The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

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.

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

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.

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.

Convergence on chromatin of non-genomic and genomic pathways of hormone signaling.

Gene regulation by steroid hormones involves genomic and non-genomic signaling pathways and the relationship between these two pathways is unknown. Genomic actions are often mediated by binding of the ligand-activated hormone receptors to hormone responsive elements (HREs) followed by recruitment of co-regulators, remodeling of chromatin and formation of the transcription initiation complex. The non-genomic effects of steroid hormones involve the rapid and transient activation of several kinase cascades often mediated by a subpopulation of "nuclear" receptors located in the cytoplasmic side of the cell membrane. The progesterone effect on breast cancer cell proliferation involves activation of the Src/Ras/Erk cascade mediated by a specific interaction between two domains of the N-terminal half of PR and the ligand-binding domain of ERalpha. Unexpectedly, selective inhibition of Erk, or its target kinase Msk1, interferes with chromatin remodeling and blocks MMTV transcriptional activation. A complex of activated PR, Erk and Msk1 is recruited to promoter already 5 min after hormone treatment and phosphorylates histone H3 at serine 10, leading to displacement of HP1gamma, as a requisite for recruitment of Src1, chromatin remodeling complexes (hSnf2h and Brg1) and RNA polymerase II. Thus, activation of signaling cascades in the cytoplasm is essential for chromatin remodeling and transcriptional activation of a subset of steroid hormone target genes.

Specific immunotherapy for rhinitis and asthma with a subcutaneous hypoallergenic high-dose house dust mite extract: results of a 9-month therapy.

AIM: Effectiveness of a 9-month specific immunotherapy with a subcutaneous hypoallergenic high-dose house dust mite extract to reduce allergic symptoms as perceived by patients and physicians was assessed. PATIENTS & METHODS: An observational, retrospective, multicenter study was carried out in patients diagnosed with asthma and/or rhinitis caused by house dust mites having started specific immunotherapy with Acaroid(®). Primary end point was perceived effectiveness. RESULTS: A total of 409 patients were included. According to physician-completed visual analogue scale, a 58.1% clinical improvement was observed. Patient-completed visual analogue scale showed a 69.8% clinical improvement. The need for unscheduled/emergency healthcare, as an indication of poor quality of life, showed a significant reduction. CONCLUSION: Our results confirm in a real-world setting the findings from randomized clinical trials of high-dose house dust mites allergoid immunotherapy with a subcutaneous hypoallergenic high-dose house dust mite extract.

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

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.

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.

Progesterone induction of the 11beta-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 11beta-hydroxysteroid dehydrogenase type 2 (11beta-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 11beta-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 11beta-HSD2. On the other hand, the DBD-mutated PR fully supports 11beta-HSD2 expression. These results, along with data from a deletion analysis, indicate that the distal region is crucial for hormone regulation of 11beta-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 11beta-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.

Induction of progesterone target genes requires activation of Erk and Msk kinases and phosphorylation of histone H3.

How genes are regulated in the context of chromatin is a central question of biology. Steroid hormones control gene expression via interaction of their receptors with target sequences on DNA but can also activate cytoplasmic signaling cascades. Here we report that rapid Erk activation by progestins participates in induction of target genes by preparing the chromatin for transcription. Five minutes after hormone treatment, Erk activation leads to phosphorylation of the progesterone receptor (PR), activation of Msk1, and recruitment of a complex of the three proteins to a nucleosome on the MMTV promoter. Msk1 phosphorylates histone H3, leading to displacement of HP1gamma and recruitment of Brg1 and RNA polymerase II. Cell-free experiments show a direct interaction between PR, Erk, and Msk1 and support the importance of H3 phosphorylation for nucleosome remodeling. Inhibition of Msk1 activation blocks recruitment of the kinase complex, H3 phosphorylation, and HP1gamma displacement, thus precluding remodeling and induction of the promoter.