Sam Thiagalingam

Histone Deacetylases: Unique Players in Shaping the Epigenetic Histone Code

Abstract: The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones. This histone code determines the expression status of individual genes dependent upon their localization on the chromatin. The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3. In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3. The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin. Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD‐dependent enzymes that are similar to the yeast SIR2 proteins. Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer. Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy. Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized. Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made. With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

Mad-related genes in the human

Resistance to the growth inhibitory effects of TGF-β is common in human cancers1,2. However, the mechanism(s) by which tumour cells become resistant to TGF-β are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-β family members3–5. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-β-like ligands.

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: A preliminary report

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short‐term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post‐mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post‐mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol–chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein‐1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty‐three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = −5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post‐mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi‐quantitative RT‐PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

Mechanisms underlying losses of heterozygosity in human colorectal cancers

Losses of heterozygosity are the most common molecular genetic alteration observed in human cancers. However, there have been few systematic studies to understand the mechanism(s) responsible for losses of heterozygosity in such tumors. Here we report a detailed investigation of the five chromosomes lost most frequently in human colorectal cancers. A total of 10,216 determinations were made with 88 microsatellite markers, revealing 245 chromosomal loss events. The mechanisms of loss were remarkably chromosome-specific. Some chromosomes displayed complete loss such as that predicted to result from mitotic nondisjunction. However, more than half of the losses were associated with losses of only part of a chromosome rather than a whole chromosome. Surprisingly, these losses were due largely to structural alterations rather than to mitotic recombination, break-induced replication, or gene conversion, suggesting novel mechanisms for the generation of much of the aneuploidy in this common tumor type.

Differential DNA Hypermethylation of Critical Genes Mediates the Stage-Specific Tobacco Smoke-Induced Neoplastic Progression of Lung Cancer

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non–small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fishers exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fishers exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer–associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

Epigenetic dysregulation of HTR2A in the brain of patients with schizophrenia and bipolar disorder

INTRODUCTION HTR2A gene has been the subject of numerous studies in psychiatric genetics because LSD, which resembles serotonin causes psychosis and atypical antipsychotic drugs target the HTR2A receptor. However, evidence for the role of HTR2A polymorphism(s) in schizophrenia (SCZ) and bipolar disorder (BD) has been elusive. We hypothesized that epigenetic dysregulation of HTR2A may be involved in psycho-pathogenesis and analyzed promoter DNA methylome and expression of HTR2A in SCZ, BD and control subjects. METHOD DNA derived from post-mortem brains of patients with SCZ and BD and matched control subjects (each 35) were obtained from the Stanley Medical Research Institute. While bisulfite DNA sequencing was used to screen and quantify cytosine methylation in the HTR2A promoter, corresponding gene expression was analyzed by qRT-PCR. RESULTS We found strong evidence for epigenetic fine-tuning of HTR2A expression. In general, the expression of HTR2A in individuals carrying the C allele of T102C (or G allele of -1438A/G polymorphism) was higher than TT genotype. Interestingly, promoter DNA of HTR2A was hypermethylated at and around the -1438A/G polymorphic site, but was hypomethylated at and around T102C polymorphic site in SCZ and BD compared to the controls. Furthermore, epigenetic down-regulation of HTR2A was associated with early age of disease onset in SCZ and BD. CONCLUSION Epigenetic dysregulation of HTR2A may contribute to SCZ, BD and earlier age of disease onset. Further research is required to delineate the dysregulation of other components of serotoninergic pathway to design new therapeutics based on the downstream effects of serotonin.

Genetics and epigenetics in major psychiatric disorders: dilemmas, achievements, applications, and future scope.

No specific gene has been identified for any major psychiatric disorder, including schizophrenia, in spite of strong evidence supporting a genetic basis for these complex and devastating disorders. There are several likely reasons for this failure, ranging from poor study design with low statistical power to genetic mechanisms such as polygenic inheritance, epigenetic interactions, and pleiotropy. Most study designs currently in use are inadequate to uncover these mechanisms. However, to date, genetic studies have provided some valuable insight into the causes and potential therapies for psychiatric disorders.There is a growing body of evidence suggesting that the understanding of the genetic etiology of psychiatric illnesses, including schizophrenia, will be more successful with integrative approaches considering both genetic and epigenetic factors. For example, several genes including those encoding dopamine receptors (DRD2, DRD3, and DRD4), serotonin receptor 2A (HTR2A) and catechol-O-methyltransferase (COMT) have been implicated in the etiology of schizophrenia and related disorders through meta-analyses and large, multicenter studies. There is also growing evidence for the role of DRD1, NMDA receptor genes (GRIN1, GRIN2A, GRIN2B), brain-derived neurotrophic factor (BDNF), and dopamine transporter (SLC6A3) in both schizophrenia and bipolar disorder. Recent studies have indicated that epigenetic modification of reelin (RELN), BDNF, and the DRD2 promoters confer susceptibility to clinical psychiatric conditions.Pharmacologic therapy of psychiatric disorders will likely be more effective once the molecular pathogenesis is known. For example, the hypoactive alleles of DRD2 and the hyperactive alleles of COMT, which degrade the dopamine in the synaptic cleft, are associated with schizophrenia. It is likely that insufficient dopaminergic transmission in the frontal lobe plays a role in the development of negative symptoms associated with this disorder. Antipsychotic therapies with a partial dopamine D2 receptor agonist effect may be a plausible alternative to current therapies, and would be effective in symptom reduction in psychotic individuals. It is also possible that therapies employing dopamine D1/D2 receptor agonists or COMT inhibitors will be beneficial for patients with negative symptoms in schizophrenia and bipolar disorder. The complex etiology of schizophrenia, and other psychiatric disorders, warrants the consideration of both genetic and epigenetic systems and the careful design of experiments to illumine the genetic mechanisms conferring liability for these disorders and the benefit of existing and new therapies.

Loss of heterozygosity as a predictor to map tumor suppressor genes in cancer: molecular basis of its occurrence.

High frequency of chromosomal deletions elicited as losses of heterozygosity is a hallmark of genomic instability in cancer. Functional losses of tumor suppressor genes caused by loss of heterozygosity at defined regions during clonal selection for growth advantage define the minimally lost regions as their likely locations on chromosomes. Loss of heterozygosity is elicited at the molecular or cytogenetic level as a deletion, a gene conversion, single or double homologous and nonhomologous mitotic recombinations, a translocation, chromosome breakage and loss, chromosomal fusion or telomeric end-to-end fusions, or whole chromosome loss with or without accompanying duplication of the retained chromosome. Because of the high level of specificity, loss of heterozygosity has recently become invaluable as a marker for diagnosis and prognosis of cancer. The molecular defects for the occurrence of loss of heterozygosity are derived from disabled caretaker genes, which protect the integrity of DNA, or chromosome segregator genes, which mediate faithful chromosome disjunction.

Hypomethylation of the serotonin receptor type-2A Gene (HTR2A) at T102C polymorphic site in DNA derived from the saliva of patients with schizophrenia and bipolar disorder.

Several lines of evidence indicate that dysfunction of serotonin signaling and HTR2A receptor are involved in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BD). DNA methylation of HTR2A at T102C polymorphic site influences HTR2A expression and aberrant DNA methylation of HTR2A promoter was reported in postmortem brain of patients with SCZ and BD. Hypothesizing that the brains epigenetic alteration of HTR2A may also exist in peripheral tissues that can be used as a diagnostic/therapeutic biomarker, we analyzed HTR2A promoter DNA methylation in DNA extracted from the saliva of patients with SCZ and BD, and their first degree relatives versus normal controls. Bisulfite sequencing was used to screen DNA methylation status of the HTR2A promoter CpGs and qMSP was used to quantify the degree of cytosine methylation at differentially methylated sites. Most of the cytosines of the HTR2A promoter were unmethylated. However, CpGs of the −1438A/G polymorphism site, −1420 and −1223 were >95% methylated. The CpG at T102C polymorphic site and neighboring CpGs were ∼70% methylated both in the patients and controls. qMSP analysis revealed that the cytosine of the T102C polymorphic site was significantly hypo‐methylated in SCZ, BD, and their first degree relatives compared to the controls. Cytosine methylation of HTR2A at T102C polymorphic site in DNA derived from the saliva can potentially be used as a diagnostic, prognostic, and/or therapeutic biomarker in SCZ and BD. However, these preliminary observations need to be replicated in other populations with a larger sample size to be considered for clinical applications.

Loss of Heterozygosity Patterns Provide Fingerprints for Genetic Heterogeneity in Multistep Cancer Progression of Tobacco Smoke–Induced Non–Small Cell Lung Cancer

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non-small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P >0.05, Fishers exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P <0.05, Fishers exact test) occur at the later stages of non-small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P <0.05, Fishers exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.