Structural basis of specific DNA binding by the transcription factor ZBTB24

Abstract

ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes—the other three being DNMT3B, CDCA7 and HELLS—that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.

Zbtb24 binding protects promoter activity by antagonizing DNA methylation in mESCs

“…In the absence of a ChIP-seq suitable endogenous Zbtb24 antibody we used Ty1-tagged Zbtb24 to investigate genome-wide binding in mESCs (Figure 2A). Consistent with previous studies (Thompson et al 2018;Ren et al 2019), we found that most Zbtb24 peaks localized to promoters, exons and introns, and some overlapped with intergenic regions (Figure 2A; Table S2). The promoters of Cdca7, Ostc, Rnf187 and Cdc40 were among the strongest peaks (Table S2).…”

“…Intriguingly, promoters with aberrant DNA methylation gain included Zbtb24 targets previously identified in mESCs, such as Cdca7 and Arid5b Ren et al 2019) ( Table S1). We also confirmed ZBTB24-associated promoter hypermethylation by analysis of published Illumina 450K datasets from controls and ICF2 patients (Velasco et al 2018) ( Figure 1I; Figure S2), and again, some of these sites are known ZBTB24 targets in HCT116 cells (Thompson et al 2018).…”

“…Local binding of transcription factors can contribute to maintain hypomethylated states of CGI (Krebs et al 2014). Since Zbtb24 is often found at unmethylated CpG rich promoters (Thompson et al 2018;Ren et al 2019), we considered that promoter methylation gain could be a consequence of loss of Zbtb24 binding. In the absence of a ChIP-seq suitable endogenous Zbtb24 antibody we used Ty1-tagged Zbtb24 to investigate genome-wide binding in mESCs (Figure 2A).…”

“…A combined function of ZBTB24 and DNMT3B has been suggested for mediating some gene body methylation in HCT116 cells (Thompson et al 2018), but the mechanisms by which ZBTB24 influences DNA methylation at other genomic regions remain unclear. ZBTB24 also functions as a transcriptional activator/repressor, and with its C2H2 zinc finger domain, ZBTB24 can bind to DNA in a sequence-specific manner (Thompson et al 2018;Aktar et al 2019;Ren et al 2019).…”

Identification of ZBTB24 protein domains and motifs for heterochromatin localization and transcriptional activation

“…None of the reporter constructs were activated by EGFP–ZBTB24_ΔZFs. A 12‐bp ZBTB24 binding consensus motif [5′‐CT(G/T)NCAGGACCT‐3′] was recently identified by chromatin immunoprecipitation‐sequencing (ChIP‐seq) for both mouse and human ZBTB24 (Ren et al, ; Thompson et al, ). The two regions identified by the reporter assay, respectively, contained a 12‐bp sequence ( −225 CGCTCAGGACCG −214 and −48 CTTGCAGGACCT −37 ) showing complete or partial match with the consensus motif (Figure a).…”