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.

Production and Purification of Antibodies Against Histone Modifications.

Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

A Method for Large-Scale Screening of Random Sequence Libraries to Determine the Function of Unstructured Regions from Essential Proteins.

In this chapter we present a method allowing the screening of random sequences to discover essential aspects of unstructured protein regions in yeast. The approach can be applied to any protein with unstructured peptide sequences for which functions are difficult to decipher, for example the N-terminal tails of histones. The protocol first describes the building and preparation of a large library of random peptides in fusion with a protein of interest. Recent technical advances in oligonucleotide synthesis allow the construction of long random sequences up to 35 residues long. The protocol details the screening of the library in yeast for sequences that can functionally replace an unstructured domain in an essential protein in vivo. Our method typically identifies sequences that, while being totally different from the wild type, retain essential features allowing yeast to live. This collection of proteins with functional synthetic sequences can subsequently be used in phenotypic tests or genetic screens in order to discover genetic interaction.

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.

H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation.

Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me).

Structure of the Rtt109-AcCoA/Vps75 complex and implications for chaperone-mediated histone acetylation.

Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ∼12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.

[H2A.Z: a histone variant that decorates gene promoters]. / H2A.Z: un variant d'histone qui orne les promoteurs des gènes.

The nucleosome, fundamental unit of chromatin, is composed of four basic histones, H2A, H2B, H3 and H4, around which DNA is wrapped. In order to have access to DNA, cells must modify the structure of chromatin by different known mechanisms. One such mechanism is by replacing canonical histones in the nucleosome with variants, which can confer special functions to chromatin. H2A.Z is an evolutionary conserved variant of H2A that has both a positive and a negative role on gene transcription. The mechanisms by which H2A.Z acts are still poorly understood. However, recent reports have shed some light on this subject. H2A.Z is found associated with almost 2/3 of the promoters of genes in yeast, suggesting that this histone could have a global role on gene expression by poising chromatin for activation. We review here recent literature and discuss different aspects of the biology of this histone variant.

Reuniting the contrasting functions of H2A.Z.

It is now well established that cells modify chromatin to set transcriptionally active or inactive regions. Such control of chromatin structure is essential for proper development of organisms. In addition to the growing number of histone post-translational modifications, cells can exchange canonical histones with different variants that can directly or indirectly change chromatin structure. Moreover, enzymatic complexes that can exchange specific histone variants within the nucleosome have now been identified. One such variant, H2A.Z, has recently been the focus of many studies. H2A.Z is highly conserved in evolution and has many different functions, while defining both active and inactive chromatin in different contexts. Advanced molecular techniques, such as genome-wide binding assays (chromatin immunoprecipitation on chip) have recently given researchers many clues as to how H2A.Z is targeted to chromatin and how it affects nuclear functions. We wish to review the recent literature and summarize our understanding of the mechanisms and functions of H2A.Z.

Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning.

H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions--typically one or two nucleosomes wide--decorated with H2A.Z. Those "Z loci" are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed.

BRCA1 can modulate RNA polymerase II carboxy-terminal domain phosphorylation levels.

A high incidence of breast and ovarian cancers has been linked to mutations in the BRCA1 gene. BRCA1 has been shown to be involved in both positive and negative regulation of gene activity as well as in numerous other processes such as DNA repair and cell cycle regulation. Since modulation of the RNA polymerase II carboxy-terminal domain (CTD) phosphorylation levels could constitute an interface to all these functions, we wanted to directly test the possibility that BRCA1 might regulate the phosphorylation state of the CTD. We have shown that the BRCA1 C-terminal region can negatively modulate phosphorylation levels of the RNA polymerase II CTD by the Cdk-activating kinase (CAK) in vitro. Interestingly, the BRCA1 C-terminal region can directly interact with CAK and inhibit CAK activity by competing with ATP. Finally, we demonstrated that full-length BRCA1 can inhibit CTD phosphorylation when introduced in the BRCA1(-/-) HCC1937 cell line. Our results suggest that BRCA1 could play its ascribed roles, at least in part, by modulating CTD kinase components.

Immunohistochemical localization of type 2 inositol 1,4,5-trisphosphate receptor to the nucleus of different mammalian cells.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a ligand-gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti-InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS-7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co-localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity-binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed.