Figure 2. Identification of direct MIXL1 transcriptional targets by ChIP-Sequencing.

(A) Venn diagram 1MIXL-Flag normalized to control-Flag and control-IgG (Set 1), 2MIXL-Flag normalized to control-Flag and control-IgG (Set 2), and 1MIXL-Flag and 2MIXL-Flag combined and normalized to control-Flag and control-IgG (Set 3). A total of 179 peaks shared by the three groups is denoted. (B) Pie chart depicting localization of MIXL1 in the human genome. Peaks were classified according to distance from the nearest transcribed gene using the following criteria: upstream was 5–25 kbp 5′ of the transcription start site, promoter was 0–5 kbp upstream of the transcription start site, body was between the transcription start site and end, TSE was 0–5 kbp downstream of the transcriptional end, downstream was 5–25 kbp downstream of the transcriptional end, and distant peaks were those not allocated to a gene. Note that the majority of peaks (64%) localized to gene promoters. (C) ChIP of five candidate peaks identified by ChIP-Se1. FLAG antibodies were used and ChIP-Seq (EIF1, c-REL, SLC39A13, SMYD5, and ZP3) showed specific MIXL1 binding to both 1MIXL and 2MIXL clones by ChIP normal mouse IgG served as control. Error bars represent standard deviation between triplicates. (D) The most common motif in the ChIP-seq peaks are Zinc-Finger binding sites. Motif1 and Motif2 were the two most statistically significant generated using the Multiple EM for Motif Elicitation [MEME] against the peak regions identified in the ChIP-seq analysis. Both motifs are C/G-heavy regions with similarity to known zinc finger motifs.