Large DNA Methylation Canyons Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. In order to understand how the DNA methylation is connected with nuclear architecture and can vary between cell types and during cell differentiation, we began to explore the 3D architecture of human hematopoietic stem and progenitor cells (HSPCs) by performing in situ Hi-C experiments at 5kb resolution. We found that large (~10kb) DNA methylation canyons can form long loops connecting anchor loci that may be dozens of megabases apart. These canyons also can form interchromosomal links (Fig.1a and 1b). We further confirmed these long-range interactions by performing 3D-FISH using two color fluorescent labeled probes that spanned the HOXA locus loop anchor (green) and the SP8 locus loop anchor (red), which are ~7MB apart (Fig. 1c).

In order to begin to investigate mechanisms that may regulate these long loops and how they relate to commonly studied loops that are mediated by CTCF-extrusion, we examined their properties systematically. Interestingly, the anchors of long loops exhibited minimal enrichment for CTCF (1.04-fold), and, even when CTCF was bound, they did not obey the convergent rule. The data suggest these loops are formed by phase separation of the interacting loci to form a genomic subcompartment, rather than by CTCF-mediated extrusion. Next, we sought to determine whether other features correlated with these long loops. By aligning DNA methylation profiles with the Hi-C data, we observed that anchors often corresponded to regions of very low DNA methylation, and thus sought to analyze the relationship in detail. We found that the anchor position of the long loops had lower average DNA methylation levels than standard loop anchors and very often overlapped with DNA methylation canyons. Canyons are typically decorated with either active or repressive histone marks. We considered whether a particular group of canyons was associated with the long loops. Our findings further indicate that repressed regions marked by the polycomb-mediated histone modification H3K27me3 at DNA methylation canyons generally mediate the formation of canyon loops.

Next, we considered whether the long loops associated with repressive grand canyons that we had annotated in HSPCs were present in other cell types. Using Aggregate Peak Analysis (APA), a computational strategy in which the Hi-C submatrices from the vicinity of multiple putative loops are superimposed, we examined 19 human cell types and 10 murine cell types in which loop-resolution Hi-C maps are available. Interestingly, unlike previously characterized genomic subcompartments, these long-range loops are only present in stem and progenitor cells, but not in differentiated cell types, such as T cells and erythroid progenitors (Fig. 1d).

Further, we identified one particular loop anchor that lay at the anchor of a long loop and contained no apparent genes ("geneless" canyon, or "GLS"). The GLS harboring this anchor is 17 kb long, lies 1.4 Mb upstream of the HOXA1 gene, and forms long loops with a 28 kb grand canyon in the HOXA region. In order to understand the role of the GLS region in hematopoietic stem cells (HSCs), we deleted the GLS in HSPCs using Cas9-mediated editing and assayed the edited cells for their ability to form colonies. Strikingly, after deleting the GLS, the number of colonies and their size was greatly reduced in edited cells compared to control experiments using either random guide RNAs or electroporation only (Fig. 1e). After ex vivo culture, the overwhelming majority of GLS-knock out HSPCs acquired the marker CD38, indicating that they were differentiating. Similarly, HOXA gene expression, an indicator of HSPC function, was greatly diminished after GLS deletion compared to control cells. These data indicate that the GLS identified in our study is functionally associated with maintenance of the HSC state. Overall, our work reveals long-range interactions between H3K27me3-marked DNA methylation canyons comprising a novel microcompartment associated with cellular identity.

Figure 1.

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Figure 1.

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