Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Abstract

Eukaryotic gene expression is regulated by enhancer–promoter interactions but the molecular mechanisms that govern specificity have remained elusive. Genome-wide studies utilizing STARR-seq identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). We hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer–promoter contacts. By evaluating ChIP-seq occupancy of architectural proteins, typical enhancer-associated proteins, and histone modifications, we determine that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, we map the interactions of each enhancer class utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer–promoter interaction specificity.

Evolutionarily Conserved Principles Predict 3D Chromatin Organization

“…Visualization of these domains in Drosophila also requires heatmaps at a smaller genomic scale than in humans due to their differences in size (Figure 2AB). Similar to CTCF loops found in human cells, we also found 458 interaction peaks in Drosophila enriched in various architectural proteins, but unlike in humans, we did not see an enrichment of CTCF at the anchors of these loops (Cubeñas-Potts et al, 2016; Rao et al, 2014). Importantly, these interaction peaks do not occur at domain corners (Figure S2A).…”

“…To gain further insights into the principles controlling the establishment of 3D chromatin organization in D. melanogaster , we combined Hi-C datasets acquired in Kc167 cells to obtain nearly a billion uniquely mapped reads (Cubeñas-Potts et al, 2016). In comparison to the ultra-high resolution Hi-C dataset in humans (Rao et al, 2014), this is equivalent to 12-fold higher contacts at short distances (<10kb) (Figure S1A).…”

“…Identification of Drosophila TADs and domains has been described previously (Cubeñas-Potts et al, 2016; Hou et al, 2012; Ulianov et al, 2016) as were GM12878 TADs and smaller contact domains (Moore et al, 2015; Rao et al, 2014). Hi-C directionality index (DI) was calculated as previously described (Dixon et al, 2012) using the equation: $$\text{italicDI}=false(\frac{B-A}{false|B-Afalse|}false)false(\frac{{(A-E)}^2}{E}+\frac{{(B-E)}^2}{E}false)$$To compensate for the smaller genome and smaller domain structures seen in D. melanogaster , we calculated A and B using interactions more than 5 kb but less than 100 kb from each 250 bp bin throughout the genome.…”

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization

“…Patterns of genome folding have been scrutinized with ever-increasing precision, but the identity and roles of the underlying molecular actors are still poorly understood, limiting our functional understanding of chromosome architecture. While genome organization and molecular actors differ between distant species (Cubeñas-Potts et al, 2016; Dekker and Heard, 2015; Ea et al, 2015), here we focus on mammals.…”

Long-range control of gene expression via RNA-directed DNA methylation

“…In Drosophila , where enhancers have been identified genome-wide, gene expression correlates well with promoter—enhancer contacts [60]. In human cells, Capture Hi-C also found that promoters of active genes participate in chromatin interactions more than inactive genes [61].…”