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A.C. Glembotsky et al.
TREML1 and ITGA2 are novel RUNX1 targets
To investigate whether TREML1 and ITGA2 represent direct RUNX1 targets, we searched for RUNX1 and acti- vating histone mark H3K27Ac enrichment across the entirety of these two genes by ChIP-sequencing in mature MK. Although no significant RUNX1 enrichment was shown in promoter regions, it was detected in intronic regions of both genes (Figure 2A and C). RUNX1 putative binding sites overlapping H3K27Ac were then identified
by in silico analysis. Using ChIP, we confirmed 3-4-fold enrichment for RUNX1 in two RUNX1 sites identified in TREML1 and more than 5-fold enrichment in four sites identified in ITGA2 (Figure 2B and D). To assess whether these sites are functional, we cloned these intragenic reg- ulatory regions (TREML1_RR and ITGA2_RR) upstream of TREML1 and ITGA2 promoters into lentivirus allowing mCherry reporter expression under these promoters (Online Supplementary Figure S2A and B) and tested them in
AB
CD
EF
Figure 2. TREML1 and ITGA2 are novel RUNX1 targets. (A and C) Illustration of chromatin immunoprecipitation (ChIP)-sequencing peaks across TREML1 and ITGA2 genes in primary megakaryocytes (MK) differentiated from CD34+ cells isolated from leukapheresis samples (RUNX1 and H3K27Ac). (B and D) ChIP-polymerase chain reaction (PCR) assays performed in primary MK confirming that RUNX1 directly binds TREML1 and ITGA2 intragenic regions. Genomic location of putative RUNX1 binding sites (RUNX1 BS) based on the University of California Santa Cruz (UCSC) database (CGCh37/hg19). ChIP experiments show RUNX1 binding in both (A) and (B) RUNX1 BS identified in the intragenic region of the TREML1 gene and in (A-D) RUNX1 BS identified in the intragenic region of the ITGA2 gene. RUNX1+23 enhancer was used as a positive control. Amplification of regions without RUNX1 putative BS was performed as a negative control. Data represent Mean±Standard Deviation (SD) of three independent experiments; *P<0.05; **P<0.01. (E and F) Functional analysis of identified RUNX1 BS. Intragenic TREML1 and ITGA2 regula- tory regions were cloned upstream of TREML1 and ITGA2 promoters (RRwt_prom), respectively, in lentivirus allowing mCherry expression under these promoters and also encoding a PGK-GFP cassette, which allows selection of transduced MK. Mutagenesis was performed to delete all RUNX1 BS (RRmut_prom). MK were trans- duced on day 6 of culture and analyzed on day 10. (E) Results for TREML1. (F) Results for ITGA2. An identical gate was set for all conditions for each gene, including cells transduced with lentivirus carrying the promoter alone (right panel) or coupled with the wild-type (left) and mutated (middle) regulatory regions. For TREML1, Cherry expression was assessed in GFP-high CD41+ cells. Representative histograms are shown for one of four independent experiments. Median mCherry fluores- cence intensity (MFI) was calculated relative to that obtained for ITGA2_prom (n=4) and TREML1_prom (n=4) constructs, respectively. Data represent Mean±SD of four independent experiments; *P<0.05; **P<0.01.
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haematologica | 2019; 104(6)