Structure of Nascent Chromatin Is Essential for Hematopoietic Lineage Specification

Abstract

The role of chromatin structure in lineage commitment of multipotent hematopoietic progenitors (HPCs) is presently unclear. We show here that CD34⁺ HPCs possess a post-replicative chromatin globally devoid of the repressive histone mark H3K27me3. This H3K27-unmodified chromatin is required for recruitment of lineage-determining transcription factors (TFs) C/EBPα, PU.1, and GATA-1 to DNA just after DNA replication upon cytokine-induced myeloid or erythroid commitment. Blocking DNA replication or increasing H3K27me3 levels prevents recruitment of these TFs to DNA and suppresses cytokine-induced erythroid or myeloid differentiation. However, H3K27me3 is rapidly associated with nascent DNA in more primitive human and murine HPCs. Treatment of these cells with instructive cytokines leads to a significant delay in accumulation of H3K27me3 in nascent chromatin due to activity of the H3K27me3 demethylase UTX. Thus, HPCs utilize special mechanisms of chromatin modification for recruitment of specific TFs to DNA during early stages of lineage specification.

Keywords: DNA replication; H3K27me3; HMTs; KDMs; hematopoietic progenitors; myeloid and erythroid differentiation; nascent DNA; nascent chromatin; transcription factors.

Figure 1. Kinetics of H3K27me3 accumulation following DNA replication in cytokine-treated CD34+ HPCs

(A) Left, accumulation of H3K27me3 on nascent DNA of G-CSF-mobilized CD34+ HPCs. DNA was labeled with EdU for 15 min and chased to 1, 2 and 4 hr. Following conjugation with biotin, CAA was performed between nascent DNA (biotin) and H3K27me3. PLA, red, EdU (biotin), green, DAPI, blue. Lower panels show PLA signals only. Right, quantification of the results of CAA experiments shown to the left by counting the number of PLA signals per EdU-labeled nuclei in 50 cells/each of the three independent experiments. (B) Left, accumulation of post-replicative H3K27me3 during differentiation of CD34+HPCs. Cells were induced for myeloid differentiation with either G-CSF (upper panels) or M-CSF (lower panels) for 0, 6, 12 and 24 hr. DNA was labeled with EdU for 15 min and CAA performed between EdU (biotin) and H3K27me3. PLA, red, EdU (biotin), green, DAPI, blue. Lower panels show PLA signals only. Right, quantification of the results of CAA experiments shown to the left by counting the number of PLA signals per EdU-labeled nuclei in 50 cells/each of the three independent experiments. Error bars represent +/− standard deviation. p Values were determined by ANOVA. ns, non-significant; *, p<0.05.

Figure 2. Kinetics of recruitment of lineage-determining TFs C/EBPα, PU.1 and GATA-1 to DNA

(A) In vitro differentiation of G-CSF-mobilized CD34+ cells. Cells were treated with G-CSF, M-CSF or EPO for the indicated times. Differentiation was assessed by flow cytometry for lineage specific markers. (B) G-CSF-mobilized CD34+ HPCs were induced toward myeloid or erythroid differentiation by treatment with G-CSF, M-CSF, or EPO respectively, for 6, 12 and 24 hr. DNA was pulse-labeled with EdU for 15 min and chased to 1 hr. CAA was performed between nascent DNA (biotin) and C/EBPα, PU.1, or GATA-1. 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. Error bars represent +/− standard deviation. p Values were determined by ANOVA. *, p<0.05.

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.

Figure 4. C/EBPα is recruited to DNA only during post-replicative period

(A) Cord blood CD34+ HPCs were labeled with EdU for 30 min and induced with G-CSF for 24 hr in the absence (Cont) or presence of 2.5 mM thymidine. After release from thymidine block, CAA was performed between nascent DNA (biotin) and C/EBPα at 0, 2 and 4 hr. PLA, red, EdU (biotin), green, DAPI, blue. Lower panel shows PLA signals only. A graph below shows quantification of the results of CAA experiments by counting the number of PLA signals per EdU-labeled nuclei in 50 cells/each of the three independent experiments. (B) Following DNA labeling with EdU for 30 min and induction with G-CSF, CD34+ HPCs were grown for 24 hr in the presence of thymidine. Thymidine block was released for 0, 2 and 4 hr, and cells were labeled for 20 min with BrdU. Cells were double-immunostained for EdU (green) and BrdU (red). Error bars represent +/− standard deviation. p Values were determined by ANOVA. *, p<0.05.

Figure 5. H3K27me3-conatining nascent chromatin undergoes dynamic changes in cytokine-treated primitive HPCs

(A) CD34+ HPCs were labeled with EdU for 15 min. CAA was performed for H3K27me3 antibody. Cells were then immunostained for CD90 (green) and EdU (blue). (B) Induction of CD34+CD38− HPCs with G-CSF (top) or M-CSF (bottom) leads to de-methylation of H3K27me3. Purified CD34+CD38− HPCs were induced with G-CSF (upper panels) or M-CSF (lower panels) for 0, 12 and 24 hr. Cells were labeled with EdU for 15 min. CAA was performed between nascent DNA (biotin) and H3K27me3 antibodies. PLA, red, EdU (biotin), green, DAPI, blue. Lower panel shows PLA signals only. Quantification of the results of CAA experiments is shown under images and was estimated by counting the number of PLA signals per EdU-labeled nuclei in 50 cells/each of the three independent experiments. Error bars represent +/− standard deviation. p Values were determined by ANOVA. *, p<0.05.

Figure 6. Kinetics of H3K27me3 accumulation at nascent DNA of specific genes during erythroid differentiation of CD34+ HPCs

(A) ChIP assays with the H3K27me3 antibody. Uninduced and 24 hr EPO-induced CD34+ cells were labeled with BrdU for 15 min or 15 min and chased to 60 min. Chromatin was immunoprecipitated with H3K27me3 and IgG antibodies. (B) ChIP assays with the BrdU antibody. DNA from the first immunoprecipitation step with H3K27me3 antibody (A) was denatured and immunoprecipitated with the BrdU antibody. Q-PCR was performed at genes repressed before and after EPO induction (GPR85, WNT16, HOXB8, HOXB13) and at a non-repressed control gene (CD11B) (Cui et al., 2009). Percent of input for re-ChIP experiments was calculated using the amount of material eluted from the beads following immunoprecipitation with H3K27me3 as 100% input. Data are represented as mean ± SD of technical replicates of one of the independent experiments. p-values were calculated by a one-way ANOVA with Bonferroni’s post-hoc test. The statistical significance was defined as p<0.05.

Figure 7. Induction of H3K27me3 KDMs UTX and JMJD3 during lineage specification

(A) Kinetics of EZH2 (left panels) panels and UTX (right panels) accumulation during induction of CD34+CD38− HPCs with G-CSF (upper panels) or M-CSF (lower panels). Purified CD34+CD38− HPCs were induced with G-CSF (left) or M-CSF (right) for 0 and 12 hr. Cells were labeled with EdU for 15 min and chased to 1 hr. CAA was performed between nascent DNA (biotin) and EZH2 (upper panels) or UTX (lower panel) antibodies. PLA, red, EdU (biotin), green, DAPI, blue. Lower panel shows PLA signals only. Graphs represent 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. Error bars represent +/− standard deviation. p Values were determined by ANOVA. *, p<0.05. (B) CD34+CD38− or CD34+CD38+ HPCs were uninduced or induced by treatment with G-CSF or M-CSF. Cells were analyzed by Western blotting with antibody to UTX protein. (C) CD34+ HPCs were immunostained with antibodies to UTX (red), CD90 (green, indicated by arrows) and EZH2 (blue). (D) CD34+ HPCs were pulse-labeled with EdU for 1 hr. CAA was performed for UTX and JMJD3 antibodies and EdU (biotin). Cells were then immunostained for CD90 (green, indicated by arrows) and EdU (blue). (E) A model for opening of post-replicative chromatin and recruitment of TFs to nascent DNA upon induction of primitive HPCs with lineage-specific cytokines. Primitive (CD34+CD38− or CD34+CD90+) HPCs exhibit the H3K27me3 repressive mark on post-replicative chromatin; treatment with instructive cytokines induces H3K27me3 demethylase activity (UTX in the model) and expression of lineage-determining TFs. H3K27me3-unmodified post-replicative chromatin allows binding of lineage-determining TFs to nascent DNA. Upon commitment to unilineage differentiation, H3K27me3 accumulates fast on nascent DNA preventing binding of additional TFs thus restricting transcriptional program and differentiation potential of these cells.