Figure 3. H3K27me3-unmodified post-replicative chromatin is essential for DNA binding of lineage-determining TFs.

(A) Top, accumulation of post-replicative H3K27me3 leads to decreased recruitment of lineage-determining TFs. Cord blood CD34+ cells were induced toward myeloid differentiation with G-CSF (left) or M-CSF (middle), or erythroid differentiation with EPO (right) for 12 hr. During the induction period cells were left untreated (upper panels) or treated with the GSKJ4 inhibitor of KDMs UTX and JMJD3 (lower panels). DNA was pulse-labeled with EdU for 15 min and chased to 1 hr. CAA was performed between nascent DNA (biotin) and C/EBPα (left), PU.1 (middle) or GATA-1 (right). PLA, red, EdU (biotin), green, DAPI, blue. Lower panel shows PLA signals only. Bottom, quantification of the results of CAA experiments shown above by counting the number of PLA signals per EdU-labeled nuclei in 50 cells/each of the three independent experiments.

(B, C) G-CSF-mobilized CD34+ cells were cultured for 24 h in the presence of the CC100 cytokine cocktail (CTRL), or with EPO only (EPO) or with EPO and GSKJ4 (EPO+GSKJ4). B, Cells were immunostained with antibody to GATA-1 (red). DAPI, blue. Lower panel shows GATA-1 signals only. C, Western blot analysis with GATA1 and Actin antibodies

(D, E) Effect of UTX/JMJD3 inhibition on EPO-induced erythroid differentiation of CD34+ HPCs. Cells were cultured for 72 hr in the presence of the CC100 cytokine cocktail (CTRL), or in the presence of EPO only (EPO) or in the presence of EPO only and GSKJ4 (EPO + GSKJ4). D, real time PCR analysis (performed in triplicate) of GATA-1 target genes in untreated, EPO-, or EPO plus GSKJ4-treated CD34+CD38+ cells; E, erythroid differentiation monitored by flow cytometry. Error bars represent +/− standard deviation. p Values were determined by Student’s T test. ***, p<0.001.