Rabbit Anti-Human Histone H3 (phospho Ser28) (a.a. 13-62) Polyclonal antibody

Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets. Moreover, reader proteins of trimethyllysine K36 on the histone H3 (H3K36me3) not only bind the methyllysine but also the nucleosomal DNA. Here, we sought to find peptide-based binders of H3K36me3 reader PSIP1, which relies on DNA interactions to tightly bind H3K36me3 modified nucleosomes. We designed several peptides that mimic the nucleosomal context of H3K36me3 recognition by including negatively charged Glu-rich regions. Using a detailed NMR analysis, we find that addition of negative charges boosts binding affinity up to 50-fold while decreasing binding to the trimethyllysine binding pocket. Since screening and selection of compounds for reader domains is typically based solely on affinity measurements due to their lack of enzymatic activity, our case highlights the need to carefully control for the binding mode, in particular for the challenging case of H3K36me3 readers.

Thyroid hormone (T-3) plays pivotal roles in vertebrate development, acting via nuclear T-3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T-3 dependent developmental process, caused us to hypothesize that T-3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T-3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3).Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T-3 response elements within the DNA methyltransferase 3a and Kruppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T-3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T-3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T-3 response element. Our findings show thatT(3) can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes.