Kn Harikrishnan

Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.

Deep sequencing reveals increased DNA methylation in chronic rat epilepsy

Epilepsy is a frequent neurological disorder, although onset and progression of seizures remain difficult to predict in affected patients, irrespective of their epileptogenic condition. Previous studies in animal models as well as human epileptic brain tissue revealed a remarkably diverse pattern of gene expression implicating epigenetic changes to contribute to disease progression. Here we mapped for the first time global DNA methylation patterns in chronic epileptic rats and controls. Using methyl-CpG capture associated with massive parallel sequencing (Methyl-Seq) we report the genomic methylation signature of the chronic epileptic state. We observed a predominant increase, rather than loss of DNA methylation in chronic rat epilepsy. Aberrant methylation patterns were inversely correlated with gene expression changes using mRNA sequencing from same animals and tissue specimens. Administration of a ketogenic, high-fat, low-carbohydrate diet attenuated seizure progression and ameliorated DNA methylation mediated changes in gene expression. This is the first report of unsupervised clustering of an epigenetic mark being used in epilepsy research to separate epileptic from non-epileptic animals as well as from animals receiving anti-convulsive dietary treatment. We further discuss the potential impact of epigenetic changes as a pathogenic mechanism of epileptogenesis.

Disparity of histone deacetylase inhibition on repair of radiation-induced DNA damage on euchromatin and constitutive heterochromatin compartments

Epigenetic regulation of chromatin structure is central to the process of DNA repair. A well-characterized epigenetic feature is the dynamic phosphorylation of the histone H2AX (γH2AX) and mobilization of double strand break (DSB) recognition and repair factors to the site. How chromatin structure is altered in response to DNA damage and how such alterations influence DSB repair mechanisms are currently relevant issues. Despite the clear link between histone deacetylases (HDACs) and radiosensitivity, how histone hyperacetylation influence DSB repair remains poorly understood. We have determined the structure of chromatin is a major factor determining radiosensitivity and repair in human cells. Trichostatin A (TSA) enhances radiosensitivity with dose modification factors of 1.2 and 1.9 at 0.2 and 1 μM, respectively. Cells treated with TSA causing hyperacetylation and remodelling on euchromatic alleles coexist with γH2AX accumulation in radiosensitized cells. Formation of γH2AX on heterochromatin was significantly reduced even when cells were treated with TSA, suggesting that chromatin structure and histone hyperacetylation are pronounced features of radiation sensitivity and repair in euchromatic regions.

Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain

BackgroundThe Rett Syndrome (RTT) brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected.ResultsUsing microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (clusterin and cytochrome c oxidase subunit 1) or are involved in synaptic vesicle cycling (dynamin 1). RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP) analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression.ConclusionsThis study has shed more light on the subset of aberrantly expressed genes that result from MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP 2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.

Effect of valproic acid on radiation-induced DNA damage in euchromatic and heterochromatic compartments

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced γH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.

The histone deacetylase inhibitor, trichostatin A, enhances radiation sensitivity and accumulation of gammaH2A.X

Histone deacetylase inhibitors have been shown to induce numerous biologic effects including, altering cell cycle distribution, cytostasis and in certain cases apoptosis. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that histone deacetylase inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent histone deacetylase inhibitor, Trichostatin A, modulates the radiation sensitivity of human erythroleukemic K562 cells. The endpoints we used were clonogenic survival, apoptosis and ?H2AX immunoprecipitations of soluble chromatin. The findings from clonogenic survival assays indicated that incubation with Trichostatin A 24 hours prior to irradiation enhances the radiation sensitivity of K562 cells. The dose modification factors ranged from 1.1 when cells were incubated with 0.1 ?M Trichostatin A to 2.3 at 1 ?M Trichostatin A. Similarly, caspase-3 and caspase-7 assays indicated that Trichostatin A potentiates radiation-induced apoptosis in K562 cells, in a concentration dependent manner. Our results suggest the modulation of radiation effects observed at the lower Trichostatin A concentrations was associated with histone hyperacetylation and changes in phosphorylated ?H2A.X formation on euchromatin. In contrast, at the higher Trichostatin A concentrations mechanisms such as drug-mediated cytotoxicity and G1 cell cycle arrest, contributed to the sensitization effect. More generally, our findings are consistent with those from recent studies and support the development of histone deacetylase inhibitors for use as radiation sensitizers, particularly for targeting radioresistant cancers.

Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex

Reply to “Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex”

Epigenetic Modification of the Norepinephrine Transporter Gene in Postural Tachycardia Syndrome

Objective—The postural tachycardia syndrome (POTS) has multiple symptoms, chief among which are tachycardia, weakness, and recurrent blackouts while standing. Previous research has implicated dysfunction of the norepinephrine transporter. A coding mutation in the norepinephrine transporter gene (SLC6A2) sequence has been reported in 1 family kindred only. The goal of the present study was to further characterize the role and regulation of the SLC6A2 gene in POTS. Methods and Results—Sympathetic nervous system responses to head-up tilt were examined by combining norepinephrine plasma kinetics measurements and muscle sympathetic nerve activity recordings in patients with POTS compared with that in controls. The SLC6A2 gene sequence was investigated in leukocytes from POTS patients and healthy controls using single nucleotide polymorphisms genotyping, bisulphite sequencing, and chromatin immunoprecipitation assays for histone modifications and binding of the transcriptional regulatory complex, methyl-CpG binding protein 2. The expression of norepinephrine transporter was lower in POTS patients compared with healthy volunteers. In the absence of altered SLC6A2 gene sequence or promoter methylation, this reduced expression was directly correlated with chromatin modifications. Conclusion—We propose that chromatin-modifying events associated with SLC6A2 gene suppression may constitute a mechanism of POTS.

Expression analysis of the epigenetic methyltransferases and methyl-CpG binding protein families in the normal B-cell and B-cell chronic lymphocytic leukemia (CLL).

The importance of epigenetic modifications in carcinogenesis has been a source of controversy for some time. There is little doubt that changes in genomic hypermethylation contribute to the silencing of tumour suppressor genes. Furthermore, recent studies have also identified the significance of genomic hypomethylation associated with chromosomal instability and tumourigenesis. One of the most perplexing questions regarding epigenetic modifications and leukemogenesis is the relationship with DNA methyltransferases (DNMT’s). The primary function of the DNMT enzymes is to methylate genomic DNA, whereas the methyl-CpG binding domain proteins (MBD) interpret this methylation signal and regulate gene expression and chromatin behaviour. In this study we analyse these gene families by quantitative real-time PCR to investigate whether expression levels and the B-cell chronic lymphocytic leukemia (B-CLL) phenotype are associated. Furthermore, given the epigenetic crosstalk between genome stability and the histone chromatin code we have analysed eukaryotic histone methyltransferase (Eu-HMTaseI). Surprisingly, we did not observe significant changes in DNMT1 expression in B-CLL cases when compared to normal lymphocytes, regardless of whether we normalise against GAPDH or PCNA as reference standards. Indeed, expression of the maintenance and de novo methylases were independently regulated. Of particular note was the significant down regulation of DNMT3b. Furthermore, we observed a positive correlation between HMTaseI expression levels and stage of leukemia suggesting that changes in the methylation patterns in B-CLL may represent deregulation of the epigenetic repertoire that also include the methylation dependent binding proteins, MBD2 and MeCP2. We envisage changes in the epigenetic program are multifactorial in nature and postulate that the prevalent genomic methylases just one component of a larger epigenetic repertoire.

Alleviating Transcriptional Inhibition of the Norepinephrine Slc6a2 Transporter Gene in Depolarized Neurons

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increased Slc6a2 mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on the Slc6a2 promoter sequence by chromatin immunopurification strategies. Increased Slc6a2 gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on the Slc6a2 promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism of Slc6a2 gene regulation.