A Gene Depleted DNA Methylation Canyon Maintains Hematopoietic Stem Cell Self-Renewal and NPM1c+ Leukemia By Regulating the Gene Expression of HOXA Cluster
Authors: Zhang Xiaotian , Goodell Margaret , Jeong Mira , Gore Haley , Zhou Wanding 
Published: October 2019

Abstract
DNA methylation Canyons (DMC in short, also referred to as DNA methylation Valleys) are long unmethylated regions (UMR) over 3.5kb, in the mammalian genome. DMCs are associated with homeotic genes and can be classified into active DMCs marked by H3K4me3 and repressive DMCs marked by H3K27me3.
We performed high resolution in situ HiC on human hematopoietic stem and progenitor cells (HSPC) and differentiated red blood cell (RBC) progenitors derived from HSPC. We found that DMCs over 7.3kb form significant 3D micro-compartment interactions with each other. These interactions are extremely long range and can occur between two loci separated by 60Mb. Thus, we name these DMCs over 7.3kb as Grand DNA methylation Canyon (GDMC). GDMCs are repressive DMCs and bear the highest level of H3K27me3 in the HSPC compared with the remaining UMRs under 7.3kb. Additionally, we found that the interacting GDMCs is organized by Polycomb mediated long range interaction but not cohesion loop extrusion.
We also found GDMC interactions disappear in differentiated RBC progenitors derived from HSPC. This suggests a function of GDMC interactions in stem cell maintenance. We thus set out to test the function of GDMC interactions in stem cell self-renewal by deleting GDMC loci. We found one GDMC that lacks genes and transcription activity and enhancer activity marked by H3K4me3 and H3K27ac, is interacting with a repressive part (covered by H3K27me3) of the HOXA cluster only in HSPC. This GDMC is thus named "Geneless Canyon"- GLC in short. By deleting GLC, we found that HSPC self-renewal is impaired significantly. Moreover, expression of active HOXA9 and HOXA10 gene adjacent to the repressive part of HOXA cluster also decreased after deletion. When we checked the 3D genomic interactions around the HOXA region after deletion, we found the long range interactions with GLC disappear, and the enhancer interactions with active HOXA cluster gene promoters are also weakened. This suggests that GDMC interactions can act as the scaffold for the enhancer-promoter interactions to maintain active gene expression.In the detailed examination of regulatory elements in GLC, we found that CTCF binding sites are at the boundary of neighboring Lamin associated domain (LAD) and GLC (Figure 1A). The CTCFs are forming cohesion extrusion loops to include the whole LAD region. Deletion of the CTCF sites also result in the loss of HOXA9 and HOXA10 expression as well as the compromise of self-renewal.
Since HOXA9-10 genes are important transcriptional factors for leukemias carrying NPM1c+ and MLL-X mutations. We thus performed CTCF deletions in cell lines with OCI-AML3 and MV4:11 as leukemia cell line models carrying NPM1c+ and MLL-X mutations. We surprisingly found the NPM1c+ cell leukemia cell line display the growth arrest with CTCF deletion, while the MLL-X cell line MV4:11 don't display such effect (Figure 1B). We perform in situ HiC on OCI-AML3 and MV4:11 and found in OCI-AML3 cells GLC forms interaction with HOXA9-HOTTIP regions, while in MV4:11 cells there is no such interaction (Figure 1C).Further examination on epigenomic profiles identified that GLC is activated as super enhancer in OCI-AML3 cells with the loss of Polycomb binding. This indicates that NPM1c+ leukemia may utilize the GLC region as enhancer to boost active gene expression in from HOXA9 to Hottip, with a different mechanism than HSPC. Gene expression analysis after the CTCF deletion further validates that after the CTCF deletion the expression of HOXA9, HOXA10 and HOXA11 is decreased. Interestingly, GLC is also hypermethylated in the OCI_AML3 cells.
Thus, we have discovered an important DNA methylation Canyon that regulates the hematopoietic stem cell self-renewal via the structural organization of HOXA region that act as the scaffold for the enhancer-promoter interaction. This Canyon can also act as a super enhancer to activate the HOXA expression in the NPM1c+ leukemia. This suggests the versatile roles of Polycomb targeted Canyon in normal hematopoiesis and leukemia development.
Figure. 1 Figure. 1.

Disclosures
No relevant conflicts of interest to declare.