Fig. 2: ZBTB43 is a DNA binding protein with affinity to PPRs.
From: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome
a, Structure of the ZBTB43 protein. Location of the BTB domain and the three ZF domains are indicated. The full-length protein (ZBTB43-FL), its ZF domain (ZBTB43-ZF) and its BTB domain (ZBTB43-BTB) were purified, as shown in the protein gels to the right. Protein purification and protein gel testing was done twice for ZBTB-FL and ZBTB-ZF and once for ZBTB43-BTB. b, Outline of the affinity sequencing experiment. MBP tag was used for the capture. c, ZBTB43 protein has affinity to methylated and unmethylated genomic DNA. Heatmap analysis of MBP-ZBTB43-FL binding is shown to genomic DNA, either DNMT-TKO ES cell DNA16 or DNA fully methylated by SssI bacterial CpG methyltransferase centred at the TKO DNA peaks. Background level binding by MBP is shown on the right. d, Consensus binding sequences of the affinity peaks in TKO determined by RSAT43 (top), significance 4.5e−13, BaMM44 (middle), dataset performance 0.096, motif performance 0.84, and MEME45 (bottom), significance 4.7e−2775. e, Heatmap shows the match between ZBTB43 affinity binding and predicted Z-DNA sites17. f, IGV browser images of selected specific MBP-ZBTB43 peaks are shown in TKO DNA samples at four genomic regions. Control samples show the background of MBP capture. The tracks for transcripts and predicted Z-DNA are displayed at the bottom. The affinity-sequencing results shown represent two independent biological replicates in c, e and f. g, EMSA confirm the binding of ZBTB43-FL or ZBTB43-ZF to the regions detected by affinity sequencing. The FAM-labelled probes (CACG)8, Rps6kl1, and Ago2, marked as a, b, and c, respectively, were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. (h) EMSA confirms the binding of ZBTB43-ZF to the consensus PPR sequences. The FAM-labelled probes (CA)16 and (CACG)8, marked as d and a, respectively, resulted in specific shift; they were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. The BTB domain of ZBTB43 (ZBTB43-BTB) lacked binding activity. Data shown represent three independent experiments in panels g and h.
