The chromatin remodeller CHD8 is required for E2F-dependent transcription activation of S-phase genes

The precise regulation of S-phase-specific genes is critical for cell proliferation. How the repressive chromatin configuration mediated by the retinoblastoma protein and repressor E2F factors changes at the G1/S transition to allow transcription activation is unclear. Here we show ChIP-on-chip studies that reveal that the chromatin remodeller CHD8 binds ∼2000 transcriptionally active promoters. The spectrum of CHD8 target genes was enriched in E2F-dependent genes. We found that CHD8 binds E2F-dependent promoters at the G1/S transition but not in quiescent cells. Consistently, CHD8 was required for G1/S-specific expression of these genes and for cell cycle re-entry on serum stimulation of quiescent cells. We also show that CHD8 interacts with E2F1 and, importantly, loading of E2F1 and E2F3, but not E2F4, onto S-specific promoters, requires CHD8. However, CHD8 recruiting is independent of these factors. Recruiting of MLL histone methyltransferase complexes to S-specific promoters was also severely impaired in the absence of CHD8. Furthermore, depletion of CHD8 abolished E2F1 overexpression-dependent S-phase stimulation of serum-starved cells, highlighting the essential role of CHD8 in E2F-dependent transcription activation

Genome-wide study of chromatin remodeling factor CHD8 role in transcription

1 página. Cold Spring Harbor Laboratory (CSHL) Meeting on Mechanisms of Eukaryotic Trasncription 2011. August 30 - september 3, 2011.CHD8 (Chromodomain-Helicase-DNA binding protein 8) is a member of the chromodomain helicase DNA-binding (CHD) subfamily of enzymes, which also belongs to the SNF2 family of ATP-dependent chromatin remodelers.Peer reviewe

To cross or not to cross the nucleosome, that is the elongation question...

The natural template for transcription is chromatin. In vitro and in vivo experiments demonstrate that positioned nucleosomes are obstacles for RNA polymerase II (RNAPII) elongation, raising the question of how RNAPII crosses a nucleosome. In fact, transcription elongation is accompanied by chromatin remodeling in the body of the genes. Numerous results evidence that chromatin remodelers such as histone chaperones and histone acetyl transferases contribute to transcription elongation. Recent data indicate that the SWI/SNF complex, an ATP-dependent chromatin remodeling machine, also helps RNAPII to overcome a nucleosomal barrier during elongation. Finally, the idea that remodeling of positioned nucleosomes in the coding regions would alter RNAPII elongation rate and therefore, would regulate gene expression at different levels is discussed. © 2011 Landes Bioscience.This work was supported by Ministerio de Educación y Ciencia (BFU2008-00238, CSD2006-00049) and by Junta de Andalucía (P09-CVI-4844).Peer Reviewe

BRG1 helps RNA polymerase II to overcome a nucleosomal barrier during elongation, in vivo

7 páginas, 5 figuras.RNA polymerase II (RNAPII) transcribes genes in a chromatin context. We have designed a system to investigate the role of chromatin remodelling during elongation in vivo, which involves inserting a strong nucleosome-positioning sequence between a promoter and a reporter gene. Our data indicate that a nucleosome positioned in the body of a transcription unit impairs RNAPII progression, provokes RNAPII accumulation upstream to the positioned nucleosome and reduces transcription. By using this system, we show that BRG1, the enzymatic motor of the SWI–SNF chromatin-remodelling complex, is recruited to the positioned nucleosome in a transcription elongation-dependent manner and facilitates traversal of the nucleosome by RNAPII.This study was supported by Ministerio de Educación y Ciencia (BFU2008-00238, CSD2006-00049) and by Junta de Andalucía (P06-CVI-01400).Peer reviewe

The chromatin remodeller CHD8 is required for E2F-dependent transcription activation of S-phase genes

The precise regulation of S-phase-specific genes is critical for cell proliferation. How the repressive chromatin configuration mediated by the retinoblastoma protein and repressor E2F factors changes at the G1/S transition to allow transcription activation is unclear. Here we show ChIP-on-chip studies that reveal that the chromatin remodeller CHD8 binds ∼2000 transcriptionally active promoters. The spectrum of CHD8 target genes was enriched in E2F-dependent genes. We found that CHD8 binds E2F-dependent promoters at the G1/S transition but not in quiescent cells. Consistently, CHD8 was required for G1/S-specific expression of these genes and for cell cycle re-entry on serum stimulation of quiescent cells. We also show that CHD8 interacts with E2F1 and, importantly, loading of E2F1 and E2F3, but not E2F4, onto S-specific promoters, requires CHD8. However, CHD8 recruiting is independent of these factors. Recruiting of MLL histone methyltransferase complexes to S-specific promoters was also severely impaired in the absence of CHD8. Furthermore, depletion of CHD8 abolished E2F1 overexpression-dependent S-phase stimulation of serum-starved cells, highlighting the essential role of CHD8 in E2F-dependent transcription activationSpanish Ministerio de Ciencia e Innovacion [BFU2011- 23442, CSD2006-00049 and an FPU fellowship to E.V.C.]; Junta de Andalucía [P06-CVI-4844]; and Fundación Ramón Areces. JAE grant from C.S.I.C (to A.S.R.). Funding for open access charge: Spanish Ministerio de Ciencia e Innovacion [BFU2011-23442].Peer Reviewe

Mutational analysis of progesterone receptor functional domains in stable cell lines delineates sets of genes regulated by different mechanisms

Steroid hormone receptors act directly in the nucleus on the chromatin organization and transcriptional activity of several promoters. Furthermore, they have an indirect effect on cytoplasmic signal transduction pathways, including MAPK, impacting ultimately on gene expression. We are interested in distinguishing between the two modes of action of progesterone receptor (PR) on the control of gene expression and cell proliferation. For this, we have stably expressed, in PR-negative breast cancer cells, tagged forms of the PR isoform B mutated at regions involved either in DNA binding (DNA-binding domain) or in its ability to interact with the estrogen receptor and to activate the c-Src/MAPK/Erk/Msk cascade (estrogen receptor-interacting domain). Both mutants impair PR-mediated activation of a well-understood model promoter in response to progestin, as well as hormone-induced cell proliferation. Additional mutants affecting transactivation activity of PR (activation function 2) or a zinc-finger implicated in dimerization (D-box) have also been tested. Microarrays and gene expression experiments on these cell lines define the subsets of hormone-responsive genes regulated by different modes of action of PR isoform B, as well as genes in which the nuclear and nongenomic pathways cooperate. Correlation between CCND1 expression in the different cell lines and their ability to support cell proliferation confirms CCND1 as a key controller gene. Copyright © 2009 by The Endocrine Society.This work was supported by grants from the Catalan Department for Universities, Research and the Information Society, and the Spanish Ministry of Science and Technology and Fondo Europeo de desarrollo regional (SAF2002-03320, BFU2008-00359). A.J. was recipient of a ‘Ramón y Cajal’ appointment from the Spanish Ministry of Science and Technology. I.Q. was recipient of a Formación personal universitario predoctoral fellowship from the Spanish Ministry of Education. L.M-A. was recipient of a predoctoral fellowship funded by Fundación para la investigación y prevención del SIDA en EspañaPeer Reviewe

Progesterone Induction of the 11β-Hydroxysteroid Dehydrogenase Type 2 Promoter in Breast Cancer Cells Involves Coordinated Recruitment of STAT5A and Progesterone Receptor to a Distal Enhancer and Polymerase Tracking▿ †

Steroid hormone receptors regulate gene expression, interacting with target DNA sequences but also activating cytoplasmic signaling pathways. Using the human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) gene as a model, we have investigated the contributions of both effects on a human progesterone-responsive promoter in breast cancer cells. Chromatin immunoprecipitation has identified two different mechanisms of hormone-induced progesterone receptor (PR) recruitment to the 11β-HSD2 promoter: (i) direct PR binding to DNA at the proximal promoter, abrogated when PR contains a mutated DNA binding domain (DBD), and (ii) STAT5A (signal transducer and activator of transcription 5A)-mediated recruitment of PR to an upstream distal region, impaired by AG490, a JAK/STAT pathway inhibitor. The JAK/STAT inhibitor, as well as expression of dominant-negative STAT5A, impairs hormone induction of 11β-HSD2. On the other hand, the DBD-mutated PR fully supports 11β-HSD2 expression. These results, along with data from a deletion analysis, indicate that the distal region is crucial for hormone regulation of 11β-HSD2. We show active RNA polymerase II tracking from the distal region upon PR and STAT5A binding, concomitant with synthesis of noncoding, hormone-dependent RNAs, suggesting that this region works as a hormone-dependent transcriptional enhancer. In conclusion, coordination of PR transcriptional effects and cytoplasmic signaling activation, in particular the JAK/STAT pathway, are critical in regulating progestin-induced endogenous 11β-HSD2 gene expression in breast cancer cells. This is not unique to this promoter, as AG490 also alters the expression of other progesterone-regulated genes

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

15 pages, 9 figures.Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.This work was supported by grants from Ministerio de Ciencia y Tecnologia (PM99-0113 and SAF2003-02188), Comunidad Autónoma de Madrid (08.1/0047/98), and Fondo de Investigaciones Sanitarias (01/1316, 03C03/10). J.J.A. was supported by a fellowship from Fundación Científica de la Asociación Española contra el Cancer; L.G. was supported by a fellowship from Fundación Carolina; and J.M.G.M. was supported by a fellowship from Fondo de Investigaciones Sanitarias. J.M.P. is member of the Spanish Breast Cancer Research Group, which in part supported this research.Peer reviewe