Liette Laflamme

Histone H2A.Z is essential for estrogen receptor signaling

Incorporation of H2A.Z into the chromatin of inactive promoters has been shown to poise genes for their expression. Here we provide strong evidence that H2A.Z is incorporated into the promoter regions of estrogen receptor (ERalpha) target genes only upon gene induction, and that, in a cyclic pattern. Moreover, members of the human H2A.Z-depositing complex, p400, also follow the same gene recruitment kinetics as H2A.Z. Importantly, cellular depletion of H2A.Z or p400 leads to a severe defect in estrogen signaling, including loss of estrogen-specific cell proliferation. We find that incorporation of H2A.Z within TFF1 promoter chromatin allows nucleosomes to adopt preferential positions along the DNA translational axis. Finally, we provide evidence that H2A.Z is essential to allow estrogen-responsive enhancer function. Taken together, our results provide strong mechanistic insight into how H2A.Z regulates ERalpha-mediated gene expression and provide a novel link between H2A.Z-p400 and ERalpha-dependent gene regulation and enhancer function.

The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition.

A role for variant histone H2A.Z in gene expression is now well established but little is known about the mechanisms by which it operates. Using a combination of ChIP–chip, knockdown and expression profiling experiments, we show that upon gene induction, human H2A.Z associates with gene promoters and helps in recruiting the transcriptional machinery. Surprisingly, we also found that H2A.Z is randomly incorporated in the genome at low levels and that active transcription antagonizes this incorporation in transcribed regions. After cessation of transcription, random H2A.Z quickly reappears on genes, demonstrating that this incorporation utilizes an active mechanism. Within facultative heterochromatin, we observe a hyper accumulation of the variant histone, which might be due to the lack of transcription in these regions. These results show how chromatin structure and transcription can antagonize each other, therefore shaping chromatin and controlling gene expression.

Reconciling the positive and negative roles of histone H2A.Z in gene transcription

The incorporation of variant histone H2A.Z within chromatin is important for proper gene expression and genome stability. H2A.Z is inserted at discrete loci by the Swr1 or Swr1-like remodeling complexes, although very little is known about the nature of the targeting mechanism involved. Replacement of canonical histone H2A for H2A.Z has been shown to modify nucleosome dynamics, although discrepancies still exist in the literature regarding the mechanisms. Recent experiments have shown that H2A.Z can allow nucleosomes to adopt stable translational positions as compared to H2A, which could influence the accessibility to DNA regulatory proteins. This review provides a brief overview of H2A.Z biology and presents hypotheses that could reconcile contradictory reports that are found in the literature regarding the influence of H2A.Z on nucleosome stability.

Nitric oxide, a new second messenger involved in the action of angiotensin II on neuronal differentiation of NG108-15 cells.

Nitric Oxide (NO) is a gas that diffuses freely through membranes of target cells to activate cGMP formation. NO is synthesised from arginine, by a family of Nitric Oxide Synthase (NOS). In the brain, NO influences synaptic plasticity, apoptosis and development. It has been recently shown that angiotensin II (Ang II) could mediate NO production by its two types of receptors, AT1 and AT2. Since we have shown that Ang II, via the AT2 receptor could induce neurite outgrowth and morphological differentiation of NG108-15 cells, the aim of the study was to investigate if NO could be one of the second messengers involved in the Ang II effect. Using the Griess colorimetric assay, we found that Ang II, by its AT2 receptor, induced nitrite formation from NO. This effect was abolished by the N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. We also found that treatment of the cells with S-nitroso-N-acetylpenicillamine (SNAP), an exogenous source of NO, induced the same morphological differentiation. These results demonstrate that the morphological differentiation induced by the AT2 receptor is partly due to an increase in NO production.

Posttranslational Regulation of Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor σL and Roles in Virulence and in Global Regulation of Gene Expression

ABSTRACT In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a sigL-rslA mutant strain was significantly attenuated in a mouse model of infection.

Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

Low levels of 3,3′-diindolylmethane activate estrogen receptor α and induce proliferation of breast cancer cells in the absence of estradiol

Background3,3′-diindolylmethane (DIM) is an acid-catalyzed dimer of idole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables that include broccoli, Brussels sprouts and cabbage. DIM is an aryl hydrocarbon receptor (AhR) ligand and a potential anticancer agent, namely for the treatment of breast cancer. It is also advertised as a compound that regulates sex hormone homeostasis.MethodsHere we make use of RNA expression assays coupled to Chromatin Immunoprecipitation (ChIP) in breast cancer cell lines to study the effect of DIM on estrogen signaling. We further make use of growth assays, as well as fluorescence-activated cell sorting (FACS) assays, to monitor cell growth.ResultsIn this study, we report that ‘physiologically obtainable’ concentrations of DIM (10 μM) activate the estrogen receptor α (ERα) signaling pathway in the human breast cancer cell lines MCF7 and T47D, in a 17β-estradiol (E2)-independent manner. Accordingly, we observe induction of ERα target genes such as GREB1 and TFF1, and an increase in cellular proliferation after treatment with 10 μM DIM in the absence of E2. By using an ERα specific inhibitor (ICI 182 780), we confirm that the transcriptional and proliferative effects of DIM treatment are mediated by ERα. We further show that the protein kinase A signaling pathway participates in DIM-mediated activation of ERα. In contrast, higher concentrations of DIM (e.g. 50 μM) have an opposite and expected effect on cells, which is to inhibit proliferation.ConclusionsWe document an unexpected effect of DIM on cell proliferation, which is to stimulate growth by inducing the ERα signaling pathway. Importantly, this proliferative effect of DIM happens with potentially physiological concentrations that can be provided by the diet or by taking caplet supplements.

The renin–angiotensin system in hybrid NG108-15 cells: Renin gene is from mouse neuroblastoma, angiotensinogen and angiotensin-converting enzyme genes are of rat glioma origin

Angiotensin II (Ang II) increases the level of tyrosine phosphorylation of several proteins in nondifferentiated NG108-15 cells, a hybrid derived from the fusion of mouse neuroblastoma and rat glioma cells. Conversely, incubation of NG108-15 cells with an angiotensin-converting enzyme (ACE) inhibitor decreased the basal level of tyrosine phosphorylation of proteins, suggesting that locally secreted Ang II may act as an autocrine regulator. By RT-PCR, we found that nondifferentiated NG108-15 cells contained the mRNA transcript of the rat angiotensinogen, mouse renin and rat ACE genes, thus confirming that NG108-15 cells contain all the elements of a local renin-angiotensin system.

A role for p21ras in the angiotensin II AT2 receptor transduction pathway

We have previously shown that the activation of the AT2 receptor of Ang II induced neurite outgrowth in NG108-15 cells. We also found that stimulation of NG108-15 cells with Ang II induced a rapid decrease in GTP-bound p21ras. In order to investigate the possible role of p21ras in Ang II-induced neuronal differentiation, we have established NG108-15 sublines which inducibly express a dominant inhibitory form of p21ras (p21N17Ras). We observed that IPTG-induced expression of p21N17Ras in these NG108-15 sublines induced the same morphological changes as does Ang II in control untransfected cells. Immunofluorescence labeling of beta-tubulin showed that expression of p21N17Ras induced neurite outgrowth and elongation. These observations were supported by Western blot analysis of the level of polymerized tubulin. These results strongly support the hypothesis that AT2 receptor-induced neuronal differentiation in NG108-15 cells is mediated by the inhibition of p21ras.

An Assay for Measuring Histone Variant Exchange within Nucleosomes In Vitro.

The incorporation of histone variants into specific chromatin regions is a mechanism by which cells can regulate many important biological processes. One such example is H2A.Z, a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a preassembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, have been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone H2A.Z exchange activity of recombinant human p400 on immobilized mononucleosomes in vitro. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.