Altered Histone Methylation Research Articles

Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning

Undernutrition during pregnancy is implicated in the programming of offspring for the development of obesity and diabetes. We hypothesized that maternal programming causes epigenetic changes in fetal hypothalamic pathways regulating metabolism. This study used sheep to examine the effect of moderate maternal undernutrition (60 d before to 30 d after mating) and twinning to investigate changes in the key metabolic regulators proopiomelanocortin (POMC) and the glucocorticoid receptor (GR) in fetal hypothalami. Methylation of the fetal hypothalamic POMC promoter was reduced in underfed singleton, fed twin, and underfed twin groups (60, 73, and 63% decrease, respectively). This was associated with reduced DNA methyltransferase activity and altered histone methylation and acetylation. Methylation of the hypothalamic GR promoter was decreased in both twin groups and in maternally underfed singleton fetuses (52, 65, and 55% decrease, respectively). This correlated with changes in histone methylation and acetylation and increased GR mRNA expression in the maternally underfed singleton group. Alterations in GR were hypothalamic specific, with no changes in hippocampi. Unaltered levels of OCT4 promoter methylation indicated gene-specific effects. In conclusion, twinning and periconceptional undernutrition are associated with epigenetic changes in fetal hypothalamic POMC and GR genes, potentially resulting in altered energy balance regulation in the offspring.

CS03-04 - Histone methylation landscapes in prefrontal cortex reveal epigenetic risk architectures in autism and schizophrenia

Little is known about the molecular mechanisms governing developmentally regulated changes in gene expression and neuronal function during the maturation of human prefrontal cortex (PFC), including alterations in neuropsychiatric disease. Here, we separate neuronal and non-neuronal chromatin from postmortem PFC across the lifespan and map the genome-wide distribution of histone H3-trimethyl-lysine 4 (H3K4me3), an epigenetic mark associated with transcriptional regulation. Methods Neuronal nuclei from postmortem prefrontal cortex were immunotagged with NeuN antibody, and NeuN+ and NeuN- nuclei sorted separately via fluorescence-activated nuclei sorting, and purified mononuclesomes enriched for H3K4me3 analyzed by massively parallel sequencing. Neuronal H3K4me3 epigenomes were obtained from the PFC of control subjects from late prenatal to old age, and compared to epigenomes of subjects on the autism or schizophrenia spectrum. Results We present evidence for a highly regulated,’pre-programmed’ remodeling of histone methylation landscapes in immature PFC neurons that lasts at least into the early childhood years, involving hundreds of loci and a distinct set of transcription factors. Furthermore, histone methylation alterations in prefrontal neurons of diseased subjects were highly variable. As a group, loci with disease-associated H3K4me3 alterations showed a significant, 2–3 fold enrichment for genes associated with heritable risk for neurodevelopmental disease. We estimate that less than 5% of altogether 711 loci affected in our cohort of 16 autism subjects were related to a copy number variant at that site. Conclusions Taken together, these findings highlight the’epigenetic vulnerability’ of the immature PFC and point to significant overlap between the genetic and epigenetic risk architectures of major psychiatric disease. Acknowledgements Supported by Autism Speaks, the International Mental Health Research Organization, the Brain & Behavior Research Foundation, and the National Institutes of Mental Health (U.S.A.).

CpG Array Analysis of Histone H3 Lysine 4 Trimethylation in Peripheral Blood Mononuclear Cells of Uremia Patients

Studies of the epigenome have attracted little interest in nephrology, especially in uremia. Several lines of evidence have suggested that there are links between genomic DNA hypomethylation and cardiovascular complications in uremia patients. However, to date, our knowledge about the alterations in histone methylation in uremia is unknown. H3K4me3 variations were analyzed in peripheral blood mononuclear cells from 20 uremia patients and 20 healthy subjects, using chromatin immunoprecipitation microarray (ChIP-chip) approach. ChIP-real-time polymerase chain reaction (PCR) was used to validate the microarray results. mRNA expression and DNA methylation status can be further analyzed by quantitative (q) reverse transcription (RT)-PCR and methyl-DNA immunoprecipitation (MeDIP)-qPCR, respectively. Seven hundred twenty-six increased and 218 decreased H3K4me3 genes displaying significant H3K4me3 differences were found in uremia patients compared with healthy subjects. The results of ChIP-real-time PCR coincided well with microarray results. Expression analysis by qRT-PCR revealed positive correlations between mRNA and H3K4me3 levels. Aberrant DNA methylation can also be found on selected positive genes (CNOT1 PLTP EDG1 TCF3 KIR3DL2). In addition, we even found that there is an inverse relationship between H3K4me3 and promoter DNA methylation in uremia patients. Our studies indicate that there are significant alterations of H3K4me3 in uremia patients; these significant H3K4me3 candidates may help to explain the immunological disturbance and high cardiovascular complications in uremia patients. Such novel findings show the significance of H3K4me3 as a potential biomarker or promising target for epigenetic-based uremia therapies.

201 SUPPRESSION OF Suz12 IN BOVINE PREIMPLANTATION EMBRYOS VIA CYTOPLASMIC SMALL INTERFERING RNA INJECTION

The proper removal of gametic epigenetic marks and coordinated reestablishment of the epigenome are critical to mammalian embryonic development. Suz12 is a member of Polycomb repressive complex 2, known to catalyse trimethylation of lysine 27 on histone 3 (H3K27me3; Pasini et al. 2004); Suz12 has also been shown to interact with Suv39h1 for proper trimethylation of H3K9 (de la Cruz et al. 2007). Our objective in this study was to suppress expression of Suz12 via cytoplasmic injection of small interfering RNA (siRNA) targeted at this gene, and to evaluate the effect on embryo development rates. Bovine zygotes were produced in vitro via standard laboratory procedures. Nineteen hours post-fertilization, presumptive zygotes (n = 3979) were divided into 3 treatment groups: noninjected control (CTL), or injected with a fluorescent dextran marker combined with either a nontargeting siRNA (NULL) or an Suz12-targeting siRNA (SUZ). Embryos were cultured in Bovine Evolve (Zenith Biotech, Canada) with 4 mg mL–1 of BSA (Probumin, Millipore, Billerica, MA) and collected at the 4-cell, 8-cell, morula, and blastocyst stages. Ribonucleic acid was isolated with an RNeasy® Mini Kit (Qiagen, Valencia, CA) from 3 replicates of pooled embryos at each stage (4-cell, n = 15; 8-cell, n = 20; morula, n = 10), except for the blastocyst stage [2 samples (n = 10) collected from the CTL and NULL groups, and 1 sample (n = 3) from the SUZ group]. Each RNA sample was reverse-transcribed into cDNA and diluted for use by quantitative real-time PCR. Relative gene expression levels from each sample were calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA), adjusted according to individual PCR efficiencies for each primer pair (R2 > 0.95) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA), to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). Our data indicate that Suz12 expression was suppressed to undetectable levels in SUZ-treated zygotes at all embryo stages analysed. The blastocyst rate of the SUZ group was extremely low (0.88 ± 0.16% SEM) compared with the CTL (19.87 ± 0.36% SEM) and NULL (5.09 ± 0.36% SEM) groups. Morphologically, SUZ morulae appeared fragmented with fewer larger cells than expected, whereas the NULL and CTL morulae seemed to develop normally. We presume the loss of Suz12 expression during this important developmental time is detrimental to embryo morphology and results in a decreased rate of blastocyst formation. Because of this decrease, we were able to collect only 3 SUZ blastocysts from a total of 227 injected. The microinjection procedure also contributed to significant (P < 0.05) decreases in blastocyst rates of the injected groups as compared with the CTL. Future experiments will explore potential alterations in histone methylation, as well as other epigenetic modifiers in bovine preimplantation embryos, to further elucidate the role of the epigenome in early embryonic development.

The leukemogenic AF4–MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures

Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

Epigenetics in plant tissue culture

Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at the molecular level, viz., changes in DNA methylation and alterations of histone methylation or acetylation. Various studies have been carried out with animals, and with plant cells or tissues that have grown in tissue culture but only little work has been done with shoots generated by axillary branching. We present various molecular methods that are being used to measure epigenetic variation. In micropropagated plants mostly differences in DNA methylation have been examined. Epigenetic changes are thought to underlie various well-known tissue-culture phenomena including rejuvenation, habituation, and morphological changes such as flower abnormalities, bushiness, and tumorous outgrowths in, among others, oil palm, gerbera, Zantedeschia and rhododendron.

Histone deacetylase inhibition alters histone methylation associated with heat shock protein 70 promoter modifications in astrocytes and neurons

The mood-stabilizing and anticonvulsant drug valproic acid (VPA) inhibits histone deacetylases (HDACs). The aim of the present study was to determine the effect of HDAC inhibition on overall and target gene promoter-associated histone methylation in rat cortical neurons and astrocytes. We found that VPA and other HDAC inhibitors, including sodium butyrate (SB), trichostatin A (TSA), and the Class I HDAC inhibitors MS-275 and apicidin all increased levels of histone 3 lysine 4 dimethylation and trimethylation (H3K4Me2 and H3K4Me3); these processes are linked to transcriptional activation in rat cortical neurons and astrocytes. VPA, SB, TSA, MS-275, and apicidin also upregulated levels of the neuroprotective heat shock protein 70 (HSP70) in rat astrocytes. Moreover, Class I HDAC inhibition by VPA and MS-275 increased H3K4Me2 levels at the HSP70 promoter in astrocytes and neurons. We also found that VPA treatment facilitated the recruitment of acetyltransferase p300 to the HSP70 promoter and that p300 interacted with the transcription factor NF-Y in astrocytes. Taken together, the results suggest that Class I HDAC inhibition is key to upregulating overall and gene-specific H3K4 methylation in primary neuronal and astrocyte cultures. In addition, VPA-induced activation of the HSP70 promoter in astrocytes appears to involve an increase in H3K4Me2 levels and recruitment of p300. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.

Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML

AbstractDNA hypermethylation of the p15INK4b tumor suppressor gene is commonly observed in acute myeloid leukemia (AML). Repressive histone modifications and their associated binding proteins have been implicated in the regulation of DNA methylation and the transcriptional repression of genes with DNA methylation. We have used high-density chromatin immunoprecipitation-on-chip to determine the histone modifications that normally regulate p15INK4b expression in AML cells and how these marks are altered in cells that have p15INK4b DNA methylation. In AML patient blasts without p15INK4b DNA methylation, a bivalent pattern of active (H3K4me3) and repressive (H3K27me3) modifications exist at the p15INK4b promoter. AML patient blasts with p15INK4b DNA methylation lose H3K4me3 at p15INK4b and become exclusively marked by H3K27me3. H3K27me3, as well as EZH2, extends throughout p14ARF and p16INK4a, indicating that polycomb repression of p15INK4b is a common feature in all AML blasts irrespective of the DNA methylation status of the gene. Reactivation of p15INK4b expression in AML cell lines and patient blasts using 5-aza-2′-deoxycytidine (decitabine) and trichostatin A increased H3K4me3 and maintained H3K27me3 enrichment at p15INK4b. These data indicate that AML cells with p15INK4b DNA methylation have an altered histone methylation pattern compared with unmethylated samples and that these changes are reversible by epigenetic drugs.

Abstract B40: Histone H3 methylation couples DNA damage detection to activation of the Tip60 tumor suppressor

Abstract Cancer is a heterogeneous disease which arises primarily from the accumulation of mutations and deletions in the cells DNA. However, recent work has implicated both DNA methylation and histone modifications in the etiology and progression of cancer. For example, abnormal patterns of histone methylation have been identified in breast cancer, raising the possibility that altered patterns of histone methylation can be exploited both as a biomarker to monitor disease status and as a novel therapeutic target. Here, we report on a novel role for histone methylation in regulating the ability of the Tip60 acetyltransferase to repair DNA double strand breaks (DSBs). Tip60 is a haplo-insufficient tumor suppressor, and loss of heterozygosity at the Tip60 locus and reduced levels of nuclear Tip60 have been detected in both breast and prostate cancer. Tip60 is an acetyltransferase, and can acetylate several DNA repair proteins implicated in breast cancer, including myc, p53 and ATM. Previously, we demonstrated that Tip60 acetylates and activates the ATM protein, a key component of the cells DSB repair mechanism. Here, we demonstrate that methylation of histones creates docking sites for the recruitment of the Tip60 DNA repair complex to sites of DNA damage. Further, interaction between Tip60 and methylated histones serves to specifically activate Tip60'fs acetyltransferase activity at DSBs. Reduction in histone methylation levels compromises Tip60 activation, leading to reduced DNA repair and increased genomic instability. These results indicate that the level and distribution of specific histone methylation marks across the chromatin is a critical determinant of cells ability to repair and survive DNA damage. Thus histone methylation may regulate the genomic stability of cells and their intrinsic sensitivity to agents, including radio- and chemo-therapy, which directly damage DNA. Altered histone methylation associated with breast cancer may therefore contribute to both genomic instability and to resistance to therapy. Finally, since histone methylation is reversible, the enzymes controlling histone methylation represent an exciting new target for therapy in breast cancer. Citation Information: Cancer Res 2009;69(23 Suppl):B40.

Embryonic stem cells lacking the epigenetic regulator Cfp1 are hypersensitive to DNA-damaging agents and exhibit decreased Ape1/Ref-1 protein expression and endonuclease activity

Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1(-/-) ES cells accumulate more DNA damage and exhibit decreased protein expression and endonuclease activity of AP endonuclease (Ape1/Ref-1), an enzyme involved in DNA base excision repair. Expression in CXXC1(-/-) ES cells of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) restores normal Ape1/Ref-1 protein expression and rescues the hypersensitivity to DNA-damaging agents, demonstrating that Cfp1 contains redundant functional domains. Furthermore, retention of either the DNA-binding activity of Cfp1 or interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes is required to restore normal sensitivity of CXXC1(-/-) ES cells to DNA-damaging agents. These results implicate Cfp1 as a regulator of DNA repair processes.

Mandatory fortification with folic acid in the United States appear to have adverse effects on histone methylation in women with pre‐cancer but not in women free of pre‐cancer

The objective of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in histone methylation in cells involved in cervical carcinogenesis. Cervical specimens (free of pre‐cancer or diagnosed with pre‐cancer cervical intraepithelial neoplasia grade 3 [CIN 3]) obtained before (1990‐92) or after mandatory folic acid fortification (2000‐02) were used to examine the degree of histone methylation (H3 Lys‐9) by immunohistochemistry. Age‐ and race‐adjusted mean H3 Lys‐9 score was significantly higher in CIN 3 specimens obtained in the post‐fortification period compared to pre‐fortification period (P < 0.05). In contrast, in specimens obtained from women free of CIN, H3 Lys‐9 scores were significantly lower in post‐fortification specimens compared to pre‐fortification specimens (P < 0.05). Altered histone methylation has emerged as key to DNA methylation‐related gene silencing and this type of gene silencing in cancer is accompanied by an increase in H3 Lys9 methylation. Therefore, higher Lys‐9 methylation in cervical tissues of women diagnosed with CIN 3 in the post‐fortification period compared to pre‐fortification period suggest that fortification may have adverse effects on histone methylation in already initiated cells. Lower Lys‐9 methylation in cervical cells of women free of CIN in the post fortification period compared to pre fortification on the other hand suggest that fortification is likely to protect against the initiation of carcinogenic process in the cervix.Supported by NCI R03 CA 102893

An Epigenetic Clue to Diabetic Vascular Disease

See related article, pages 615–623 The cardiovascular complications associated with diabetes and the closely related metabolic syndrome are of increasing clinical importance throughout the world and especially in the Western hemisphere.1 Although insulin resistance and hyperglycemia are thought to play a pathogenic role during the progression of atherosclerosis, the precise mechanisms involved are not completely clear.2 One factor that has been implicated is enhanced inflammatory signaling leading to the recruitment of monocytes/macrophages to the vessel wall. Evidence suggests that high glucose (HG)-mediated increases in oxidative stress and the formation of AGEs (advanced glycation end products) result in the upregulation of cytokines, cell adhesion molecules, and matrix modifying genes that facilitate lymphocyte activation and invasion.3–6 Many of these genes are regulated by nuclear factor (NF)-κB, which has been shown to be activated under diabetic conditions in several cell types.7,8 Interestingly, in many instances, cells and animals that have been exposed to HG have been shown to maintain a diabetic cardiovascular phenotype even after lengthy exposure to normal glucose levels, resulting in the concept of “metabolic memory.”8–10 In this issue of Circulation Research , Reddy et al have examined this concept more closely in smooth muscle cells (SMCs) isolated from diabetic mice and SMCs subjected to HG.11 Their data suggest that the increase in cytokine expression observed in these cells may be attributable to epigenetic alterations in histone methylation that enhance the activation of these genes. Genomic DNA is organized into a basic structure called a nucleosome that consists of an octamer of histone proteins (dimers of H2A, H2B, H3, and H4) …

Lymphocytes From Patients With Type 1 Diabetes Display a Distinct Profile of Chromatin Histone H3 Lysine 9 Dimethylation

OBJECTIVE—The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications.RESEARCH DESIGN AND METHODS—We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools.RESULTS—A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-β, nuclear factor-κB, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes.CONCLUSIONS—The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes–related biological pathways and also a new understanding of the pathology of type 1 diabetes.

The EAR-motif of the Cys2/His2-type Zinc Finger Protein Zat7 Plays a Key Role in the Defense Response of Arabidopsis to Salinity Stress

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Epigenetics of prostate cancer

Prostate cancer is the most common type of cancer other than skin cancer and the second leading cause of cancer death in men in the United States. Its exact causes are unknown. Several risk factors have been associated with prostate cancer including age, race, family history and diet. Epigenetic mechanisms including DNA methylation and histone modifications are important means of gene regulation and play essential roles in diverse biological and disease processes. Recently, frequent epigenetic aberrations such as DNA hypo- and hypermethylation and altered histone acetylation and methylation have been observed in prostate cancer affecting the expression and function of a large array of genes, leading to tumorigenesis, tumor progression and metastasis. In this chapter, we examined the current literature regarding epigenetic changes in prostate cancer and discuss the clinical potential of cancer epigenetics for the diagnosis and treatment of this disease.

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.

Phenylhexyl isothiocyanate inhibits histone deacetylases and remodels chromatins to induce growth arrest in human leukemia cells

Natural isothiocyanates, present in cruciferous vegetables and synthetic phenylhexyl isothiocyanate (PHI) are chemopreventive agents which act by blocking the initiation of carcinogen-induced tumors in rodents. We have demonstrated that isothiocyanates are also growth regulators, inhibiting cell cycle cdk activity and up-regulating inhibitor p21WAF1 (p21) in cancer cells. The up-stream mechanism to modulate cell cycle progression remained to be elucidated. Here, we have demonstrated that exposure of HL-60 leukemia cells to PHI induced G1 arrest and apoptosis. The hypothesis that PHI inhibits cell growth via chromatin remodeling was investigated. PHI mediates the complex cross talk between chromatin and DNA, and it was demonstrated for the first time as an inhibitor of histone deacetylases (HDAC). Thus, the HDAC activity in PHI-exposed HL-60 cells was reduced. Additionally, PHI reduced the expression of HDAC and increased the level of acetyl transferase p300, in favor of accumulation of acetylated histones. Within hours, global acetylation of histones was enhanced. PHI further mediated selective alterations of histone methylation, with a pattern consistent to the marks of transcription competent chromatins. The relationship between acetylated histones and p21 was examined by chromatin immunoprecipitation (ChIP) assay. Chromatins from cells exposed to PHI contained more p21 DNA in the precipitates of hyperacetylated histones, indicating more accessibility of transcription machinery to the p21 promoter after chromatin unfolding. The cell cycle inhibitors were activated as a result. In contrast to the PHI-induced apoptosis in HL-60 cells, which was mediated by caspase-9 up-regulation and bcl-2 reduction, PHI did not induce significant apoptosis in the mononuclear cells from normal peripheral blood and bone marrow. The results revealed a potential selective effect of isothiocyanates to inhibit the growth of malignant cells.

Hexamethylbisacetamide Remodels the Human Immunodeficiency Virus Type 1 (HIV-1) Promoter and Induces Tat-Independent HIV-1 Expression but Blunts Cell Activation

Hexamethylbisacetamide (HMBA) induces human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected T-cell and monocytoid cell lines. We find that HMBA activation of viral expression is Tat independent but, like Tat, increases the efficiency of elongation of the HIV-1 promoter (long terminal repeat [LTR]) transcripts. Further, exposure to HMBA induces chromatin remodeling at nucleosome 1 (Nuc-1) near the start site of LTR transcription but does so without increasing histone acetylation or altering histone methylation near Nuc-1. Of note, despite enhanced proviral expression, HMBA suppressed HIV infection ex vivo in primary blood mononuclear cell (PBMC) cultures. Treatment with HMBA did not alter expression of the HIV coreceptors, CCR5 and CXCR4, in PBMCs but down-regulated CD4. Finally, HMBA interferes with cell proliferation and activation; it suppressed expression of Ki67 and CD25 and in PBMCs exposed to mitogen. As HMBA has been tested in oncology trials, its unusual properties make it a useful reagent for future studies of HIV promoter regulation and a novel prototype molecule for therapeutics that abort the latent proviral state of chronic HIV infection.

Differentiation-dependent Alterations in Histone Methylation and Chromatin Architecture at the Inducible Chicken Lysozyme Gene

It is now well established that locus-wide chromatin remodeling and dynamic alterations of histone modifications are required for the developmentally regulated activation of tissue-specific genes. However, little is known about the dynamics of these events during cell differentiation and how chromatin of an entire gene locus responds to signal transduction processes. To address this issue we investigated chromatin accessibility, linker histone distribution, and the histone methylation status at the macrophage-specific chicken lysozyme locus and the ubiquitously expressed gas41 locus in multipotent precursor cell lines and BM2 monoblast cells. The latter can be induced to go through macrophage maturation by treatment with phorbol-12-myristate acetate and can be further stimulated with bacterial lipopolysaccharide. We show that expression of the lysozyme gene in undifferentiated monoblasts is low and that a high level of gene expression requires both cell differentiation and lipopolysaccharide stimulation. However, depletion of the linker histone H1 is observed already in lysozyme non-expressing multipotent precursor cells. In undifferentiated monoblasts, the lysozyme regulatory regions are marked by the presence of monomethylated histone H3 lysine 4, which becomes increasingly converted into trimethylated H3 lysine K4 during cell differentiation. We also present evidence for extensive, differentiation-dependent alterations in nuclease accessibility at the lysozyme promoter without alterations of nucleosome and transcription factor occupancy.