07-108 Sigma-AldrichAnti-Histone H4 Antibody

Although BRCA1 function is essential for maintaining genomic integrity in all cell types, it is unclear why increased risk of cancer in individuals harbouring deleterious mutations in BRCA1 is restricted to only a select few tissues. Here we show that human mammary epithelial cells (HMECs) from BRCA1-mutation carriers (BRCA1(mut/+)) exhibit increased genomic instability and rapid telomere erosion in the absence of tumour-suppressor loss. Furthermore, we uncover a novel form of haploinsufficiency-induced senescence (HIS) specific to epithelial cells, which is triggered by pRb pathway activation rather than p53 induction. HIS and telomere erosion in HMECs correlate with misregulation of SIRT1 leading to increased levels of acetylated pRb as well as acetylated H4K16 both globally and at telomeric regions. These results identify a novel form of cellular senescence and provide a potential molecular basis for the rapid cell- and tissue- specific predisposition of breast cancer development associated with BRCA1 haploinsufficiency.

Pancreatic cancer remains a clinical challenge, thus new therapies are urgently needed. The selective Wee1 inhibitor MK-1775 has demonstrated promising results when combined with DNA damaging agents, and more recently with CHK1 inhibitors in various malignancies. We have previously demonstrated that treatment with the pan-histone deacetylase inhibitor panobinostat (LBH589) can cause down-regulation of CHK1. Accordingly, we investigated using panobinostat to down-regulate CHK1 in combination with MK-1775 to enhance cell death in preclinical pancreatic cancer models. We demonstrate that MK-1775 treatment results in increased H2AX phosphorylation, indicating increased DNA double-strand breaks, and activation of CHK1, which are both dependent on CDK activity. Combination of MK-1775 and panobinostat resulted in synergistic antitumor activity in six pancreatic cancer cell lines. Finally, our in vivo study using a pancreatic xenograft model reveals promising cooperative antitumor activity between MK-1775 and panobinostat. Our study provides compelling evidence that the combination of MK-1775 and panobinostat has antitumor activity in preclinical models of pancreatic cancer and supports the clinical development of panobinostat in combination with MK-1775 for the treatment of this deadly disease.

Aberrant epigenetic silencing of tumor suppressor genes has been recognized as a driving force in cancer. Epigenetic drugs such as the DNA methylation inhibitor decitabine reactivate genes and are effective in myeloid leukemia, but resistance often develops and efficacy in solid tumors is limited. To improve their clinical efficacy, we searched among approved anti-cancer drugs for an epigenetic synergistic combination with decitabine.We used the YB5 cell line, a clonal derivative of the SW48 colon cancer cell line that contains a single copy of a hypermethylated cytomegalovirus (CMV) promoter driving green fluorescent protein (GFP) to screen for drug-induced gene reactivation and synergy with decitabine. None of the 16 anti-cancer drugs tested had effects on their own. However, in combination with decitabine, platinum compounds showed striking synergy in activating GFP. This was dose dependent, observed both in concurrent and sequential combinations, and also seen with other alkylating agents. Clinically achievable concentrations of carboplatin at (25 μM) and decitabine reactivated GFP in 28 % of the YB5 cells as compared to 15 % with decitabine alone. Epigenetic synergy was also seen at endogenously hypermethylated tumor suppressor genes such as MLH1 and PDLIM4. Genome-wide studies showed that reactivation of hypermethylated genes by the combination was significantly better than that induced by decitabine alone or carboplatin alone. Platinum compounds did not enhance decitabine-induced hypomethylation. Rather, we found significantly inhibited HP1α expression by carboplatin and the combination. This was accompanied by increased histone H3 lysine 4 (H3K4) trimethylation and histone H3 lysine 9 (H3K9) acetylation at reactivated genes (P less than  0.0001) and reduced occupancy by methyl-binding proteins including MeCP2 and methyl-CpG-binding domain protein 2 (MBD2) (P less than  0.0001).Our results suggest that the combination of decitabine with platinum analogs shows epigenetic synergy that might be exploited in the treatment of different cancers.

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT).We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos.Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγ(null) mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells.

Williams syndrome transcription factor (WSTF) is a multifaceted protein that is involved in several nuclear processes, including replication, transcription, and the DNA damage response. WSTF participates in a chromatin-remodeling complex with the ISWI ATPase, SNF2H, and is thought to contribute to the maintenance of heterochromatin, including at the human inactive X chromosome (Xi). WSTF is encoded by BAZ1B, and is one of twenty-eight genes that are hemizygously deleted in the genetic disorder Williams-Beuren syndrome (WBS).To explore the function of WSTF, we performed zinc finger nuclease-assisted targeting of the BAZ1B gene and isolated several independent knockout clones in human cells. Our results show that, while heterochromatin at the Xi is unaltered, new inappropriate areas of heterochromatin spontaneously form and resolve throughout the nucleus, appearing as large DAPI-dense staining blocks, defined by histone H3 lysine-9 trimethylation and association of the proteins heterochromatin protein 1 and structural maintenance of chromosomes flexible hinge domain containing 1. In three independent mutants, the expression of a large number of genes were impacted, both up and down, by WSTF loss.Given the inappropriate appearance of regions of heterochromatin in BAZ1B knockout cells, it is evident that WSTF performs a critical role in maintaining chromatin and transcriptional states, a property that is likely compromised by WSTF haploinsufficiency in WBS patients.

We analyzed the activity of the histone deacetylase inhibitor (HDACi) suberoyl-anilide hydroxamic acid (SAHA) on Kasumi-1 acute myeloid leukemia (AML) cells expressing AML1/ETO. We also compared the effects of SAHA to those of valproic acid (VPA), a short-chain fatty acid HDACi. SAHA and VPA induced histone H3 and H4 acetylation, myeloid differentiation and massive early apoptosis. The latter effects were not determined by either drug in AML cell lines, such as NB4 or THP-1, not expressing AML1/ETO. SAHA was more rapid and effective than VPA in increasing H3 and H4 acetylation in total Kasumi-1 cell lysates and more effective than VPA in inducing acetylation of H4K8, H4K12, H4K16 residues. At the promoter of IL3, a transcriptionally-silenced target of AML1/ETO, SAHA was also more rapid than VPA in inducing total H4, H4K5, H4K8 and H3K27 acetylation, while VPA was more effective than SAHA at later times in inducing acetylation of total H4, H4K12, H4K16, as well as total H3. Consistent with these differences, SAHA induced the expression of IL3 mRNA more rapidly than VPA, while the effect of VPA was delayed. These differences might be exploited to design clinical trials specifically directed to AML subtypes characterized by constitutive HDAC activation. Our results led to include SAHA, an FDA-approved drug, among the HDACi active in the AML1/ETO-expressing AML cells.

Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an α,β-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function.

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.