Hyperacetylation Of H4 Research Articles

P210 Bcr‐abl tyrosine kinase interaction with histone deacetylase 1 modifies histone H4 acetylation and chromatin structure of chronic myeloid leukaemia haematopoietic progenitors

The BCR-ABL fusion gene, originating from the balanced (9;22) translocation, is the molecular hallmark and the causative event of chronic myeloid leukaemia (CML). The interactions of its p210 protein constitutively activated and improperly confined to the cytoplasm with multiple regulatory signals of cell cycle progression, apoptosis and self-renewal, induce the illegitimate enlargement of clonal haematopoiesis and genetic instability that drives its progression towards the fully transformed phenotype of blast crisis. However, its effects on the basic transcription machinery and chromatin remodelling are unknown. Our study underscored histone H4 hyperacetylation associated with p210 tyrosine kinase in vitro and in vivo and its role in BCR-ABL transcription. Histone H4 hyperacetylation proceeds, at least partly, from the 'loss of function' of histone deacetylase 1 protein, a critical component of Rb-mediated transcriptional repression, in consequence of its cytoplasmatic compartmentalisation.

The Human Alpha Hemoglobin Stabilizing Protein (AHSP) Gene Locus in EKLF-Deficient Erythroid Cells.

AHSP is an erythroid-specific protein that complexes with free α-hemoglobin, protecting it from precipitation. AHSP has been proposed as a modifier gene in β thalassemia and as a candidate gene for unexplained Heinz body anemias, thus understanding its regulation may lead to novel therapies for these disorders. Identified as an erythroid-specific, GATA-1 inducible gene, decreased AHSP mRNA has been found in the fetal livers of mice deficient in the erythroid transcription factor EKLF by both microarray and RNA subtraction analysis. In fetal livers from d13.5 EKLF-deficient mice, AHSP/α-globin mRNA ratios were decreased to 11–16% of wild type by RT-PCR and RPA. In the same fetal livers, no AHSP protein was detected on Western blots with a MoAB against AHSP. EKLF interacts with the proximal CACCC box of the β-globin gene promoter, establishing local chromatin structure and directing high-level β-globin transcription. We hypothesized that chromatin across the AHSP locus would be perturbed in erythroid cells from EKLF-deficient mice. We performed DNase I hypersensitive site (HS) mapping and chromatin immunoprecipitation (ChIP) analysis using wild type and EKLF deficient fetal liver cells. A strong HS was identified in the AHSP 5′ flanking DNA in the core promoter region, that was absent in day 13.5 fetal liver DNA from EKLF-deficient mice. Fine mapping placed this 5′ HS over a CACCC site in the core AHSP promoter. ChIP across the entire AHSP locus with d13.5 fetal liver chromatin identified 2 regions of hyperacetylation of histones H3 and H4 in wild type mice, one corresponding to the 5′ HS and the other 3′ to the AHSP coding sequence. Both of these hyperacetylated regions were hypoacetylated in EKLF-deficient fetal liver cells. ChIP across the AHSP locus with chromatin obtained from mice with an HA tag knocked into the 3′ end of the EKLF gene identified a peak of EKLF binding extending from the 5′HS to intron one, peaking over the core promoter CACCC site. The sequence of this region (ACCCACCCT) has a single mismatch compared to the EKLF consensus site (CCNCNCCCN). Using the AHSP CACCC site as probe in mobility shift assays with rEKLF protein yielded a complex that migrated at the same mobility as a complex obtained with a control β-globin promoter CACCC site probe. Both AHSP and control β-globin complexes were effectively competed by an excess of unlabeled AHSP probe, unlabeled β-globin probe, or ELKF antiserum. Mutant AHSP CACCC probes did not form DNA-protein complexes nor did they effectively displace wild type AHSP CACCC or β-globin CACCC probes in competition assays. Probes with the AHSP CACCC site mutated to the β-globin sequence (A to C) or the other 2 possibilities (A to G, A to T) yielded complexes comparable to wild type AHSP and control β-globin CACCC probes. In transfection assays in K562 cells, an AHSP promoter-luciferase reporter plasmid was transactivated by an EKLF expression plasmid to a degree comparable to a β-globin promoter-luciferase plasmid. These results support the hypotheses that the hemolytic anemia in EKLF-deficient mice is exacerbated by decreased AHSP expression and that EKLF acts as a transcription factor and a chromatin modulator for genes other than β-globin. Our data also support the hypothesis that AHSP and EKLF may be modifier genes for the β-thalassemia syndromes.

Genomic Organization, Chromatin Structure, and Transcriptional Regulation of the Murine Alpha Hemoglobin Stabilizing Protein (AHSP) Gene.

Alpha Hemoglobin Stabilizing Protein (AHSP, Eraf) is an abundant erythroid protein that binds and stabilizes alpha globin and alpha hemoglobin (Hb). In mice, loss of AHSP causes hemolytic anemia, with elevated levels of reactive oxygen species and Hb precipitation in erythrocytes. Loss of AHSP exacerbates beta thalassemia phenotypes in mice, presumably by enhancing the toxicity of excessive free alpha Hb. Based on these findings, AHSP is a candidate modifier gene for beta thalassemia in humans. No mutations in the AHSP coding region have been identified in patients to date. However, several groups reported an inverse correlation between beta thalassemia severity and erythroid AHSP expression levels, raising the possibility that AHSP is a quantitative trait modifier of beta thalassemia. To address this possibility, it is important to define the mechanisms that control expression of the AHSP gene. Transcripts of murine Ahsp are inducible by GATA-1. The goals of the current studies are to investigate the mechanisms of this induction and to define the DNA domain that regulates the locus. Using phylogenetic comparisons, we identified a hotspot for mammalian chromosomal rearrangement just downstream of the Ahsp gene. This hotspot is located at the end of a syntenic block of approximately 350 kb that is conserved in mammals and likely marks the 3′ end of the gene regulatory domain. We focused our initial functional studies on a 7 kb genomic region bounded at the 5′ (centromeric) end of Ahsp by the nearest adjacent gene, an EST expressed in multiple tissues, and at the 3′ (telomeric) end by the rearrangement hotspot. In transient transfection assays, the Ahsp promoter region conferred erythroid-specific expression to a linked reporter gene. In heterologous cells, GATA-1 transactivated the Ahsp promoter in a dose-dependent fashion. To examine GATA-1 binding and its subsequent effects on the Ahsp gene in vivo, we used G1E-ER4 cells, a GATA-1 null erythroblast line that undergoes terminal erythroid maturation after activation of an estradiol-inducible form of GATA-1. We made several findings with regards to the role of GATA-1 in Ahsp gene regulation. First, GATA-1 and its cofactor, Friend of GATA-1 (FOG-1), bind directly to the Ahsp locus at regions that contain conserved GATA consensus motifs and are predicted to be important erythroid regulatory elements by our bioinformatic studies. Second, GATA-1 induces epigenetic changes in chromatin structure that are associated with gene activation, including formation of a DNase I hypersensitive site, hyperacetylation of histones H3 and H4, and methylation of histone H3 lysine-4. Together, these findings begin to establish the DNA region and mechanisms that control Ahsp transcription, allowing for further studies to map the cis elements responsible for population variations in gene expression.

Novel Synthetic Histone Deacetylase Inhibitor (SK-7041) Potently Inhibits Proliferation in Acute Myeloid Leukemia Cell Lines.

Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, and have the ability to regulate gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. To improve the treatment outcome in AML patients, we examined the antiproliferative activity of SK-7041, a novel hybrid synthetic HDACI, in acute myelogenous leukemia (AML) cell lines (KG-1,HL-60 and HEL). This HDACI preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, compared to other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041. SK-7041 effectively induced the time-dependent hyperacetylation of histones, H3 and H4 in AML cell lines. We found that this compound exhibited potent antiproliferative activity against AML cell lines in vitro. The growth inhibitory effect of SK-7041 was related to the induction of apoptosis in AML cell lines. SK-7041(4uM) induced potent cell death, triggering apoptosis in over 60% of cells in 48h by FACS analysis. After 12h of treatment by western analysis, apoptotic cells were increased through activation of caspase-7,-9,-3 and cleaved-PARP (caspase-8 was not affected). Cyclin D1 expression was decreased, and CDK4 expression level was not affected by SK-7041. The level of p21 expression was gradually increased in KG-1 and HL-60 cell lines, whereas treated HEL cell lines showed no change. For further investigation of the genes affected by SK-7041, 20Kb oligonucleotide microarray analysis was performed. The expression of candidate genes, which we selected in chip analysis, was confirmed using real-time PCR. Fifteen genes (RGL1, FYN, CARD9, ABCA7, TNFRSF6B, CASP9, ENPP2, etc.) were consistently up-regulated and twelve genes (PTPN7, CD34, INSIG1, IL16, LHX6, TRIB3, BID, PDCD4, etc.) were down-regulated more than 4-fold by the SK-7041 compound. We suggest that the efficacy of SK-7041 was mainly mediated by the inhibition of proliferation and induction of apoptosis. Taken together, our data suggested that novel HDACI SK-7041 treatment is effective in AML cell lines, and that the further study is warranted.

Histone H4-Lysine 20 Monomethylation Is Increased in Promoter and Coding Regions of Active Genes and Correlates with Hyperacetylation

Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys(4), H3-Lys(9), H3-Lys(27), and H4-Lys(20), the degree of methylation was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys(20) mono- and trim-ethylation and H4 acetylation at induced (beta-major/beta-minor glo-bin), repressed (c-myc), and silent (embryonic beta-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the beta-major and beta-minor promoter and the beta-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys(20). Although activation of the beta-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys(20), thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys(20) mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunoprecipitation results demonstrated that in contrast to trimethylated H4-Lys(20), which was found to inversely correlate with H4 hyper-acetylation, H4-Lys(20) monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state.

Chromatin remodeling of the Th2 cytokine gene loci

Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream of the IL-13 gene, corresponding with the 5′ border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyl transferase complexes including CBP/p300, and RNA polymerase II. As for the IL-5 gene locus, CD28 costimulation selectively enhanced histone hyperacetylation through NF-κB activation and subsequent upregulation of GATA3. Chromatin remodeling of type 2 cytokine gene loci occurs also in developing Tc2 cells. IL-4 production and histone hyperacetylation in IL-4-associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells; however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1 and HDAC2. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus.

Chromatin Remodeling Is a Key Mechanism Underlying Cocaine-Induced Plasticity in Striatum

Given that cocaine induces neuroadaptations through regulation of gene expression, we investigated whether chromatin remodeling at specific gene promoters may be a key mechanism. We show that cocaine induces specific histone modifications at different gene promoters in striatum, a major neural substrate for cocaine's behavioral effects. At the cFos promoter, H4 hyperacetylation is seen within 30 min of a single cocaine injection, whereas no histone modifications were seen with chronic cocaine, consistent with cocaine's ability to induce cFos acutely, but not chronically. In contrast, at the BDNF and Cdk5 promoters, genes that are induced by chronic, but not acute, cocaine, H3 hyperacetylation was observed with chronic cocaine only. DeltaFosB, a cocaine-induced transcription factor, appears to mediate this regulation of the Cdk5 gene. Furthermore, modulating histone deacetylase activity alters locomotor and rewarding responses to cocaine. Thus, chromatin remodeling is an important regulatory mechanism underlying cocaine-induced neural and behavioral plasticity.

Lack of Telomerase Gene Expression in Alternative Lengthening of Telomere Cells Is Associated with Chromatin Remodeling of the hTR and hTERT Gene Promoters

The presence of active telomere maintenance mechanisms in immortal cells allows the bypass of senescence by maintaining telomere length. In most immortal cell lines and tumors, telomere maintenance is attributable to telomerase reactivation. However, a number of immortal cell lines and tumors can achieve telomere maintenance in the absence of detectable telomerase activity by the alternative lengthening of telomere (ALT) mechanism. Epigenetic mechanisms have been implicated in the regulation of telomerase expression. We show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumor cell lines. Lack of expression of hTR and hTERT in ALT cell lines is associated with histone H3 and H4 hypoacetylation and methylation of Lys9 histone H3. Conversely, hTR and hTERT expression in telomerase-positive cell lines is associated with hyperacetylation of H3 and H4 and methylation of Lys4 H3. Methylation of Lys20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells. This may provide an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Treatment of normal and ALT cells with 5-azadeoxycytidine in combination with Trichostatin A caused chromatin remodeling of both promoters and reactivation of hTR and hTERT expression in ALT and normal cell lines. This data establishes a definite link between the chromatin environment of the telomerase gene promoters and transcriptional activity.

Ras-ERK MAPK Cascade Regulates GATA3 Stability and Th2 Differentiation through Ubiquitin-Proteasome Pathway

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity

Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined gamma-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, gamma-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that gamma-H2AX phosphorylation can be used as a predictive marker of radioresponse.

The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation

The present experiments compared the ability of pig oocytes matured either in vivo or in vitro to structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12-14 h after in vitro fertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher for in vivo than for in vitro matured (IVM) oocytes (P < 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN (P < 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% of in vivo matured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturation in vivo (up to germinal vesicle breakdown; GVBD) and then completed the process in vitro, displayed the same PN asymmetry as oocytes matured entirely in vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.

UV irradiation stimulates histone acetylation and chromatin remodeling at a repressed yeast locus

Chromatin immunoprecipitation with anti-acetyl histone H3 (K9 and K14) and anti-acetyl histone H4 (K5, K8, K12, and K16) antibodies shows that Lys-9 and/or Lys-14 of histone H3, but not the relevant sites of histone H4 in nucleosomes at the repressed MFA2 promoter, are hyperacetylated after UV irradiation. This level of histone hyperacetylation diminishes gradually as repair proceeds. Accompanying this, chromatin in the promoter becomes more accessible to restriction enzymes after UV irradiation and returns to the pre-UV state gradually. UV-related histone hyperacetylation and chromatin remodeling in the MFA2 promoter depend on Gcn5p and partially on Swi2p, respectively. Deletion of GCN5, but not of SWI2, impairs repair of DNA damage in the MFA2 promoter. The post-UV histone modifications and chromatin remodeling at the repressed MFA2 promoter do not activate MFA2 transcriptionally, nor do they require damage recognition by Rad4p or Rad14p. Furthermore, we show that UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. These experiments indicate that chromatin at a yeast repressed locus undergoes active change after UV radiation treatment and that failure to achieve histone H3 hyperacetylation impairs the repair of DNA damage.

Targeted Deletion of the Chicken β-Globin Regulatory Elements Reveals a Cooperative Gene Silencing Activity

The chicken beta-globin locus represents a well characterized system to study the role of both proximal and distal regulatory elements in a eukaryotic multigene domain. The function of the chicken beta(A)/epsilon-intergenic enhancer and upstream regulatory elements 5'-HS1 and 5'-HS2 were studied using a gene targeting approach in chicken DT40 cells followed by microcell-mediated chromosome transfer into human erythroleukemia cells (K562). These regulatory elements all repressed expression of the rho- and beta(H)-chicken globin genes in the chromosome transfer assay. No rho- or beta(H)-globin gene expression was detected in K562 cells containing the chicken chromosome without deletions, whereas rho- and beta(H)-mRNA was activated in K562 cells containing chicken chromosomes with deletions of the intergenic enhancers, 5'-HS1 and 5'-HS2. Transcriptional activation of the rho- and beta(H)-globin genes correlated with hyperacetylation of histones H3 and H4, loss of histone H3 lysine 9 methylation, and binding of RNA polymerase II to the gene promoters. Surprisingly, the status of CpG dinucleotide methylation at the promoters did not correlate with the transcriptional status of the genes. Our results using a chromosomal transfer assay demonstrate an identical silencing function for these regulatory elements, which suggests they function as part of a common silencing pathway or complex.

A phase 1 pharmacokinetic (PK) and pharmacodynamic (PD) study of the histone deacetylase (HDAC) inhibitor PXD101 in patients (pts) with advanced solid tumours

3035 BackgroundPXD101 is a novel HDAC inhibitor with potent antiproliferative activity in vitro. PXD101 inhibits tumour growth in vivo in pre-clinical models. We present a phase 1 trial of PXD101 in pts with advanced solid tumours re fractory to standard therapy. Methods Sequential cohorts of 3–6 pts were recruited to this study to determine the maximum tolerated dose of PXD101 administered as a 30 minute IV infusion on days 1 - 5 of a 21 day cycle. The PK profile of PXD101 was calculated by non-compartmental analysis of plasma and urine from 2–4 pts at each dose level. Acetylation of histones extracted from PMN cells was measured by Western blotting. The PK profile of a single oral dose of PXD101 was evaluated in 3 pts. Results 21 pts (median age 58 years, range 39–72); 10M 11F; all ECOG PS ≤ 2) have been treated with a total of 57 cycles of PXD101 (median 2; range 1 to 7) at 5 escalating dose levels: 150mg/m2(4pts), 300 mg/m2(4pts), 600 mg/m2 (6pts), 900 mg/m2 (3pts) and 1200mg/m2 (3pts). No haematological toxicity was observed. Dose limiting toxicity was seen in 3 pts: G3 fatigue (1 pt) at 600 mg/m2, reversible atrial fibrillation (1 pt) at 1200 mg/m2and G3 diarrhoea and lethargy (1pt) at 1200mg/m2. All other adverse effects were ≤ G2. PXD101 displays linear kinetics. Dose-proportional Cmax (range 2.1 - 68.1μg/ml) was reached at the end of the infusion. The elimination half-life of 0.5–1 hr was independent of dose. PXD101 was detected in plasma at low levels after 10 hrs at 1200 mg/m2but undetectable after 8–10 hrs at lower doses. There was no drug accumulation on repeated dosing. Mean oral bioavailability of PXD101 was 33% (range 32–35%). Histone H4 hyperacetylation was observed at the end of each infusion and was sustained for 4–24 hrs in a dose-dependent manner. PXD101 induced p21, p19 and Apaf-1 expression in PMN cells. To date, stable disease has been achieved in 3pts for 2 cycles; 2pts for 4 cycles and 2 pts for 6 cycles. ConclusionsThe novel HDAC inhibitor PXD101 is well tolerated and exhibits dose-dependent PD effects. 1200mg/m2 was the maximally administered dose and dose escalation has ceased. Recruitment to a 1000mg/m2 dose level continues. Further studies of oral PXD101 are planned. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Topotarget Topotarget Topotarget

Design and synthesis of non-hydroxamate histone deacetylase inhibitors: identification of a selective histone acetylating agent

A series of suberoylanilide hydroxamic acid (SAHA)-based non-hydroxamates was designed, synthesized, and evaluated for their histone deacetylase (HDAC) inhibitory activity. Among these, methyl sulfoxide 15 inhibited HDACs in enzyme assays and caused hyperacetylation of histone H4 while not inducing the accumulation of acetylated α-tubulin in HCT116 cells.

Valproic Acid Alters Chromatin Structure by Regulation of Chromatin Modulation Proteins

Histone acetylation and deacetylation are crucial in the regulation of gene expression. Dynamic changes in gene expression may affect chromatin structure and, consequently, the interaction of chromatin with regulatory factors. In this study, the effects of the antiseizure drug valproic acid (VPA) on the expression of genes that regulate the structure of chromatin and the access of macromolecules to the DNA were investigated. Exposure of breast cancer cells to VPA resulted in rapid dose-dependent hyperacetylation of the histones H3 and H4. VPA further induced a depletion of several members of the structural maintenance of chromatin (SMC) proteins, SMC-associated proteins, DNA methyltransferase, and heterochromatin proteins. Down-regulation of these proteins was associated with chromatin decondensation. The observed alterations of chromatin structure correlated with enhanced sensitivity of DNA to nucleases and increased interaction of DNA with intercalating agents. VPA-induced chromatin decondensation led to a sequence-specific potentiation of DNA-damaging agents in cell culture and xenograft models. Modulation of heterochromatin maintenance proteins was not a direct, but a downstream, effect of histone acetylation. The effects on the chromatin structure were reversible upon drug withdrawal, but obligatory for the potentiation of DNA-damaging agents. In addition to their antitumor activity as single agents, the chromatin decondensation induced by histone deacetylase inhibitors may enhance the efficacy of cytotoxic agents that act by targeting DNA. The proposed mechanism of action suggests an effect of drug sequencing on the antitumor activity of these drugs.

MyoD Targets Chromatin Remodeling Complexes to the Myogenin Locus Prior to Forming a Stable DNA-Bound Complex

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.

Neonatal Nutrition and Consequences on Adult Health

After completing this article, readers should be able to: 1. Describe aberrations in adult health that can result from nutrition in the fetal period and in neonatal life. 2. Review the results of studies from human neonates regarding early nutritional interventions designed for “catch-up” growth. 3. Review results from animal studies regarding early nutrition and “programming” for adult metabolic syndrome. 4. Describe some of the potential epigenetic mechanisms that may be responsible for “programming.” 5. Examine methods for preventing growth delays, the consequences of undernutrition, and the metabolic syndrome related to adverse “programming.” One of the traditional goals of neonatal nutrition is to maintain growth within standardized limits. In the preterm infant, growth between the 10th and 90th percentiles of intrauterine rate has been an ideal goal. More recently, maintenance of lean body mass and bone density; prevention of complications such as chronic lung disease, necrotizing enterocolitis, and infection; and optimization of neurodevelopment and adult health through early nutritional programming have become recognized as more meaningful goals than mere somatic growth. To meet these goals, current nutritional practices require thorough scrutiny in terms of quantity and quality (composition) and how they are customized, if not for the individual patient (which would be ideal), at least for subgroups such as those who are small or appropriate for gestational age, sick or healthy, preterm or term, or male or female. Fetal nutrition and development frequently are used as a template for the low-birthweight infant. A large body of literature on the consequences of fetal undernutrition shows that infants born small for gestational age (SGA) not only have short-term morbidity, but long-term morbidity manifested in what has been termed the “metabolic syndrome.” This syndrome includes abdominal obesity, arterial hypertension, and insulin resistance. Other clinical manifestations can be observed, including thyroid dysfunction, hirsutism, ovarian hyperandrogenism and infertility, dyslipidemia (increased …

On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids1

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.

Programmed remodeling of hyperacetylated histone H4 and H3 organization on the SV40 genome during lytic infection

The presence of nucleosomes containing hyperacetylated histone H4 and H3 on the early, late, and promoter regions of the SV40 genome in chromosomes isolated 30 min, 8 h, and 48 h post-infection was determined by chromatin immunoprecipitation (ChIP) analysis with PCR amplification of fragmented SV40 chromatin using two complementary strategies. In chromosomes isolated at 30 min post-infection hyperacetylated H4 was found intermittently in all the three regions with no preference for one region over the other. In contrast, hyperacetylated H3 was organized primarily within the promoter region and occasionally elsewhere. At 8 h post-infection, nucleosomes with both hyperacetylated H4 and H3 were found regularly associated with all three regions of the SV40 genome. Finally, in SV40, chromosomes isolated 48 h post-infection hyperacetylated H4 and H3 were found frequently associated with all regions of the chromosome although hyperacetylated H4 was preferentially associated with the late region. The changing patterns of organization of hyperacetylated histones in SV40 chromosomes during the course of a lytic infection presumably reflects the different biological functions of the SV40 chromatin at each of the time points.