Displacement Of Histone H1 Research Articles

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

Prothymosin alpha interacts with C-terminal domain of histone H1 and dissociates p53-histone H1 complex

A novel mode of the tumor suppressor protein p53 regulation, mediated by recruitment of the linker histone H1 to the promoters of p53 target genes leading to specific repression of p53-dependent transcription, has recently been uncovered. Yet, how this repression could be relieved is not clear. Previously, a histone-binding nuclear protein prothymosin alpha (ProTa) was shown to trigger a p53 response. The histone-binding region of ProTa was found to be essential for this effect, raising a possibility that ProTa stimulates p53-dependent transcription by dissociating the p53-histone H1 repressive complex. Here, we have shown that ProTa interacts with the same C-terminal domain of histone H1 as p53 does and, therefore, ProTa and p53 could compete for binding to histone H1. Furthermore, ProTa, when competent for histone H1 binding, is able to liberate p53 from the histone H1-p53 complex in vitro. In vivo, stimulation of p53-dependent transcription by ProTa correlates with ability of ProTa to interact with histone H1. Ectopic expression of histone H1 or its C-terminal ProTa-binding domain specifically suppresses the stimulating effect of ProTa on transcription of the p53-responsive reporter gene in cultured cells. These results are consistent with the model that ProTa may enhance p53 transcription activity by displacement of histone H1 from p53-H1 repressive complex.

Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Although we have gained insight into how core histones and their modifications influence this process, the role of linker histones remains unclear. Here we show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation.

Potential role of a novel transcriptional coactivator PELP1 in histone H1 displacement in cancer cells.

The estrogen receptor plays an important role in breast cancer progression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions. The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known. Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1. Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions. Ligand stimulation promoted recruitment of PELP1 to 17beta-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity. Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1. Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site. The PELP1 mutant lacking histone 1-binding domain acts as a dominant-negative and blocks estrogen receptor alpha-mediated transcription. Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases. PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes. Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells.

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

Retinoid-dependent Recruitment of a Histone H1 Displacement Activity by Retinoic Acid Receptor

Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones. However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes. Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described. In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin. We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors. Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR. These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events.

SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin.

An experimental assay was developed to search for proteins capable of antagonizing histone H1-mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold-associated region (SAR) were highly selectively repressed by H1 relative to non-SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A-tracts (AT-rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. Partial, selective titration of these A-tracts by the high mobility group (HMG) protein HMG-I/Y led to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 on to non-SAR templates. SARs are associated with many highly transcribed regulated genes where they may serve to facilitate the HMG-I/Y-mediated displacement of histone H1 in chromatin. Indeed, HMG-I/Y was found to be strongly enriched in the H1-depleted subfraction which can be isolated from chromatin.

Partial displacement of histone H1 from chromatin is required before it can be phosphorylated by mitotic H1 kinase in vitro.

The massive nonselective and reversible phosphorylation of histone H1 during mitosis is a universal phenomenon among eukaryotes. The growth-associated kinase responsible for this phosphorylation is identical to the maturation promoting factor, a key regulator of the cell cycle. Here we showed that growth-associated kinase, isolated from mitotic HeLa cells which were capable of phosphorylating HeLa H1 in vitro with high activity and mostly at the same sites phosphorylated during mitosis in vivo (assayed by two-dimensional analysis of tryptic phosphopeptides), did not significantly phosphorylate chromatin-bound or nuclear H1 in vitro. Its inability to phosphorylate chromatin-bound H1 did not change when the amount of kinase was increased or the incubation was prolonged. The resistance of chromatin-bound H1 to phosphorylation did not result from chromatin aggregation. Rapid phosphorylation of H1 in vitro, as well as in a nuclear system, was restored when NaCl concentrations were raised above 200 mM where H1:DNA interactions are weakened. At 300 mM NaCl, chromatin-bound H1 was phosphorylated in a subset of the sites observed for free H1 phosphorylated in vitro. These results suggest that active displacement of H1 from chromatin DNA may take place before H1 can be fully phosphorylated during mitosis.

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Effect of exogenous histone H5 on integration of histone H1 in rat liver chromatin. Correlations with aberrant ε- N-methylation of histone H1

Binding modes of histones H1 and H5, and their competition for chromatin-binding sites in rat liver nuclei, were correlated with aberrant N-methylation of H1 histone lysine residues, induced by chicken erythrocyte histone H5, in order to gain more insight into the integration of lysine-rich histones in chromatin. Addition of approx. 2.5 molecules of histone H5 per nucleosome to rat liver nuclei increases the ratio of total basic residues in histones to DNA nucleotides ( BR NT ) in the nuclear chromatin from 1.0 to 1.5. At this concentration, approx. 0.7 molecule of histone H5 is bound per nucleosome, and there is no displacement of histone H1 from the nuclear chromatin. If S-adenosyl[ Me- 3H]methionine is present in the incubation mixture, the aberrant incorporation of labeled methyl groups into histone H1 reaches a maximum at this concentration of histone H5. The radioactivity present in histone H1 from nuclei incubated with labeled AdoMet at a total BR NT ratio of 1.5: (i) resides mainly in a histone H1 subfraction tentatively identified by Bio-Rex 70 chromatography and acrylamide gel electrophoresis as histone H1c; (ii) presents as a single spot upon peptide mapping of tryptic hydrolysates by means of two-dimensional thin-layer chromatography; and (iii) elutes in the position of mono- N-methyllysine upon ion-exchange chromatography of histone H1 hydrolysates. Upon further increase of the BR NT ratio, the following changes are produced: (i) a gradual decrease in radioactive methyl uptake into histone H1; (ii) a gradual displacement of histone H1 from the chromatin; (iii) increased binding of histone H5 in chromatin, up to a maximum of 3.4 residues per nucleosome; and (iv) a slowly increasing uptake of label into histone H5. The combined data from histone H1/H5 binding and histone H1 methylation studies suggest that upon addition of exogenous histone H5 to rat liver nuclei the binding of two lysine-rich histones per nucleosome plays a significant role in the induction of specific changes in chromatin structure, which in vivo may have important functional implications in terms of chromatin condensation and suppression of transcription.

Selective displacement of histone H1 from whole HeLa nuclei: effect on chromatin structure in situ as probed by micrococcal nuclease

Selective displacement of histone H1 from whole HeLa nuclei: effect on chromatin structure in situ as probed by micrococcal nuclease