Steroid free medium induces an E2 reversible G1 block in ERα-positive MCF-7 cells.

<p>1.5×10⁶ ERα-positive MCF-7 cells were cultivated in red medium (R) for 24 h, then medium was removed and cells were cultivated in red or white medium A) FACS analysis of MCF-7 cells after 4 days in red medium only (R), 3 days in red medium complemented with E2 10⁻⁸ M for 24 h, in white medium only for 4 days (W) or 3 days in white medium complemented with E2 10⁻⁸ M for 24 h (W+E2). For growth curves, 1×10⁶ ERα-positive MCF-7 cells were cultivated in red medium (R) for 24 h, then medium was removed and cells were cultivated in red or white medium. Cells were counted by trypan blue exclusion at different time points. B) <i>ESR1, PGR</i> and <i>CCND1</i> gene expression was analyzed by RT-qPCR.</p

OH-TAM and ICI induced G1 cell cycle block and decreased ERα target gene expression.

<p>2×10⁶ MCF-7 cells were cultivated in red medium for 24 h. OH-TAM or ICI were added to the medium at a final concentration of 1 µM. After 24 h cell cycle was analyzed by FACS and expression of ERα target genes by RT-qPCR.</p

Effect of OH-TAM and ICI on cell proliferation is enhanced in S-phase.

<p>3×10⁵ MCF-7 cells were split in 60 cm dishes and cultivated in red medium for 12 h. A) Cells were treated by lovastatin (20 µM, 32 h), thymidine (3 mM, 24 h) or nocodazol (50 ng/ml, 24 h) until T0. Cell proliferation was monitored for untreated (NT) or for OH-TAM (1 µM) or ICI (1 µM) treated cells. Cells were counted by trypan blue exclusion in triplicate at different time points. Number of cells at T0 was set to 1 and the doubling time was calculated. B-C-D). Cells were treated by lovastatin 20 µM for 32 h (B), by thymidine 3 mM, 24 h (C) or nocodazol 50 ng/ml, 24 h (D) until T0. Then, OH-TAM 1 µM or ICI 1 µM added to the medium and cells were counted by trypan blue exclusion in triplicate at different time points.</p

ERα target gene expression is cell cycle regulated in MCF-7 cells.

<p>A) FACS analysis after propidium iodide staining on asynchronous untreated cells (1), cells blocked in G1 by lovastatin treatment 20 µM for 32 h (2), in S phase by thymidine treatment 3 mM for 24 h (3) or in G2/M by nocodazol 50 ng/ml for 24 h (4). B) Real-time PCR analysis of <i>ESR1</i>, <i>PGR</i>, <i>CCND1</i> and <i>H2AFZ</i> gene expression in G1 (lovastatin), S (thymidine) and G2/M (nocodazol) cell cycle phases. 2×10⁶ of MCF-7 cells were seeded in 10 cm dishes and after 24 h submitted to specific cell cycle arrest drugs as in (A). Total RNA was extracted and reverse transcribed. The amount of analyzed genes cDNA was measured RT-qPCR divided by the amount of RLP0 cDNA. (n = 3) one representative experiment is shown. C) Real-time PCR analysis of <i>ESR1, PGR, CCND1</i> and <i>H2AFZ</i> gene expression. For lovastatin treatment cells were treated as in (B). For nocodazol/check-off treatment, 12×10⁶ cells were splited into two 140 cm dishes. After 24 h of nocodazol treatment (25 ng/ml), G2/M arrested cells are harvested by check-off.and seeded in a clean dish. After 7 hours in complete medium total RNA was extracted and reverse transcribed (n = 2).</p

Effect of OH-TAM and ICI on gene expression is cell cycle dependent.

<p>2×10⁶ cells were cultivated in red medium for 24 h. Cells were blocked in G1 by lovastatin 20 µM, 32 h (1), in S by thymidine 3 mM 24 h (2) and in G2/M by nocodazol 50 ng/ml, 24 h (3). Cell cycle arrested cells were treated by Tamoxifen (OH-TAM 1 µM, 24 h) or ICI 182.780 (ICI 1 µM, 24 h). (n = 2) one representative experiment is shown. Relative mRNA expression in G1, S or G2/M phases of ERα target with or without anti-estrogen treatment was analyzed by RT-qPCR.</p

TIP48/Reptin and H2A.Z Requirement for Initiating Chromatin Remodeling in Estrogen-Activated Transcription

<div><p>Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. The role of H2A.Z at these sites remains poorly characterized. Our study investigates changes in the chromatin environment at the <i>Cyclin D1</i> gene (<i>CCND1</i>) during transcriptional initiation in response to estradiol in estrogen receptor positive mammary tumour cells. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of <i>CCND1</i> when the gene is poorly transcribed. Stimulation of <i>CCND1</i> expression required release of H2A.Z concomitantly from both these DNA elements. The AAA+ family members TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin environment at <i>CCND1</i> as a prerequisite for binding of the estrogen receptor (ERα) in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z, which triggers a dissociation of the <i>CCND1</i> 3′ enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables the estrogen receptor to bind to the <i>CCND1</i> promoter. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Dynamic release of gene loops could be a rapid means to remodel chromatin and to stimulate transcription in response to hormones.</p></div

p400 but not Tip60 functions in <i>p21</i> expression in the absence of p53.

<p>a) Tip60 and <i>p21</i> mRNA expression. b) H2A.Z, Acetyl-H2A.Z and RNA pol II binding to the <i>p21</i> TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the <i>p21</i> TSS (fragment #4) in cells treated with TSA. d) <i>p21</i> mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e) H2A.Z, Acetyl-H2A.Z and p300 enrichment at <i>p21</i> TSS (fragment #4) in MDA-MB231 transfected with si p400.</p

TIP48 regulates estrogen-activated transcription via H2A.Z release.

<p>A, B) MCF-7 cells were cultivated 3 days in steroid free medium, transfected with, scramble (ctrl) or TIP48 siRNA for 72 h and induced by E2 10⁻⁷ M for 30 min. Association of H2A.Z (A) and histone H3 (B) with TSS and enh2 sites was analyzed by ChIP and shown as % input (n = 2). C) MCF-7 cells were cultivated 3 days in steroid free medium and transfected at day 0 with a Scramble (Ctrl), a H2A.Z siRNA or a TIP48 siRNA for 72 h and then induced by E2 10⁻⁷ M for 6 h. <i>CCND1</i> mRNA levels were determined by qRT-PCR. Control (Ctrl) without E2 treatment was set to 1. The mean and SD from three independent experiments are shown. (*) indicates a p value<0.05.</p

Release of <i>CCND1</i> gene looping requires TIP48.

<p>A) Schematic representation of the <i>CCND1</i> locus on h.s. chromosome 11 (adapted from the UCSS genome browser <a href="http://genome.ucsc.edu/cgi-bin/hgGateway" target="_blank">http://genome.ucsc.edu/cgi-bin/hgGateway</a>). DNAseI hypersensitive sites <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003387#pgen.1003387-Eeckhoute1" target="_blank">[7]</a>, Csp6I restriction sites and fragments assayed by 3C are represented. B) Quantitative PCR amplification of ligation events in a 3C assay in MCF-7 cells treated or not with E2 10⁻⁷ M for 45 min and transfected 72 h before with scramble (ctrl) or TIP48 siRNA. Crosslinked DNA was digested with the Csp6I restriction enzyme and ligated. qRT-PCR was performed with primers designed for the 4 possible ligation events (only one is shown).</p

Activation of <em>p21</em> by HDAC Inhibitors Requires Acetylation of H2A.Z

<div><p>Differential positioning of the histone variant H2A.Z in a p53 dependent manner was shown to regulate <em>p21</em> transcription. Whether H2A.Z is involved in <em>p21</em> activity in the absence of p53 is not known. The <em>p21</em> gene is repressed in estrogen receptor (ER) negative cell lines that are p53−/− and hormone independent for their growth. Here we demonstrate that class I and II pan Histone deacetylase inhibitors (HDACi) induce <em>p21</em> transcription and reduce cell proliferation of MDA-MB231, an ERα-negative mammary tumor cell line, in a H2A.Z dependent manner. H2A.Z is associated with the transcription start site (TSS) of the repressed <em>p21</em> gene. Depleting H2A.Z did not lead to transcription of <em>p21</em> but annihilated the stimulating effect of HDACi on this gene. Acetylation of H2A.Z but not of H3K9 at the <em>p21</em> promoter correlated with <em>p21</em> activation. We further show that HDACi treatment reduced the presence of the p400 chromatin remodeler at the <em>p21</em> TSS. We propose a model in which association of p400 negatively affects <em>p21</em> transcription by interfering with acetylation of H2A.Z.</p> </div