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. 2012;7(8):e43300.
doi: 10.1371/journal.pone.0043300. Epub 2012 Aug 28.

Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation

Stephanie Dumon  1 , David S Walton, Giacomo Volpe, Nicola Wilson, Emilie Dassé, Walter Del Pozzo, Josette-Renee Landry, Bryan Turner, Laura P O'Neill, Berthold Göttgens, Jon Frampton
Affiliations

Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation

Stephanie Dumon et al. PLoS One. 2012.

Abstract

Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Epigenetic regulation of the Itga2b locus at the onset of hematopoiesis.
(A) Schematic diagram of hematopoietic stem cell development. (B) ChIP on chip profiles of histone modifications across the Itga2b locus and CD41 expression in an HSC-like line (HPC7), E11.5 fetal liver cells (FL), EC (MS-1) and ES cells. ChIP assays were performed with anti-H3K9ac and anti-H3K4me3. Fold enrichments are plotted (log2) against genomic position in kilobases (kb). The position of the oligonucleotides spotted on the array (tiling array) and the Itga2b transcript are represented on the upper panels.
Figure 2
Figure 2. Transcriptional activity at the Itga2b locus in CD41- cells.
(A) Cis-element cluster analysis at the Itga2b locus. Vertical lines indicate probabilities that regulatory factors bind to cis-elements at these positions. The overlain curve indicates the overall probability of being within a cluster of cis-elements bound by their factors. (B) RNA polymerase II (PolII) recruitment on the Itga2b locus in endothelial cells (MS1). Antibody against PolII was used to perform ChIP on chip. The fold enrichments are plotted on a logarithmic scale against the position across the Itga2b locus and aligned to the cluster predictive plot. (C) Schematic representation of the alternative Itga2b TSS deduced from 5¢ RACE PCR in MS1 cells. The lower panel indicates the full-length Itga2b exons and the exons cloned by RACE PCR (full sequences available in Figure S3). (D) Cis-element cluster analysis at the Mpl locus. (E) Relative levels of transcription assayed by Q-PCR for Itga2b exons 8–9–10, 11–12 and 29–30 and for Mpl exons 6–7 and 11–12 in MS1 and ES, and HPC7 cells. PCR results were normalized to HPRT and compared to the level measured in HPC7 cells. Error bars reflect standard error or the mean (SEM).
Figure 3
Figure 3. Encode project: RNA-seq and ChIp-seq data from CD41- cells.
(A) Transcription levels assayed by RNA-seq and histone modification (H3K4me3 and H3K9ac) ChIP-seq on ITGA2B, MPL, and GPIBA in ES (H1-hESC), EC (HUVEC) and B lymphocytic (GM12878) cells. Plots are aligned to the transcript representations. CpG Islands are indicated by green boxes.
Figure 4
Figure 4. Generation of mature megakaryocytes through HPC7 in vitro differentiation.
(A) Giemsa staining of HPC7 cells and HPC7-derived megakaryocytes. (B) Differentiating cells were separated by density gradient into populations at different maturation stages which levels of ploidy were assessed by flow cytometry in the presence of propidium iodide. (C) Levels of expression of Itga2b and Mpl were measured in each cell fraction by Q-PCR, normalized to the GAPDH PCR results and standardized to the HPC7 transcript level. Results, confirmed in three different experiments, were used to determine the two-tailed p-values by equal variance t-test. Indications of p values: *<0.05, and **<0.01.
Figure 5
Figure 5. Detection of nuclease hypersensitive sites and transcription factor binding at the Itga2b promoter in CD41+ HPC and megakaryocytes.
(A) DNA content analysis of undifferentiated HPC7 cells and HPC7-derived megakaryocytes. Mature megakaryocytes were separated by density gradient and ploidy was assessed by flow cytometry in the presence of propidium iodide. (B) Detection of nuclease hypersensitives sites. Nuclei from the HPC7 cells, HPC7-derived megakaryocytes, sorted AGM CD41+ cells and FL-derived megakaryocytes were digested using 60 units of DNaseI. The corresponding DNA were analysed by Q-PCR and compared to untreated samples to determine the amount of digestion. (C) Q-PCR analysis of X-Chip material for GATA-2, PU.1 and Fli-1 in HPC7 and megakaryocytes at the HS locations and at two surrounding control locations. Enrichments are given in comparison to the IgG control ChIP. Statistical significance: ***<0.001 and **<0.01. All results were confirmed in three independent experiments.
Figure 6
Figure 6. Histone mark distribution around the Itga2b promoter in EC, ES cells, HPC and mature megakaryocytes.
(A) ChIP was performed using antibodies against H3K9ac, H3K4me3, H3K9me3, H4K8ac and H3K27me3 histone marks. ChiP material were analyzed by Q-PCR and levels of enrichment determined against the control IgG ChIP. (B) Temporal exclusion of the H3K9me3 modification with H3K4me3 and H3K9ac. Sequential ChIP experiments were performed with first a H3K4me3 antibody followed by reChIP using either a H3K9ac or H3K9me3 antibody. The pull down of H3K4me3-associated chromatin was verified by measure of enrichments against the initial input. Maximal enrichment, between +208 and +364 bp from the ATG is represented normalised to the upstream region −1048 to −846 bp (right panel). Equal amount of H3K4me3 ChIP chromatin material were used as inputs for ReChIP experiments with H3K9ac and H3K9me3 specific antibodies. The relative enrichments were determined against the input and normalized against the background as measured across the upstream region −1048 to −846 bp from the ATG. All profiles are representative of 3 independent experiments. Error bars reflect standard error or the mean (SEM).
Figure 7
Figure 7. Jmjd3 up-regulation affects Itga2b and Mpl expression during megakaryopoiesis.
(A) Measure of expression of the histone modifiers Utx, Jmjd3, Ezh2, CBP and p300 in HPC, megakaryocyte progenitors and mature cells. The levels of expression were determined by Q-PCR, normalized to HPRT and the two megakaryocytic populations were standardized to the HPC7 results. (B) JMJD3 silencing in committed cells. HPC7 cells were transfected with Jmjd3 ShRNA or control vector. After 24hours, SCF was removed from the medium and replaced by TPO to promote megakaryocytic differentiation. Puromycin was added at 48hours and cells were harvested at 72hours after transfection for cDNA preparations. The level of expression of Jmjd3, Itga2b, Mpl and Gp1ba were assessed by Q-PCR and standardized to HPRT (primers sequence provided in Table S3). Results were confirmed by 4 experiments. The two-tailed p value was determined by paired t-test. Statistical significance: ***<0.001.
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