates histone acetylation at local and global levels during Ras-induced transformation.

Carcinogenesis 2014 Oct 22;35(10):2194-202. Epub 2014 May 22.

Department of Genomic Regulation, Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Baldiri i Reixac 15-21, Parc Científic de Barcelona, E-08028 Barcelona, Spain and

Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.
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http://dx.doi.org/10.1093/carcin/bgu111DOI Listing
October 2014

An increase in MECP2 dosage impairs neural tube formation.

Neurobiol Dis 2014 Jul 19;67:49-56. Epub 2014 Mar 19.

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908L'Hospitalet, Barcelona, Catalonia, Spain; Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain. Electronic address:

Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.
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http://dx.doi.org/10.1016/j.nbd.2014.03.009DOI Listing
July 2014

Histone deacetylase 3 regulates cyclin A stability.

J Biol Chem 2013 Jul 11;288(29):21096-21104. Epub 2013 Jun 11.

From the Department of Cell Biology, Immunology and Neurosciences, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, 08036 Barcelona, Spain and. Electronic address:

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G2/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5.
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http://dx.doi.org/10.1074/jbc.M113.458323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774376PMC
July 2013

RNA polymerase II progression through H3K27me3-enriched gene bodies requires JMJD3 histone demethylase.

Mol Biol Cell 2013 Feb 14;24(3):351-60. Epub 2012 Dec 14.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Spain.

JMJD3 H3K27me3 demethylase plays an important role in the transcriptional response to different signaling pathways; however, the mechanism by which it facilitates transcription has been unclear. Here we show that JMJD3 regulates transcription of transforming growth factor β (TGFβ)-responsive genes by promoting RNA polymerase II (RNAPII) progression along the gene bodies. Using chromatin immunoprecipitation followed by sequencing experiments, we show that, upon TGFβ treatment, JMJD3 and elongating RNAPII colocalize extensively along the intragenic regions of TGFβ target genes. According to these data, genome-wide analysis shows that JMJD3-dependent TGFβ target genes are enriched in H3K27me3 before TGFβ signaling pathway activation. Further molecular analyses demonstrate that JMJD3 demethylates H3K27me3 along the gene bodies, paving the way for the RNAPII progression. Overall these findings uncover the mechanism by which JMJD3 facilitates transcriptional activation.
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http://dx.doi.org/10.1091/mbc.E12-07-0561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564542PMC
February 2013

The histone demethylase PHF8 is essential for cytoskeleton dynamics.

Nucleic Acids Res 2012 Oct 31;40(19):9429-40. Epub 2012 Jul 31.

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona, Spanish Research Council (CSIC), Barcelona Science Park, Barcelona 08028, Spain.

PHF8 is a histone demethylase associated with X-linked mental retardation. It has been described as a transcriptional co-activator involved in cell cycle progression, but its physiological role is still poorly understood. Here we show that PHF8 controls the expression of genes involved in cell adhesion and cytoskeleton organization such as RhoA, Rac1 and GSK3β. A lack of PHF8 not only results in a cell cycle delay but also in a disorganized actin cytoskeleton and impaired cell adhesion. Our data demonstrate that PHF8 directly regulates the expression of these genes by demethylating H4K20me1 at promoters. Moreover, c-Myc transcription factor cooperates with PHF8 to regulate the analysed promoters. Further analysis in neurons shows that depletion of PHF8 results in down-regulation of cytoskeleton genes and leads to a deficient neurite outgrowth. Overall, our results suggest that the mental retardation phenotype associated with loss of function of PHF8 could be due to abnormal neuronal connections as a result of alterations in cytoskeleton function.
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http://dx.doi.org/10.1093/nar/gks716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479184PMC
October 2012

Genome-wide analysis reveals that Smad3 and JMJD3 HDM co-activate the neural developmental program.

Development 2012 Aug;139(15):2681-91

Department of Molecular Genomics, Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFβ pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFβ-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFβ developmental program to proceed.
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http://dx.doi.org/10.1242/dev.078345DOI Listing
August 2012

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27 Kip1.

Nucleic Acids Res 2012 Aug 29;40(14):6520-33. Epub 2012 Apr 29.

Department of Cell Biology, Immunology and Neurosciences, Institut d'Investigacions Biomèdiques August-Pi i Sunyer (IDIBAPS), University of Barcelona, 08036-Barcelona, Spain.

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.
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http://dx.doi.org/10.1093/nar/gks343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413142PMC
August 2012

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

J Clin Invest 2012 May 16;122(5):1849-68. Epub 2012 Apr 16.

Department of Cell Biology, Barcelona Institute of Molecular Biology, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.
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http://dx.doi.org/10.1172/JCI59218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366719PMC
May 2012

H3K27me3 regulates BMP activity in developing spinal cord.

Development 2010 Sep 28;137(17):2915-25. Epub 2010 Jul 28.

Department of Genomic Regulation, Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Baldiri i Reixac 15-21, Parc Científic de Barcelona, Barcelona, Spain.

During spinal cord development, the combination of secreted signaling proteins and transcription factors provides information for each neural type differentiation. Studies using embryonic stem cells show that trimethylation of lysine 27 of histone H3 (H3K27me3) contributes to repression of many genes key for neural development. However, it remains unclear how H3K27me3-mediated mechanisms control neurogenesis in developing spinal cord. Here, we demonstrate that H3K27me3 controls dorsal interneuron generation by regulation of BMP activity. Our study indicates that expression of Noggin, a BMP extracellular inhibitor, is repressed by H3K27me3. Moreover, we show that Noggin expression is induced by BMP pathway signaling, generating a negative-feedback regulatory loop. In response to BMP pathway activation, JMJD3 histone demethylase interacts with the Smad1/Smad4 complex to demethylate and activate the Noggin promoter. Together, our data reveal how the BMP signaling pathway restricts its own activity in developing spinal cord by modulating H3K27me3 levels at the Noggin promoter.
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http://dx.doi.org/10.1242/dev.049395DOI Listing
September 2010

Characterization of structural variability sheds light on the specificity determinants of the interaction between effector domains and histone tails.

Epigenetics 2010 Feb 1;5(2):137-48. Epub 2010 Mar 1.

Institut de Medicina Preventiva i Personalitzada del Càncer, Badalona, Spain.

Structural characterization of the interaction between histone tails and effector modules (bromodomains, chromodomains, PHD fingers, etc.) is fundamental to understand the mechanistic aspects of epigenetic regulation of gene expression. In recent years many researchers have applied this approach to specific systems, thus providing a valuable but fragmentary view of the histone-effector interaction. In our work we use this information to characterize the structural features of the two main components of this interaction, histone peptides and the binding site of effector domains (focusing on those which target modified lysines), and increase our knowledge on its specificity determinants. Our results show that the binding sites of effectors are structurally variable, but some clear trends allow their classification in three main groups: flat-groove, narrow-groove and cavity-insertion. In addition, we found that even within these classes binding site variability is substantial. These results in context with the work from other researchers indicate that the there are at least two determinants of binding specificity in the binding site of effector modules. Finally, our analysis of the histone peptides sheds light on the structural transition experienced by histone tails upon effector binding, showing that it may vary depending on the local properties of the sequence stretch considered, thus allowing us to identify an additional specificity determinant for this interaction. Overall, the results of our analysis contribute to clarify the origins of specificity: different regions of the binding site and, in particular, differences in the disorder-order transitions experienced by different histone sequence stretches upon binding.
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http://dx.doi.org/10.4161/epi.5.2.11079DOI Listing
February 2010

The transcriptional co-activator PCAF regulates cdk2 activity.

Nucleic Acids Res 2009 Nov;37(21):7072-84

Department of Cell Biology, Immunology and Neurosciences, Faculty of Medicine, University of Barcelona, Spain.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G(2)/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G(2)/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.
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http://dx.doi.org/10.1093/nar/gkp777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790897PMC
November 2009

Autoacetylation regulates P/CAF nuclear localization.

J Biol Chem 2009 Jan 17;284(3):1343-52. Epub 2008 Nov 17.

Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Baldiri i Reixac 15-21, Parc Cientific de Barcelona, E-08028 Barcelona, Spain.

Acetylation is a posttranslational modification that alters the biological activities of proteins by affecting their association with other proteins or DNA, their catalytic activities, or their subcellular distribution. The acetyltransferase P/CAF is autoacetylated and acetylated by p300 in vivo. P/CAF autoacetylation is an intramolecular or intermolecular event. Intramolecular acetylation targets five lysines within the nuclear localization signal at the P/CAF C terminus. We analyzed how the subcellular distribution of P/CAF is regulated by intramolecular autoacetylation and found that a P/CAF mutant lacking histone acetyltransferase activity accumulated primarily in the cytoplasm. This cytoplasmic fraction of P/CAF is enriched for nonautoacetylated P/CAF. In addition, P/CAF deacetylation by HDAC3 and in a minor degree by HDAC1, HDAC2, or HDAC4 leads to cytoplasmic accumulation of P/CAF. Importantly, our data show that P/CAF accumulates in the cytoplasm during apoptosis. These results reveal the molecular mechanism of autoacetylation control of P/CAF nuclear translocation and suggest a novel pathway by which P/CAF activity is controlled in vivo.
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http://dx.doi.org/10.1074/jbc.M806075200DOI Listing
January 2009

The functional modulation of epigenetic regulators by alternative splicing.

BMC Genomics 2007 Jul 25;8:252. Epub 2007 Jul 25.

Instituto de Biología Molecular de Barcelona, CID, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

Background: Epigenetic regulators (histone acetyltransferases, methyltransferases, chromatin-remodelling enzymes, etc) play a fundamental role in the control of gene expression by modifying the local state of chromatin. However, due to their recent discovery, little is yet known about their own regulation. This paper addresses this point, focusing on alternative splicing regulation, a mechanism already known to play an important role in other protein families, e.g. transcription factors, membrane receptors, etc.

Results: To this end, we compiled the data available on the presence/absence of alternative splicing for a set of 160 different epigenetic regulators, taking advantage of the relatively large amount of unexplored data on alternative splicing available in public databases. We found that 49 % (70 % in human) of these genes express more than one transcript. We then studied their alternative splicing patterns, focusing on those changes affecting the enzyme's domain composition. In general, we found that these sequence changes correspond to different mechanisms, either repressing the enzyme's function (e.g. by creating dominant-negative inhibitors of the functional isoform) or creating isoforms with new functions.

Conclusion: We conclude that alternative splicing of epigenetic regulators can be an important tool for the function modulation of these enzymes. Considering that the latter control the transcriptional state of large sets of genes, we propose that epigenetic regulation of gene expression is itself strongly regulated by alternative splicing.
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http://dx.doi.org/10.1186/1471-2164-8-252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949830PMC
July 2007

Involvement of chromatin and histone deacetylation in SV40 T antigen transcription regulation.

Nucleic Acids Res 2007 6;35(6):1958-68. Epub 2007 Mar 6.

Instituto de Biología Molecular de Barcelona, CID, Consejo Superior de Investigaciones Científicas, Parc Cientific de Barcelona, Barcelona, Spain.

Simian Virus 40 (SV40) large T antigen (T Ag) is a multifunctional viral oncoprotein that regulates viral and cellular transcriptional activity. However, the mechanisms by which such regulation occurs remain unclear. Here we show that T antigen represses CBP-mediated transcriptional activity. This repression is concomitant with histone H3 deacetylation and is TSA sensitive. Moreover, our results demonstrate that T antigen interacts with HDAC1 in vitro in an Rb-independent manner. In addition, the overexpression of HDAC1 cooperates with T antigen to antagonize CBP transactivation function and correlates with chromatin deacetylation of the TK promoter. Finally, decreasing HDAC1 levels with small interfering RNA (siRNA) partially abolishes T antigen-induced repression. These findings highlight the importance of the histone acetylation/deacetylation balance in the cellular transformation mediated by oncoviral proteins.
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http://dx.doi.org/10.1093/nar/gkl1113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874590PMC
May 2007

The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway.

Biochem J 2006 Sep;398(2):215-24

Instituto de Biología Molecular de Barcelona, CID, Consejo Superior de Investigaciones Científicas, Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.
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http://dx.doi.org/10.1042/BJ20060052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550303PMC
September 2006

Role of histone modifications in marking and activating genes through mitosis.

J Biol Chem 2005 Dec 30;280(52):42592-600. Epub 2005 Sep 30.

Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.

The global inhibition of transcription at the mitotic phase of the cell cycle occurs together with the general displacement of transcription factors from the mitotic chromatin. Nevertheless, the DNase- and potassium permanganate-hypersensitive sites are maintained on potentially active promoters during mitosis, helping to mark active genes at this stage of the cell cycle. Our study focuses on the role of histone acetylation and H3 (Lys-4) methylation in the maintenance of the competency of these active genes during mitosis. To this end we have analyzed histone modifications across the promoters and coding regions of constitutively active, inducible, and inactive genes in mitotic arrested cells. Our results show that basal histone modifications are maintained during mitosis at promoters and coding regions of the active and inducible RNA polymerase II-transcribed genes. In addition we have demonstrated that, together with H3 acetylation and H3 (Lys-4) methylation, H4 (Lys-12) acetylation at the coding regions contributes to the formation of a stable mark on active genes at this stage of the cell cycle. Finally, analysis of cyclin B1 gene activation during mitosis revealed that the former occurs with a strong increase of H3 (Lys-4) trimethylation but not H3 or H4 acetylation, suggesting that histone methyltransferases are active during this stage. These data demonstrate a critical role of histone acetylation and H3 (Lys-4) methylation during mitosis in marking and activating genes during the mitotic stage of the cell cycle.
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http://dx.doi.org/10.1074/jbc.M507407200DOI Listing
December 2005

Do protein motifs read the histone code?

Bioessays 2005 Feb;27(2):164-75

Institut Català per la Recerca i Estudis Avançats, Barcelona, Spain.

The existence of different patterns of chemical modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) of the histone tails led, some years ago, to the histone code hypothesis. According to this hypothesis, these modifications would provide binding sites for proteins that can change the chromatin state to either active or repressed. Interestingly, some protein domains present in histone-modifying enzymes are known to interact with these covalent marks in the histone tails. This was first shown for the bromodomain, which was found to interact selectively with acetylated lysines at the histone tails. More recently, it has been described that the chromodomain can be targeted to methylation marks in histone N-terminal domains. Finally, the interaction between the SANT domain and histones is also well documented. Overall, experimental evidence suggests that these domains could be involved in the recruitment of histone-modifying enzymes to discrete chromosomal locations, and/or in the regulation their enzymatic activity. Within this context, we review the distribution of bromodomains, chromodomains and SANT domains among chromatin-modifying enzymes and discuss how they can contribute to the translation of the histone code.
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http://dx.doi.org/10.1002/bies.20176DOI Listing
February 2005

Mechanisms of P/CAF auto-acetylation.

Nucleic Acids Res 2003 Aug;31(15):4285-92

Instituto de Biología Molecular de Barcelona, CID, Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain.

P/CAF is a histone acetyltransferase enzyme which was originally identified as a CBP/p300-binding protein. In this manuscript we report that human P/CAF is acetylated in vivo. We find that P/CAF is acetylated by itself and by p300 but not by CBP. P/CAF acetylation can be an intra- or intermolecular event. The intermolecular acetylation requires the N-terminal domain of P/CAF. The intramolecular acetylation targets five lysines (416-442) at the P/CAF C-terminus, which are in the nuclear localisation signal (NLS). Finally, we show that acetylation of P/CAF leads to an increment of its histone acetyltransferase (HAT) activity. These findings identify a new post-translation modification on P/CAF which may regulate its function.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC169960PMC
http://dx.doi.org/10.1093/nar/gkg655DOI Listing
August 2003

The SV40 T antigen modulates CBP histone acetyltransferase activity.

Nucleic Acids Res 2003 Jun;31(12):3114-22

Instituto de Biología Molecular de Barcelona, CID, Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, E-08034 Barcelona, Spain.

Histone acetyltransferases (HATs) play a key role in transcription control, cell proliferation and differentiation by modulating chromatin structure; however, little is known about their own regulation. Here we show that expression of the viral oncoprotein SV40 T antigen increases histone acetylation and global cellular HAT activities. In addition, it enhances CREB-binding protein HAT activity and modulates its transcriptional activity. Finally, we show that inhibition of cellular histone deacetylases by trichostatin A increases the SV40 infectivity rate. These findings highlight the importance of histone acetylation in the regulation of the cell cycle by oncoviral proteins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC162251PMC
http://dx.doi.org/10.1093/nar/gkg418DOI Listing
June 2003