Epigenetic Regulatory Factors Research Articles

Identifying Candidate Oocyte Reprogramming Factors Using Cross-Species Global Transcriptional Analysis

There is mounting evidence to suggest that the epigenetic reprogramming capacity of the oocyte is superior to that of the current factor-based reprogramming approaches and that some factor-reprogrammed induced pluripotent stem cells (iPSCs) retain a degree of epigenetic memory that can influence differentiation capacity and may be linked to the observed expression of immunogenicity genes in iPSC derivatives. One hypothesis for this differential reprogramming capacity is the "chromatin loosening/enhanced reprogramming" concept, as previously described by John Gurdon and Ian Wilmut, as well as others, which postulates that the oocyte possesses factors that loosen the somatic cell chromatin structure, providing the epigenetic and transcriptional regulatory factors more ready access to repressed genes and thereby significantly increasing epigenetic reprogramming. However, to empirically test this hypothesis a list of candidate oocyte reprogramming factors (CORFs) must be ascertained that are significantly expressed in metaphase II oocytes. Previous studies have focused on intraspecies or cross-species transcriptional analysis of up to two different species of oocytes. In this study, we have identified eight CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO, and KDM6B) based on unbiased global transcriptional analysis of oocytes from three different species (human, rhesus monkey, and mouse) that both demonstrate significant (p<0.05, FC>3) expression in oocytes of all three species and have well-established roles in loosening/opening up chromatin structure. We also identified an additional 15 CORFs that fit within our proposed "chromatin opening/fate transformative" (COFT) model. These CORFs may be able to augment Shinya Yamanaka's previously identified reprogramming factors (OCT4, SOX2, KLF4, and cMYC) and potentially facilitate the removal of epigenetic memory in iPSCs and/or reduce the expression of immunogenicity genes in iPSC derivatives, and may have applications in future personalized pluripotent stem cell based therapeutics.

Gene expression of epigenetic regulatory factors related to primary silencing mechanism is less susceptible to lower doses of bisphenol A in embryonic hypothalamic cells

DNA methyltransferases (DNMTs) are associated with epigenetic regulation of gene expression, and methyl-CpG binding protein 2 (MECP2) acts as a long-range regulator of methylated genes. We evaluated the effects of bisphenol A (BPA) on embryonic mouse hypothalamic cells, with particular emphasis on the gene expression of Dnmts (Dnmt1, Dnmt3a, and Dnmt3b) and Mecp2 isoforms. In a dose-dependent (0.02-200 µM BPA) 3-hr experiment, real-time reverse transcription polymerase chain reaction revealed that gene expression of both Dnmts and Mecp2_e2 was affected at 200 µM and that of Mecp2_e1 was affected at > 20 µM. These results suggest that gene expression of Dnmts and Mecp2 are less susceptible to lower doses of BPA in developing hypothalamic cells. However, as BPA concentration increases, this agent has the potential to alter gene expression of key players that provide stability and flexibility of epigenetic gene regulation, which could disrupt the normal development of hypothalamic functions.

Genetic and epigenetic factors in regulation of microRNA in colorectal cancers

Studies on miRNA profiling revealed that a large number of them are significantly deregulated in human cancers. The molecular mechanisms of this deregulation are not totally clarified, even if genetics and epigenetics are frequently involved.Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in the human genome. A SNP into miRNA gene might affect the transcription of primary miRNA, its processing and miRNA–mRNA interaction. We investigated the distribution of sequence variants of miR-146a, miR-196a2, miR-499 and miR-149 in colorectal cancer (CRC) and their effect on miRNA expression. Each variant was identified with HRM. For miR-499 we demonstrated a significant reduction of its expression in CRC connected to a specific genotype.To evaluate the epigenetic effects on miRNA genes in CRC, we investigated the influence of DNA methylation on miR-34b, miR-34c and miR-9-1 expression. We aimed to verify the relationship between the methylation status of these miRNA genes and their relative expression in tumor samples. For the quantification of DNA methylation we adopted a method based on Differential High Resolution Melting (D-HRM).

Epigenetic Profiling of Primary DLBCLs Reveals Novel DNA Methylation-Based Clusters and New Underlying Mechanisms of Lymphomagenesis

Abstract 556Epigenetic profiling of primary DLBCLs reveals novel DNA methylation-based clusters and new underlying mechanisms of lymphomagenesis. Nyasha Chambwe, Matthias Kormaksson, Subhajyoti De, Franziska Michor, Nathalie Johnson, David W. Scott, Randy D. Gascoyne, Ari Melnick, Fabien Campagne and Rita Shaknovich. Diffuse Large B Cell Lymphomas (DLBCLs) are a heterogeneous group of diseases from the clinical, molecular and genetic standpoints. While gene expression profiling has identified clinically and biologically relevant DLBCL subtypes, there is still considerable heterogeneity beyond what has been resolved through transcriptional and genetic profiling. It is increasingly clear that lesions in epigenetic regulatory proteins and transcription factors are a hallmark of DLBCL, which suggests that aberrant epigenetic programming is likely to be a significant factor in these tumors. Our recent preliminary data suggests that aberrant DNA methylation is also widespread in DLBCL and contributes to the abnormal expression patterns of GCB and ABC DLBCLs. Moreover recent data in the setting of AML show that DNA methylation profiles delineate disease subtypes not captured through transcriptional or genetic profiling. We hypothesized that DNA methylation profiles would allow us to identify new, biologically significant DLBCL subtypes. We therefore examined the DNA methylation of over 140 patients with DLBCL using the HELP assay covering multiple CpGs at over 14,000 gene loci. We next performed an unbiased (unsupervised) analysis of probesets that display significant variability (n=3,005), using K-means consensus clustering. This procedure identified four robust DLBCL subtypes based on epigenetic profiles. To identify the genes that define these four clusters we next performed supervised analysis of the DLBCL subtypes including the normal counterpart germinal center B-cells as a normal control, using three independent statistical methods. 46 genes defined cluster A, 236 genes defined cluster B, 376 genes defined cluster C and 1271 genes defined cluster D (selected genes displayed change in methylation of at least 30% at BH corrected p-value < 0.05). Each of these epigenetically defined DLBCL subtypes featured aberrant DNA methylation of genes and pathways with potential relevance to pathogenesis. For example cluster A was notable for aberrant DNA methylation of REL, STAT3, CD30; cluster B for aberrant methylation of the TNFa and IFN1 networks; cluster C of IDH2, FOXG1 genes; and cluster D of CDKN2A, ATF3, FOXL3 genes. Other defining characteristics of Cluster D was enrichment for ABC DLBCLs (Fisher exact test, p=0.007) and most remarkably, marked intra-tumor and inter-individual heterogeneity of DNA methylation patterning. This latter feature is suggestive of potential epigenetic clonal complexity and failure to properly control the boundaries of methylated regions of the genome in these tumors. Clusters A and B revealed enrichment for GC features and cluster B had increased expression of MUM1 (Fisher exact test, p=0.007 and p=0.002). Furthermore, we noted that higher expression of DNMT3B and DNMT3L were associated with hypermethylation in DLBCL samples, while higher expression level of AICDA was associated with aberrant hypomethylation in DLBCLs as compared to normal GC B-cells. Higher expression levels of epigenetic modifiers EZH2 and MBD4 was associated with greater heterogeneity of DNA methylation patterning compared to the normal methylation pattern in germinal center B cells. Collectively, the data indicate that DLBCLs are composed of entities defined by specific DNA methylation profiles that only partially overlap with the ABC and GCB classification. The DLBCL subtypes display perturbation of genes likely to play significant biological roles, and aberrant methylation patterning can be traced in part to aberrant expression of epigenetic regulators including DNA methyltransferases, AICDA and EZH2. Disclosures:No relevant conflicts of interest to declare.

Histone Methyltransferase PR-Set7 and Histone Variant H2A.Z, Induced during Hepatocarcinogenesis, Repress the Promoter Activity of the Tumor Marker Gene and the Ras-Induced Colony Formation Activity

Genetic and epigenetic studies are required to understand molecular mechanisms of carcinogenesis and tumorigenesis. Although alterations of DNA methylation and histone modification profiles are observed in cancer cells, the knowledge of the epigenetic regulatory factors involved in carcinogenesis is insufficient. In this study, we showed that the histone variant, H2a.z, and the histone methyltransferase, Pr-set7/Set8/KMT5a, were up-regulated during chemically induced hepatocarcinogenesis. During this process, the glutathione S-transferase placental form (GST-P), which is completely repressed in normal liver, is strongly induced and is therefore an excellent tumor marker. Reporter analysis performed using the regulatory region of the Gst-p gene revealed that H2A.Z and PRSET7 repressed Gst-p promoter activity. The enhancer element responsible for hepatocarcinogenic-specific gene expression was required for repression by H2A.Z, and the negative regulation by PR-SET7 mediated the regulatory element but not the enhancer. Furthermore, we examined the effects of overexpression of H2A.Z and PR-SET7 on the colony formation activity of mouse NIH-3T3 cells grown on a soft agarose medium. Colony formation based on anchoring independent cell growth is a feature of malignant transformed cells. These factors did not exhibit colony formation activity but repression of the ras oncogene induced this activity. PR-SET7 suppressed RAS val12 -mediated colony formation through methyltransferase activity. These results suggest that H2a.z and Pr-set7 that are induced during hepatocarcinogenesis may function as carcinogenesis suppressors.

REST and CoREST Modulate Neuronal Subtype Specification, Maturation and Maintenance

BackgroundThe repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST functions as a modular scaffold for dynamic recruitment of epigenetic regulatory factors including its primary cofactor, the corepressor for element-1-silencing transcription factor (CoREST), to genomic loci that contain the repressor element-1 (RE1) binding motif. While REST was initially believed to silence RE1 containing neuronal genes in neural stem cells (NSCs) and non-neuronal cells, emerging evidence shows an increasingly complex cell type- and developmental stage-specific repertoire of REST target genes and functions that include regulation of neuronal lineage maturation and plasticity.Methodology/Principal FindingsIn this study, we utilized chromatin immunoprecipitation on chip (ChIP-chip) analysis to examine REST and CoREST functions during NSC-mediated specification of cholinergic neurons (CHOLNs), GABAergic neurons (GABANs), glutamatergic neurons (GLUTNs), and medium spiny projection neurons (MSNs). We identified largely distinct but overlapping profiles of REST and CoREST target genes during neuronal subtype specification including a disproportionately high percentage that are exclusive to each neuronal subtype.Conclusions/SignificanceOur findings demonstrate that the differential deployment of REST and CoREST is an important regulatory mechanism that mediates neuronal subtype specification by modulating specific gene networks responsible for inducing and maintaining neuronal subtype identity. Our observations also implicate a broad array of factors in the generation of neuronal diversity including but not limited to those that mediate homeostasis, cell cycle dynamics, cell viability, stress responses and epigenetic regulation.

Genomic (epigenetic) DNA methylation in patients with open‐angle glaucoma

Abstract Purpose DNA methylation occurs by transfer of a methyl group to cytosine residues in the CpG1 islands. The extent of DNA methylation positively correlates with the extent of gene inactivation. The present study was performed to investigate whether there is an altered global DNA methylation in patients with open‐angle glaucoma. Methods This prospectice case control study included 59 patients with primary open‐angle glaucoma (POAG, age: 68 (SD 8) years), 54 patients with secondary open‐angle glaucoma due to pseudoexfoliation syndrome (PEXG, age: 72 (SD 8) years), and 53 patients with cataract as controls (age: 69 (SD 11) years). Total DNA was extracted from frozen EDTA‐blood using QIAmp DNA Blood Mini Kit (Qiagen). Global methylation status [DNA methylation in %, 1‐(HpaII/MspI)] was measured with a modified non‐radioactive assay. Statistics: Mann‐Whitney‐Test (2‐tailed), the results are presented as means (SD), significance level p&lt;0.05. Results There was a significantly elevated genomic DNA methylation (in %) 1‐(HpaII/MspI) in peripheral mononuclear cells in patients with POAG (68%, SD 18; U=868, Z = ‐2.79, p=0.005), but not in PEXG (55%, SD 17; U=1049, Z=‐0.57, p=0.57) when compared with healthy controls (55%, SD 24). Thus, the difference between POAG and PEXG was exactly 13% of methylated HpaII/MspI restriction sites. Conclusion Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have implications for a possible subsequent derangement of epigenetic control in patients with POAG. Further studies including gene‐specific analyses are needed to clarify the differences between POAG and PEXG.

Histone Modification Enzymes Induced during Chemical Hepatocarcinogenesis

Aberrant methylation patterns of genomic DNA are well-studied epigenetic mutations in cancer. Hypermethylation of CpG islands in tumor-suppressor genes promotes oncogenesis and hypomethylation of global genomic DNA affects genomic stability. Cancer is recognized as a genetic and epigenetic disease. However, it is not clear how epigenetic regulatory factors, including histone modification enzymes, chromatin components and other factors are involved in carcinogenesis. To gain insights into the molecular mechanisms mediated by these factors at the early stage of hepatocarcinogenesis and hepatotoxicity induced by chemicals, we investigated gene expression profiles by DNA microarray and Western blot analyses. We prepared RNA and nuclear extracts from livers with hyperplastic nodules expressing Glutathione S-transferase placental form (GST-P) and compared findings with those of normal liver. GST-P is a phase II detoxification enzyme and a well-known tumor marker. We identified several epigenetic regulatory factors that showed dysregulated expression during chemically induced hepatocarcinogenesis. Here I review the characterization and functions of these factors and discuss the mechanisms of tumor marker gene expression during chemical hepatocarcinogenesis.

Epigenetics of autism spectrum disorders

The autism spectrum disorders (ASD) comprise a complex group of behaviorally related disorders that are primarily genetic in origin. Involvement of epigenetic regulatory mechanisms in the pathogenesis of ASD has been suggested by the occurrence of ASD in patients with disorders arising from epigenetic mutations (fragile X syndrome) or that involve key epigenetic regulatory factors (Rett syndrome). Moreover, the most common recurrent cytogenetic abnormalities in ASD involve maternally derived duplications of the imprinted domain on chromosome 15q11-13. Thus, parent of origin effects on sharing and linkage to imprinted regions on chromosomes 15q and 7q suggest that these regions warrant specific examination from an epigenetic perspective, particularly because epigenetic modifications do not change the primary genomic sequence, allowing risk epialleles to evade detection using standard screening strategies. This review examines the potential role of epigenetic factors in the etiology of ASD.

Lowered DNA methyltransferase (DNMT-3b) mRNA expression is associated with genomic DNA hypermethylation in patients with chronic alcoholism

DNA methyltransferases (DNMTs) are involved within the epigenetic control of DNA methylation processes. Recently, it has been shown that the genomic DNA methylation in patients with alcoholism is increased. In the present controlled study we observed a significant decrease of mRNA expression of DNMT-3a and DNMT-3b when comparing alcoholic patients (n = 59) with healthy controls (n = 66): DNMT-3a (t = -2.38, p = 0.019), DNMT-3b (t = -2.65, p = 0.008). No significant differences were seen for DNMT-1 and Mbd-2 (Methyl-CpG-Binding-Domain protein 2) expression. Additionally, we observed a significant negative correlation between DNMT-3b expression and the blood alcohol concentration (r = -0.45, p = 0.003) which might explain the decrease of DNMT-3b mRNA expression in alcoholic patients. Using a multivariate model we observed that the increase (10%) of genomic DNA methylation in patients with alcoholism was significantly associated with their lowered DNMT-3b mRNA expression (multiple linear regression, p = 0.014). Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have important implications for a possible subsequent derangement of epigenetic control in these patients.

Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism

Higher plasma homocysteine concentrations can influence genomic DNA methylation in peripheral blood cells. In the present controlled study we observed a significant increase (10%) of genomic DNA methylation in patients with alcoholism (t = -3.16, df = 158, p = 0.002) which was significantly associated with their elevated homocysteine levels (multiple linear regression, p < 0.001). Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have important implications for a possible subsequent derangement of epigenetic control these patients.

Expression of PRV-1 Decreases Dependency of Cells on Growth Factors for Proliferation.

Polycythemia rubra vera-1 (PRV-1) is a GPI-linked protein expressed on surface of neutrophils. Polycythemia vera and essential thrombocythemia were found to be associated with an increase in the level of PRV-1 mRNA; however, the function of PRV-1 remains unknown. We have studied the functional effect of expression of PRV-1, both in heterologous cell line and in neutrophils. cDNA encoding PRV-1 was subcloned from a leukocyte cDNA library and expressed in Chinese hamster ovary cells (CHO). After seeding equal numbers of CHO cells stably transfected with PRV-1 or empty plasmid, cell count was conducted at different time intervals and under different concentration of fetal bovine serum (FBS) in growth media. The numbers of PRV-1 transfected cells were higher than sham-transfected cells at 24, 48, 96 and 144 hrs after seeding, and this difference was enhanced with cells growing in a medium with a reduced concentration of serum with the maximal effect seen in serum-free medium. The percentage of apoptotic cells was similar between PRV-1 and sham-transfected cells. These results are suggestive of that CHO cells expressing PRV-1 proliferate faster than sham-transfected cells. The difference in proliferation rate was more significant at lower concentrations of serum and was the highest in serum-free medium. An immunoblot analysis with anti-phosphotyrosine antibody on the whole cell lysate demonstrated that the presence of PRV-1 alters cell signaling. A 60kDa protein band that disappeared after removal of serum in growth medium of sham-transfected cells continued to be present in the PRV-1 transfected cell lysate regardless of the presence or absence of FBS in the media. In order to understand the function of PRV-1 in neutrophils, we used the fact that in 85% of individuals PRV-1 is expressed only by a subgroup of neutrophils. We separated PRV-1 expressing neutrophils of an individual from those lacking this protein in the same donor, using anti-PRV-1 monoclonal antibody. We then compared the gene expression profile of the two groups of neutrophil using DNA microarray technique. Expression of PRV-1 was associated with several folds increase in expression of growth factors (fracture callus-1, X 14.9 times; Insulin-like 3, X 6.7; neural proliferation differentiation, and control-1 or NPDC1, X 3.4 times) and post-translational modifying enzyme of cell surface protein (N-acetylase N sulfotransferase-1, X 18.9 times). Under physiologic conditions, transcriptional regulation of the PRV-1 gene limits its expression to only a certain percentage of neutrophils. In order to investigate the role of epigenetic factors in regulation of transcription of the PRV-1 gene, we analyzed methylation of CpG dinucleotide in promoter of the PRV-1 gene in the genomic DNA obtained from neutrophils expressing PRV-1 to those that don't express this protein in the same individual. Expression of PRV-1 was associated with a decrease in methylation of the CpG dinucleotide located 2937 bp before initiation codon (45% methylated in PRV-1 negative neutrophils compared to 30% methylated in PRV positive neutrophils). We are currently studying the methylation pattern of CpG islands inside the PRV-1 gene. Studying the effect of DNA methylation on expression of PRV-1 in physiologic conditions, may shed light to the mechanism of overexpression of PRV-1 in MPD.

The role of RNA interference in heterochromatic silencing.

Soon after its discovery 75 years ago, heterochromatin, a dense chromosomal material, was found to silence genes. But its importance in regulating gene expression was controversial. Long thought to be inert, heterochromatin is now known to give rise to small RNAs, which, by means of RNA interference, direct the modification of proteins and DNA in heterochromatic repeats and transposable elements. Heterochromatin has thus emerged as a key factor in epigenetic regulation of gene expression, chromosome behaviour and evolution.