High Order Chromatin Structure Regulates Gene Expression in Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation

High order chromatin structure is implicated in multiple developmental processes and disease. However, a global picture of chromosomal looping interaction alterations during stem cell self-renewal and differentiation is lacking. Hematopoietic stem cell (HSCs) and their differentiated progenitors (HSPCs) offer a system in which to examine this. Of the key differentiated lineages, the erythroid lineage undergoes a unique nuclear condensation process during a well-characterized differentiation process which can be induced in vitro from CD34+ HSPCs. Thus erythroid differentiation offers an ideal model system to study differentiation-associated changes in high order chromatin structure.

We have thus generated the in situ Hi-C contact map for human cord blood CD34+ CD38- HSPC (CD34+) and erythroid progenitors undergoing differentiation in vitro at day 7 from CD34+ HSPCs (EryD7). In our 5kb resolution map, we identified over 2000 chromosomal loop interactions in both CD34+ and Day 7 erythroid respectively . The EryD7 sample exhibited higher random intra-chromosomal interactions in comparison with CD34+, presumably due to nuclear condensation.

By comparing the chromosomal loop interactions in the 2 cell types. We identified self-renewal and erythroid differentiation-specific looping patterns in the two cell types. Strikingly, we found that a gene depleted region (GDR) 2MB upstream of the HOXA cluster forms a strong chromosome loop with the HOXA cluster exclusively in the HSPCs (Fig1A). Within this GDR site, we identified two conserved CTCF sites, which are thought to organized chromosome looping. Utilizing the CRISPR-mediated deletion of each of the two CTCF sites, we found that deletion of either site reduce the colony forming ability of CD34+, indicating a loss of stem cell self-renewal. (Fig 1B) Gene expression analysis showed that HOXA9 expression was compromised the CTCF site deletion. These data suggest that the GDR is forming a distant regulatory loop which controls the expression of HOXA9 in HSPCs. Because the GDR is implicated in controlling HOXA9 expression, a key gene in leukemogenesis, we then tested the importance of this looping site in different leukemia cell lines that are dependent on HOXA9. Of those cell lines, we found the deletion of the CTCF sites inhibit the growth of DNMT3A and NPM1 mutated OCI-AML3 and promote the apoptosis. In contrast, growth of the MLL translocation cell line MV 4:11 was not abrogated by their deletion (Fig 1C). As a control cell line which doesn't express HOXA9, HL60 cells were not sensitive to the deletion of the GDR CTCF sites. Together, these data indicate leukemic cells may adopt different strategies to activate HOXA9. MLL translocation leukemias activates HOXA9 by the direct binding of the MLL fusion protein, while the NPM1 mutated leukemia is more likely to utilize the stem cell looping to activate HOXA9 expression.

Among EryD7 specific interactions, we found the β-globin locus specifically forms chromatin loops at Day7 that are not evident in the CD34+ HSPCs. Detailed examination showed that Dnase I hypersensitivity sites HS5 and 3'HS1 both contains CTCF site and form chromosomal loops. Two other loop-forming CTCF sites, both on the telomeric and centromeric side of β-globin locus were also identified. Interestingly, we found a CTCF binding site adjacent to OR52A5 gene which forms a chromosomal loop with HS5 and is not well studied. To test the role of the chromosomal looping in the regulation of hemoglobin gene expression in β-globin locus, we deleted the OR52A5-CTCF site and the 3'HS1 CTCF site in K562 and adult CD34+ HSPCs. We found the deletion of OR52A5-CTCF resulted in a decrease of HBE and increase of HBB expression in K562 cells, which suggest the OR52A5 CTCF also plays a role in regulating hemoglobin gene expression in the β-globin locus (Fig 1D). Furthermore, we found the deletion of 3'HS1 CTCF resulted in a 4-fold increase of HBG2 expression in adult CD34+ HSPC during erythroid differentiation (Fig1 E). Thus this indicating the 3'HS1 and OR52A5-CTCF CTCF sites in β-globin locus are forming loops that regulate the β-globin locus gene expression.

In summary, we have mapped the higher order chromatin structure alterations during stem cell differentiation and identified the critical looping interaction essential for the self-renewal and differentiation specific functions.

Figure 1

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Figure 1

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