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.

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

BACKGROUND: 3,3'-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. METHODS: Here 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. RESULTS: In 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 17ß-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. CONCLUSIONS: We 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.

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.

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.

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.

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.

p21 transcription is regulated by differential localization of histone H2A.Z.

In yeast cells, H2A.Z regulates transcription and is globally associated within a few nucleosomes of the initiator regions of numerous promoters. H2A.Z is deposited at these loci by an ATP-dependent complex, Swr1.com. Here we show that H2A.Z suppresses the p53 --> p21 transcription and senescence responses. Upon DNA damage, H2A.Z is first evicted from the p21 promoter, followed by the recruitment of the Tip60 histone acetyltransferase to activate p21 transcription. p400, a human Swr1 homolog, is required for the localization of H2A.Z, and largely colocalizes with H2A.Z at multiple promoters investigated. Notably, the presence of sequence-specific transcription factors, such as p53 and Myc, provides positioning cues that direct the location of H2A.Z-containing nucleosomes within these promoters. Collectively, this study strongly suggests that certain sequence-specific transcription factors regulate transcription, in part, by preferentially positioning histone variant H2A.Z within chromatin. This H2A.Z-centered process is part of an epigenetic process for modulating gene expression.

Identification and characterization of RanBPM, a novel coactivator of thyroid hormone receptors.

Thyroid hormone receptors (TRs) are transcription factor members of the nuclear receptor superfamily. The transcriptional activity of TRs is controlled by thyroid hormones and cell-specific coregulators. Using the yeast two-hybrid system, we identified RanBPM as a new protein partner for TRs. RanBPM was initially discovered as an interacting partner for Ran, and was also shown to be a protein partner and coactivator of the androgen receptor. The novel interaction between RanBPM and TR isoforms was addressed by glutathione-S-transferase (GST) pull-down assays and co-immunoprecipitation in intact mammalian cells, where RanBPM was shown to bind TRs in a ligand-independent fashion. We also studied the regions implicated in the interaction with deletion mutants: the principal interacting region of RanBPM is comprised within its carboxyl-terminal end and the TR DNA-binding domain is sufficient to mediate the interaction. To investigate the potential role of RanBPM in thyroid hormone action, transient transfections with luciferase reporter genes were performed in CV-1 cells. We found that the over-expression of RanBPM increases the activation of TRETK- and DR+4-positive thyroid hormone response elements. Interestingly, over-expression of the truncated protein RanBPM55, which lacks the N-terminal polyglutaminated region but binds TRs, decreased the fold activation by almost 80%. Furthermore, we performed competition assays using transient transfection of RanBPM and increasing amounts of RanBPM55. This revealed that the stimulating effect on TR transactivation by the full-length protein is inhibited in a dose-dependent fashion by RanBPM55. This suggests that although the polyglutaminated region of RanBPM is not required for the binding to TRs, it is required for the stimulation of TR transactivation. Taken together, our results provide evidence that RanBPM is a potent novel coactivator for thyroid hormone receptors.

Posttranslational regulation of Mycobacterium tuberculosis extracytoplasmic-function sigma factor sigma L and roles in virulence and in global regulation of gene expression.

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.