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  • Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16INK4A and p14ARF expression. 21245331

    Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA3A are each essential for EBV conversion of primary human B lymphocytes into continuously proliferating lymphoblast cell lines (LCLs) and for maintaining LCL growth. We now find that EBNA3C and EBNA3A's essential roles are to repress p16(INK4A) and p14(ARF). In the absence of EBNA3C or EBNA3A, p16(INK4A) and p14(ARF) expression increased and cell growth ceased. EBNA3C inactivation did not alter p16(INK4A) promoter CpG methylation, but reduced already low H3K27me3, relative to resting B cells, and increased H3K4me3 and H3-acetylation, linking EBNA3C inactivation to histone modifications associated with increased transcription. Importantly, knockdown of p16(INK4A) or p14(ARF) partially rescued LCLs from EBNA3C or EBNA3A inactivation-induced growth arrest and knockdown of both rescued LCL growth, confirming central roles for p16(INK4A) and p14(ARF) in LCL growth arrest following EBNA3C or EBNA3A inactivation. Moreover, blockade of p16(INK4A) and p14(ARF) effects on pRb and p53 by human papilloma virus type 16 E7 and E6 expression, sustained LCL growth after EBNA3C or EBNA3A inactivation. These data indicate that EBNA3C and EBNA3A joint repression of CDKN2A p16(INK4A) and p14(ARF) is essential for LCL growth.
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  • Regulation of cyclin B2 expression and cell cycle G2/m transition by menin. 20404349

    Multiple endocrine neoplasia type 1 (MEN1) results from mutations in tumor suppressor gene Men1, which encodes nuclear protein menin. Menin up-regulates certain cyclin-dependent kinase inhibitors through increasing histone H3 lysine 4 (H3K4) methylation and inhibits G(0)/G(1) to S phase transition. However, little is known as to whether menin controls G(2)/M-phase transition, another important cell cycle checkpoint. Here, we show that menin expression delays G(2)/M phase transition and reduces expression of Ccnb2 (encoding cyclin B2). Menin associates with the promoter of Ccnb2 and reduces histone H3 acetylation, a positive chromatin marker for gene transcription, at the Ccnb2 locus. Moreover, Men1 ablation leads to an increase in cyclin B2 expression, histone H3 acetylation at the Ccnb2 locus, and G(2)/M transition. In contrast, knockdown of cyclin B2 diminishes the number of cells at M phase and reduces cell proliferation. Furthermore, menin interferes with binding of certain positive transcriptional regulators, such as nuclear factor Y (NF-Y), E2 factors (E2Fs), and histone acetyltransferase CREB (cAMP-response element-binding protein)-binding protein (CBP) to the Ccnb2 locus. Notably, MEN1 disease-related mutations, A242V and L22R, abrogate the ability of menin to repress cyclin B2 expression and G(2)/M transition. Both of the mutants fail to reduce the acetylated level of the Ccnb2 locus. Together, these results suggest that menin-mediated repression of cyclin B2 is crucial for inhibiting G(2)/M transition and cell proliferation through a previously unrecognized molecular mechanism for menin-induced suppression of MEN1 tumorigenesis.
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  • Acetylation of EKLF is essential for epigenetic modification and transcriptional activation of the beta-globin locus. 18710946

    Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the beta-like globin locus chromatin structure and activate transcription of the adult beta-globin gene. By establishing an EKLF-null erythroid line whose closed beta-locus chromatin structure and silent beta-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult beta-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult beta-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.
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  • Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. 22837004

    Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.
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  • Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats. 21310085

    Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.
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  • Lysine-specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) synergistically repress proinflammatory cytokines and classical complement pathway components. 22542627

    Histone modifying enzymes confer epigenetic marks, directing the changes in gene expression required for diverse cellular processes. Lysine-specific demethylase 1 (LSD1) functions as a transcriptional coregulator by demethylating histone H3 on lysine 4 and lysine 9. Analyzing transcriptomes on microarrays, we identified genes which represent inflammatory-related targets of LSD1. We demonstrate a repressive role of LSD1 in proinflammatory cytokine expression such as IL1α, IL1β, IL6 and IL8 and classical complement components. Consistently, LSD1 occupies and regulates the promoter of these genes. In addition, we demonstrate that HDAC1 and LSD1 synergistically regulate these inflammatory-related genes. Our data reveal a novel role for LSD1 in suppressing immune responses.
    Document Type:
    Reference
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    17-615
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    ChIPAb+ Acetyl-Histone H3 - ChIP Validated Antibody and Primer Set

  • p53 mutants induce transcription of NF-κB2 in H1299 cells through CBP and STAT binding on the NF-κB2 promoter and gain of function activity. 22198284

    Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 and show gain of function (GOF) phenotypes such as increased growth rate, enhanced resistance to chemotherapeutic drugs, increased cell motility and tumorigenicity; although the mechanism for this function remains unknown. In this communication we report that p53-mediated NF-κB2 up-regulation significantly contributes to the aggressive oncogenic behavior of cancer cells. Lowering the level of mutant p53 in a number of cancer cell lines resulted in a loss of GOF phenotypes directly implicating p53 mutants in the process. RNAi against NF-κB2 in naturally occurring cancer cell lines also lowers GOF activities. In H1299 cells expressing mutant p53, chromatin immunoprecipitation (ChIP) assays indicate that mutant p53 induces histone acetylation at specific sites on the regulatory regions of its target genes. ChIP assays using antibodies against transcription factors putatively capable of interacting with the NF-κB2 promoter show increased interaction of CBP and STAT2 in the presence of mutant p53. Thus, we propose that in H1299 cells, mutant p53 elevates expression of genes capable of enhancing cell proliferation, motility, and tumorigenicity by inducing acetylation of histones via recruitment of CBP and STAT2 on the promoters causing CBP-mediated histone acetylation.
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    Reference
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    17-615
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    ChIPAb+ Acetyl-Histone H3 - ChIP Validated Antibody and Primer Set

  • Dynamics of plant histone modifications in response to DNA damage. 22574698

    DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.
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  • Histone deacetylase complexes promote trinucleotide repeat expansions. 22363205

    Expansions of DNA trinucleotide repeats cause at least 17 inherited neurodegenerative diseases, such as Huntington's disease. Expansions can occur at frequencies approaching 100% in affected families and in transgenic mice, suggesting that specific cellular proteins actively promote (favor) expansions. The inference is that expansions arise due to the presence of these promoting proteins, not their absence, and that interfering with these proteins can suppress expansions. The goal of this study was to identify novel factors that promote expansions. We discovered that specific histone deacetylase complexes (HDACs) promote CTG•CAG repeat expansions in budding yeast and human cells. Mutation or inhibition of yeast Rpd3L or Hda1 suppressed up to 90% of expansions. In cultured human astrocytes, expansions were suppressed by 75% upon inhibition or knockdown of HDAC3, whereas siRNA against the histone acetyltransferases CBP/p300 stimulated expansions. Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions. Expansion assays with nuclease mutants indicated that Sae2 is one of the relevant factors regulated by Rpd3L and Hda1. The causal relationship between HDACs and expansions indicates that HDACs can promote mutagenesis at some DNA sequences. This relationship further implies that HDAC3 inhibitors being tested for relief of expansion-associated gene silencing may also suppress somatic expansions that contribute to disease progression.
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  • Biomechanical stimulation of osteoblast gene expression requires phosphorylation of the RUNX2 transcription factor. 22337141

    Bone can adapt its structure in response to mechanical stimuli. At the cellular level, this involves changes in chromatin organization, gene expression, and differentiation, but the underlying mechanisms are poorly understood. Here we report on the involvement of RUNX2, a bone-related transcription factor, in this process. Fluid flow shear stress loading of preosteoblasts stimulated translocation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) to the nucleus where it phosphorylated RUNX2 on the chromatin of target genes, and increased histone acetylation and gene expression. MAPK signaling and two RUNX2 phosphoacceptor sites, S301 and S319, were critical for this response. Similarly, in vivo loading of mouse ulnae dramatically increased ERK and RUNX2 phosphorylation as well as expression of osteoblast-related genes. These findings establish ERK/MAPK-mediated phosphorylation of RUNX2 as a critical step in the response of preosteoblasts to dynamic loading and define a novel mechanism to explain how mechanical signals induce gene expression in bone.
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