ZBTB38 requires an extended N-terminal zinc finger network to read mCpG- and discriminate TpG-containing DNA sequences.

Methylation of cytosine bases in the CpG context (mCpG) is an essential regulatory mechanism cells use to spatially and temporally orchestrate access to genomic regions and mediate transcription. In many diseases, DNA methylation patterns become inappropriately distributed leading to aberrant transcriptional outcomes. Methyl-CpG binding proteins (MBPs) are key epigenetic mediators that selectively recognize mCpG sites, translating these signals into discrete transcriptional responses. ZBTB38 is a zinc finger (ZF) MBP that uniquely harbors two sets of five ZF clusters; each capable of selectively distinguishing mCpG sites. While the cognate DNA sequence and molecular basis for selective mCpG recognition have been defined for the ZBTB38 C-terminal (C-term) ZF domain, the molecular basis for differentiating DNA targets by the N-terminal (N-term) ZF domain remained uncharacterized. Here we report the mCpG-containing consensus sequence for the ZBTB38 N-term ZFs and demonstrate that unlike the other two ZBTB MBP family members ZBTB33 (Kaiso) and ZBTB4, the three shared core ZF domain discriminates against binding to TpG-containing DNA, and that at least one additional N-term ZF is required to stabilize DNA engagement. In addition, we demonstrate that each ZBTB38 ZF domain exhibits preferential target recognition for their respective cognate methylated DNA consensus motif. These findings expand understanding for how ZBTB38 differentially mediates epigenetic-based transcriptional process in normal and disease-state cells by providing new insight into the molecular basis by which the ZBTB38 N-term ZF domain differentiates DNA targets and offering further insight into the interplay between the N- and C-term ZF domains in directing cellular activities.