MIRA-seq for DNA methylation analysis of CpG islands.

AIM: To develop a reliable method for whole genome analysis of DNA methylation. MATERIALS & METHODS: Genome-scale analysis of DNA methylation includes affinity-based approaches such as enrichment using methyl-CpG-binding proteins. One of these methods, the methylated-CpG island recovery assay (MIRA), is based on the high affinity of the MBD2b-MBD3L1 complex for CpG-methylated DNA. Here we provide a detailed description of MIRA and combine it with next generation sequencing platforms (MIRA-seq). RESULTS: We assessed the performance of MIRA-seq and compared the data with whole genome bisulfite sequencing. CONCLUSION: MIRA-seq is a reliable, genome-scale DNA methylation analysis platform for scoring DNA methylation differences at CpG-rich genomic regions. The method is not limited by primer or probe design and is cost effective.

TGF-ß induces acetylation of chromatin and of Ets-1 to alleviate repression of miR-192 in diabetic nephropathy.

MicroRNAs (miRNAs), such as miR-192, mediate the actions of transforming growth factor-ß1 (TGF-ß) related to the pathogenesis of diabetic kidney diseases. We found that the biphasic induction of miR-192 expression by TGF-ß in mouse renal glomerular mesangial cells initially involved the Smad transcription factors, followed by sustained expression that was promoted by acetylation of the transcription factor Ets-1 and of histone H3 by the acetyltransferase p300, which was activated by the serine and threonine kinase Akt. In mesangial cells from Ets-1-deficient mice or in cells in which Ets-1 was knocked down, basal amounts of miR-192 were higher than those in control cells, but sustained induction of miR-192 by TGF-ß was attenuated. Furthermore, inhibition of Akt or ectopic expression of dominant-negative histone acetyltransferases decreased p300-mediated acetylation and Ets-1 dissociation from the miR-192 promoter and prevented miR-192 expression in response to TGF-ß. Activation of Akt and p300 and acetylation of Ets-1 and histone H3 were increased in glomeruli from diabetic db/db mice compared to nondiabetic db/+ mice, suggesting that this pathway may contribute to diabetic nephropathy. These findings provide insight into the regulation of miRNAs through signaling-mediated changes in transcription factor activity and in epigenetic histone acetylation under normal and disease states.

5-hydroxymethylcytosine and its potential roles in development and cancer.

Only a few years ago it was demonstrated that mammalian DNA contains oxidized forms of 5-methylcytosine (5mC). The base 5-hydroxymethylcytosine (5hmC) is the most abundant of these oxidation products and is referred to as the sixth DNA base. 5hmC is produced from 5mC in an enzymatic pathway involving three 5mC oxidases, Ten-eleven translocation (TET)1, TET2, and TET3. The biological role of 5hmC is still unclear. Current models propose that 5hmC is an intermediate base in an active or passive DNA demethylation process that operates during important reprogramming phases of mammalian development. Tumors originating in various human tissues have strongly depleted levels of 5hmC. Apparently, 5hmC cannot be maintained in proliferating cells. Furthermore, mutations in the TET2 gene are commonly observed in human myeloid malignancies. Since TET proteins and many lysine demethylases require 2-oxoglutarate as a cofactor, aberrations in cofactor biochemical pathways, including mutations in isocitrate dehydrogenase (IDH), may affect levels of 5hmC and 5mC in certain types of tumors, either directly or indirectly. We discuss current data and models of the function of 5hmC in general, with special emphasis on its role in mechanisms of development and cancer.

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine.

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.