ABSTRACT
During development, a small but significant number of CpG islands (CGIs) become methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here, we used genome-wide DNA methylation microarrays to identify epigenetic changes during human embryonic stem cell (hESC) differentiation. We discovered a group of CGIs associated with developmental genes that gain methylation after hESCs differentiate. Conversely, erasure of methylation was observed at the identified CGIs during subsequent reprogramming to induced pluripotent stem cells (iPSCs), further supporting a functional role for the CGI methylation. Both global gene expression profiling and quantitative reverse transcription-PCR (RT-PCR) validation indicated opposing effects of CGI methylation in transcriptional regulation during differentiation, with promoter CGI methylation repressing and 3' CGI methylation activating transcription. By studying diverse human tissues and mouse models, we further confirmed that developmentally programmed 3' CGI methylation confers tissue- and cell-type-specific gene activation in vivo. Importantly, luciferase reporter assays provided evidence that 3' CGI methylation regulates transcriptional activation via a CTCF-dependent enhancer-blocking mechanism. These findings expand the classic view of mammalian CGI methylation as a mechanism for transcriptional silencing and indicate a functional role for 3' CGI methylation in developmental gene regulation.
Subject(s)
CpG Islands , DNA Methylation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Transcriptional Activation , Animals , CCCTC-Binding Factor , Cell Differentiation , Cell Line , DNA/genetics , Epigenesis, Genetic , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Methylation , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Repressor Proteins/metabolismABSTRACT
Caytaxin is a highly-conserved protein, which is encoded by the Atcay/ATCAY gene. Mutations in Atcay/ATCAY have been identified as causative of cerebellar disorders such as the rare hereditary disease Cayman ataxia in humans, generalized dystonia in the dystonic (dt) rat, and marked motor defects in three ataxic mouse lines. While several lines of evidence suggest that Caytaxin plays a critical role in maintaining nervous system processes, the physiological function of Caytaxin has not been fully characterized. In the study presented here, we generated novel specific monoclonal antibodies against full-length Caytaxin to examine endogenous Caytaxin expression in wild type and Atcay mutant mouse lines. Caytaxin protein is absent from brain tissues in the two severely ataxic Atcay(jit) (jittery) and Atcay(swd) (sidewinder) mutant lines, and markedly decreased in the mildly ataxic/dystonic Atcay(ji-hes) (hesitant) line, indicating a correlation between Caytaxin expression and disease severity. As the expression of wild type human Caytaxin in mutant sidewinder and jittery mice rescues the ataxic phenotype, Caytaxin's physiological function appears to be conserved between the human and mouse orthologs. Across multiple species and in several neuronal cell lines Caytaxin is expressed as several protein isoforms, the two largest of which are caused by the usage of conserved methionine translation start sites. The work described in this manuscript presents an initial characterization of the Caytaxin protein and its expression in wild type and several mutant mouse models. Utilizing these animal models of human Cayman Ataxia will now allow an in-depth analysis to elucidate Caytaxin's role in maintaining normal neuronal function.