Increased Histone H3 Lysine Research Articles

Hypoxic silencing of tumor suppressor RUNX3 by histone modification in gastric cancer cells

RUNX3 is a tumor suppressor that is silenced in cancer following hypermethylation of its promoter. The effects of hypoxia in tumor suppressor gene (TSG) transcription are largely unknown. Here, we investigated hypoxia-induced silencing mechanisms of RUNX3. The expression of RUNX3 was downregulated in response to hypoxia in human gastric cancer cells at the transcriptional level. This downregulation was abolished following treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cytosine methylation inhibitor 5-aza-2-deoxycytidine (5-Aza), suggesting that an epigenetic regulatory mechanism may be involved in RUNX3 silencing by hypoxia. DNA methylation PCR and bisulfite-sequencing data revealed that hypoxia did not affect the methylation of RUNX3 promoter. A chromatin immunoprecipitation (ChIP) assay revealed increased histone H3-lysine 9 dimethylation and decreased H3 acetylation in the RUNX3 promoter following hypoxia. Hypoxia resulted in the upregulation of G9a histone methyltransferase (HMT) and HDAC1; additionally, overexpression of G9a and HDAC1 attenuated RUNX3 expression. The overexpression of G9a and HDAC1, but not their mutants, inhibited the nuclear localization and expression of RUNX3. Diminished mRNA expression and nuclear localization of RUNX3 during hypoxia was abolished by siRNA-mediated knockdown of G9a and HDAC1. This study suggests that hypoxia silences RUNX3 by epigenetic histone regulation during the progression of gastric cancer.

Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation—a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

SIRT1 inhibition alleviates gene silencing in Fragile X mental retardation syndrome.

Expansion of the CGG•CCG-repeat tract in the 5′ UTR of the FMR1 gene to >200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS), the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase, SIRT1, plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I, II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase, hMOF. DNA methylation, on the other hand, is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However, since DNA methylation inhibitors require DNA replication in order to be effective, SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious.

Histone Modifications at the ABCG2 Promoter following Treatment with Histone Deacetylase Inhibitor Mirror Those in Multidrug-Resistant Cells

ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in pharmacology and may play a role in stem cell biology and clinical drug resistance. To study the mechanism(s) regulating ABCG2 expression, we used ChIP to investigate the levels of acetylated histone H3, histone deacetylases (HDAC), histone acetyltransferases, and other transcription regulatory proteins associated with the ABCG2 promoter. Following selection for drug resistance and the subsequent overexpression of ABCG2, an increase in acetylated histone H3 but a decrease in class I HDACs associated with the ABCG2 promoter was observed. Permissive histone modifications, including an increase in histone H3 lysine 4 trimethylation (Me(3)-K4 H3) and histone H3 serine 10 phosphorylation (P-S10 H3), were observed accompanying development of the resistance phenotype. These changes mirrored those in some cell lines treated with a HDAC inhibitor, romidepsin. A repressive histone mark, trimethylated histone H3 lysine 9 (Me(3)-K9 H3), was found in untreated parental cells and cells that did not respond to HDAC inhibition with ABCG2 up-regulation. Interestingly, although all five studied cell lines showed global histone acetylation and MDR1 up-regulation upon HDAC inhibition, only those cells with removal of the repressive mark, and recruitment of RNA polymerase II and a chromatin remodeling factor Brg-1 from the ABCG2 promoter, showed increased ABCG2 expression. In the remaining cell lines, HDAC1 binding in association with the repressive Me3-K9 H3 mark apparently constrains the effect of HDAC inhibition on ABCG2 expression. These studies begin to address the differential effect of HDAC inhibitors widely observed in gene expression studies.

Concordant Epigenetic Silencing of Transforming Growth Factor-β Signaling Pathway Genes Occurs Early in Breast Carcinogenesis

Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.

Genome-wide dynamics of SAPHIRE, an essential complex for gene activation and chromatin boundaries.

In this study, we characterize a four-protein nucleosome-binding complex from Schizosaccharomyces pombe, termed SAPHIRE, that includes two orthologs of human Lsd1, a histone demethylase. The SAPHIRE complex is essential for cell viability, whereas saphire mutants lacking key conserved catalytic residues are viable but thermosensitive, suggesting that SAPHIRE has both an important enzymatic function and an essential nonenzymatic function. SAPHIRE is present in (or adjacent to) particular heterochromatic loci and also in the transcription start site regions of many highly active polymerase II genes. However, ribosomal protein genes are notably SAPHIRE deficient. SAPHIRE promotes activation, as target genes are selectively attenuated in saphire mutants. Interestingly, saphire mutants display increased histone H3 lysine 4 dimethylation, a modification typically associated with euchromatin. SAPHIRE localization is dynamic, as activated genes rapidly acquire SAPHIRE. Furthermore, saphire mutants dramatically shift a heterochromatin-euchromatin boundary in Chr1, suggesting a novel role in boundary regulation.

Cooperation of SRC-1 and p300 With NF-κB and CREB in Angiotensin II-Induced IL-6 Expression in Vascular Smooth Muscle Cells

The purpose of this study was to evaluate the role of coactivator histone acetyltransferases (HATs) p300 and SRC-1 in angiotensin II (Ang II)-induced interleukin-6 (IL-6) gene expression in vascular smooth muscle cells (VSMCs). Ang II increased IL-6 mRNA expression via NF-kappaB and CREB in an extracellular signal-regulated kinase (ERK)-dependent manner in rat VSMCs. It was also significantly enhanced by the histone deacetylase inhibitor, Trichostatin A. Chromatin immunoprecipitation (ChIP) assays showed that Ang II increased Histone H3 Lysine (K9/14) acetylation on the IL-6 promoter. Ang II-induced IL-6 promoter transactivation was significantly enhanced by p300 and SRC-1, with maximal activation in cells cotransfected with NF-kappaB (p65) and SRC-1. Nucleofection of VSMCs with either an ERK phosphorylation site mutant of SRC-1 or p300/CBP HAT deficient mutants significantly blocked Ang II-induced IL-6 expression. ChIP assays revealed that Ang II enhanced coordinate occupancy of p65, CREB, p300, and SRC-1 at the IL-6 promoter. An ERK pathway inhibitor blocked Ang-induced IL-6 promoter SRC-1 occupancy and histone acetylation. Ang II-induced IL-6 expression requires NF-kappaB and CREB as well as ERK-dependent histone acetylation mediated by p300 and SRC-1. These results provide new insights into nuclear chromatin mechanisms by which Ang II regulates inflammatory gene expression.

SUPPRESSOR OF FRI 4encodes a nuclear-localized protein that is required for delayed flowering in winter-annualArabidopsis

The floral inhibitor FLOWERING LOCUS C (FLC) is a crucial regulator of flowering time in Arabidopsis, and is positively regulated by the FRIGIDA (FRI) gene in late-flowering winter-annual accessions. In rapid-cycling accessions, FLC expression is suppressed by the autonomous floral-promotion pathway (AP); thus AP mutants contain high levels of FLC and are late flowering. Previous work has shown that the upregulation of FLC in FRI- or AP-mutant backgrounds is correlated to an increase in histone H3 lysine 4 (H3K4) trimethylation at the FLC locus. This increase in trimethylation requires a PAF1-like complex and EARLY FLOWERING IN SHORT DAYS (EFS), a putative histone H3 methyltransferase. We have identified a putative zinc-finger-containing transcription factor, SUF4, that is required for the upregulation of FLC by FRI. suf4 mutations strongly suppress the late-flowering phenotype of FRI, but only weakly suppress AP mutants. As with mutants in efs or the PAF1-like complex, suf4 mutants show reduced H3K4 trimethylation at FLC. An interesting distinction between the phenotypes of suf4 mutants and mutants in efs or the PAF1-like complex is observed in the expression of genes that are adjacent to FLC or FLC-like genes. In efs and PAF1-like-complex mutants, the expression of FLC, FLC-like genes and adjacent genes is suppressed. In suf4 mutants, however, only FLC expression is suppressed. These data are consistent with a model in which SUF4 may act to specifically recruit EFS and the PAF1-like complex to the FLC locus.

Gfi1b alters histone methylation at target gene promoters and sites of γ-satellite containing heterochromatin

Gfi1b is a 37 kDa nuclear protein with six C2H2 zinc-finger domains that can silence transcription upon binding to specific target gene promoters. Here we show by using a chromatin immunoprecipitation and cloning protocol that Gfi1b also binds to gamma-satellite sequences that mainly occur in pericentric heterochromatin. Immuno-FISH experiments demonstrated that Gfi1b is localized at foci of pericentric heterochromatin identified by DAPI staining. Elevated levels of Gfi1b correlated with increased histone H3 lysine 9 dimethylation at sites neighboring gamma-satellite sequences but also at Gfi1b target gene promoters. In Gfi1b-deficient cells, however, a decrease of histone H3 lysine 9 trimethylation and a loss of heterochromatic structures was observed. Strikingly, we found that Gfi1b binds to both SUV39H1 and G9A histone methyl transferases, which provides a direct link between histone methylation and Gfi1b at heterochromatic and euchromatic sites. We propose that Gfi1b functions in heterochromatin formation and silencing of euchromatic transcription by recruiting histone methyl transferases to either gamma-satellite sequences or specific target gene promoters.

Coordinated changes of histone modifications and HDAC mobilization regulate the induction of MHC class II genes by Trichostatin A

The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the Major Histocompatibility Class II (MHC II) DRA gene in a way independent of the master coactivator CIITA. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHC II expression, we used chromatin immunoprecipitation (ChIP) assays to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. We found that a dramatic increase in promoter linked histone acetylation is followed by an increase in Histone H3 lysine 4 methylation and a decrease of lysine 9 methylation. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA increases the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHC II family and the adjacent histone cluster that are located in chromosome 6p21-22 locus are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response.

RIZ1 Tumor Suppressor Regulates IGF-1 Autocrine Loop To Control Cell Proliferation, Apoptosis, and Differentiation in Chronic Myeloid Leukemia Blast Crisis Cell Lines.

RIZ1 (PRDM2) is a tumor suppressor gene whose expression and activity are reduced by genetic and epigenetic aberrations in many cancers. In chronic myeloid leukemia (CML), blastic transformation is associated with loss of heterozygosity at 1p36, the region where RIZ1 is located, suggesting that RIZ1 has an essential role in CML pathogenesis. In CML patients and in the CML blast crisis cell line K562, we observed aberrant RIZ1 promoter methylation. To further characterize RIZ1 tumor suppressor properties that are related to CML, we analyzed RIZ1-induced changes in proliferation, apoptosis, and differentiation in CML myeloid blast crisis (CML-BC) cell lines (K562, ERY-1, YN-1, and JURL-MK1) that express low levels of endogenous RIZ1.Forced RIZ1 expression in CML-BC cell lines substantially decreased proliferation, increased apoptosis, and increased the population of cells in G2/M phase of the cell cycle. RIZ1 expression also promoted differentiation as assessed by benzidine staining in CML-BC cell lines expressing immature erythroid cell features (K562, ERY-1, YN-1) and by CD117 and CD33 expression in JURL-MK1, a CML-BC cell line expressing megakaryoblastic features. To identify genes and pathways influenced by RIZ1 expression, we used 42k cDNA microarrays to globally monitor how RIZ1 expression changes the gene expression profile of K562 cells. We discovered 25 RIZ1-regulated genes that are involved in a variety of biological processes with a subgroup of genes involved in IGF-1 signaling. The most strongly RIZ1 down-regulated genes (IGF1) and up-regulated genes (SPARC, IGFBP2) are involved in IGF-1 signaling. Using chromatin immunoprecipitation (ChIP), we determined that RIZ1 associates with IGF-1 and SPARC promoters. RIZ1 contains a PR domain that has Histone H3 lysine 9 methylation activity, which is implicated in gene repression. Using ChIP assays, we found that RIZ1 expression in K562 cells increased Histone H3 lysine 9 methylation of the IGF-1 promoter. The increased Histone H3 lysine 9 methylation is associated with decreased IGF-1 expression as monitored by RT-PCR and Western analysis. We observed autocrine production of IGF-1 in K562 cells, by culturing cells in serum free media and monitoring IGF-1 production and signaling. We detected receptor bound IGF-1 by flow cytometry, and compared the growth properties of sorted IGF-1 positive and negative cells. IGF-1 positive cells have increased number of cells in G2/M and S phase, a reduced number of cells in G1 and higher numbers of mitoses and Ki-67 positive nuclei compared with IGF-1 negative cells. RIZ1 expression in K562 decreased the amount of receptor bound IGF-1, reduced IGF-1 receptor activation, and reduced the activity of downstream IGF-1 signaling pathways. RIZ1 expression in K562 substantially reduced AKT1 and ERK1/2 phosphorylation. Our study demonstrates that RIZ1 reduces cell proliferation, increases apoptosis, and enhances differentiation of CML blast crisis cell lines. RIZ1 also controls autocrine production of IGF-1 and blocks the activity of IGF-1 signaling pathways. These activities may in part be responsible for RIZ1 tumor suppressor activity and point to the therapeutic potential of IGF-1 pathway inhibition in the acute phase of CML.

Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.

Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system.

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation.

ARHI has been identified as a maternally imprinted tumor suppressor gene that maps to chromosome 1p31 and whose expression is markedly down-regulated in breast cancer. To explore possible mechanisms that could silence ARHI expression, we have tested the importance of DNA methylation, histone acetylation and histone methylation in regulating ARHI expression. We found that treatment with CpG demethylating agents and/or histone deacetylase inhibitors could reactivate both the silenced and the imprinted alleles of this tumor suppressor gene. Reactivation of ARHI expression by these reagents is related to the methylation status of the CpG islands in the ARHI promoter, especially CpG island II. Chromatin immunoprecipitation assays revealed that histone H3 lysine 9/18 acetylation levels associated with ARHI in normal cells were significantly higher than those in breast cancer cell lines that lacked ARHI expression. Treatment with a CpG demethylating agent and/or histone deacetylase inhibitor could increase ARHI expression in breast cancer cells, with a corresponding increase in histone H3 lysine 9/18 acetylation and decrease in histone H3 lysine 9 methylation.