Characterizing Intracellular Localization and Chromatin Remodeling Role of CHD6 in Human Fetal Development and in Glioblastoma

CHD6, a member of the CHD family of DNA-binding, chromatin-modifying proteins, has been shown to be upregulated in glioblastoma. Using immunofluorescence the intracellular region to which CHD6 localizes can be determined. Through the use of chromatin Immunoprecipitation (ChIP), candidate genomic loci enriched for CHD6 can be determined. Immunofluorescence and ChIP results for CHD6 using a current protocol are presented. Notably, immunofluorescence of fetal germinal matrix and adult subventricular zone showed principally cytoplasmic localization of CHD6, contrary to expectation. CHD6 ChIP showed partial enrichment in certain genes of possible significance to glioblastoma in fetal tissue relative to the positive control. A higher sample size is needed to test and evaluate the significance of these findings. The results presented here reveal new insights into the changes in the epigenetic landscape contributing to glioblastoma and the manipulation of CHD6 expression could be considered for treating specific incidents of glioblastoma. Introduction The set of proteins that constitute epigenetic regulators include so-called ‘writers,’ ‘editors’ and ‘readers’ of DNA methylation or histone post-translational modifications5. Whereas ‘writers’ are responsible for de novo modifications to DNA or DNA-bound histone proteins and ‘editors’ are responsible for chemically altering or reverting the modification placed by ‘writers’, ‘readers’ modulate and mediate interaction between proteins and protein complexes, including the transcriptional machinery5. Dysfunction or deregulation of epigenetic ‘writers,’ ‘editors’ or ‘readers,’ is widely implicated in stem cell activation and tumorigenesis5. Among the epigenetic ‘readers’ is a subset of chromodomain-containing proteins, the Chromodomain Helicase DNA-binding, or CHD, family of chromatin modifiers1,4,5,8,12. The CHD proteins are distinguished from other chromodomain-containing chromatin readers by their tandem chromodomains1,4,5,8, which have been shown to interact with methylated lysine residues correlated with either repressed transcription or active transcription (H3K27me3 and H3K4me3)1,4,11,12. Within the larger family of CHD proteins, are three subfamilies distinguished by their varying functional or conserved domains. Class I CHD proteins contain CHD1 and CHD2. The Class I CHD proteins are the best characterized in terms of function and structure4,8,12. Chromatin Immunoprecipitation (ChIP) experiments, which utilize antibodies to selectively precipitate proteins bound to specific genomic regions, have been conducted to elucidate the DNA regions enriched for CHD112. Class II CHD proteins are distinguished by their dual PHD fingers, or plant homeodomains, and are composed of CHD3, CHD4 and CHD51,4,8,12. Class III CHD proteins are composed of CHD6, CHD7, CHD8 and CHD91,4,8,12. They are distinguished from the other two classes by their SANT (Switching-defective protein 3, Adaptor 2, Nuclear-receptor corepressor,