Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression Kaixiang Cao,1 Clayton K. Collings,1 Stacy A. Marshall,1 Marc A. Morgan,1 Emily J. Rendleman,1 Lu Wang,1 Christie C. Sze,1 Tianjiao Sun,1 Elizabeth T. Bartom,1 and Ali Shilatifard1,2 1Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA; 2Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in de- velopment, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis- regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyl- transferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions inde- pendently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development. [Keywords: Hox genes; enhancer; COMPASS; SET1A; chromatin structure; epigenetic marks] Supplemental material is available for this article. Received December 15, 2016; revised version accepted April 12, 2017. In mammals, the 39 Hox genes, organized into four clus- Chromatin decondensation coincides with the activation ters on different chromosomes, encode helix–turn–helix sequences of HoxB and HoxD clusters during embryonic DNA-binding transcription factors (TFs) that are critical stem cell (ESC) differentiation and embryonic develop- for specifying the anterior–posterior body plan during an- ment (Chambeyron and Bickmore 2004; Chambeyron imal development (Alexander et al. 2009; Mallo et al. et al. 2005; Morey et al. 2007). The genome architecture 2010). Knockout experiments in mouse models indicate at the HoxD cluster is also dynamically remodeled at dif- that Hox genes are important for the development of the ferent development stages (Noordermeer et al. 2011, hindbrain, axial skeleton, and limbs (Wellik 2007; Zakany 2014). Moreover, multiple regulatory sequences are and Duboule 2007; Alexander et al. 2009). Hox genes in found to be critical for the activation of Hox clusters. each cluster are activated sequentially in both time and Retinoic acid (RA) response elements (RAREs) located space in the developing embryo according to their geno- at the 3′ of HoxA and HoxB clusters are responsible for mic position within the cluster (Kmita and Duboule the activation of 3′ Hox genes triggered by RA, but genet- 2003). Despite being extensively studied, the mechanisms ic studies and single-cell analyses suggest that additional underlying the spatial and temporal colinearity of Hox regulatory sequences are involved (Studer et al. 1994; genes remain mysterious. Popperl et al. 1995; Dupe et al. 1997; Maamar et al. Accumulating evidence demonstrates the impact of 2013). Indeed, a group of regulatory regions located cis-regulation on the activation of Hox gene clusters. © 2017 Cao et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http://creati- online at http://www.genesdev.org/cgi/doi/10.1101/gad.294744.116. vecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 31:787–801 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 787 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Cao et al. >500 kb upstream of the HoxD cluster are necessary for formed RNA sequencing (RNA-seq) in undifferentiated the expression of 5′ HoxD genes in developing mouse and RA-treated ESCs at two different time points. Upon digits and genitals (Montavon et al. 2011; Lonfat et al. RA treatment, genes within the HoxA cluster, including 2014). On the other hand, distal enhancers downstream the lncRNA genes Halr1 and Hotairm1, were highly ele- from the HoxD cluster regulate the expression of 5′ vated, while the expression of Skap2, a neighboring gene HoxD genes in the forelimb (Andrey et al. 2013). Further- of the HoxA cluster, remained unchanged (Fig. 1A). En- more, DNA elements within the Hox gene clusters, hancers harboring RAREs contribute to the activation of which include CTCF-binding sites, have been shown to Hox genes (Studer et al. 1994; Frasch et al. 1995; Morrison serve as topological boundaries separating active and re- et al. 1996); however, deletion of the Hoxa1 proximal en- pressive domains during stem cell differentiation (Naren- hancer with RAREs significantly reduces, but does not si- dra et al. 2015). Nevertheless, distal regulatory elements lence, the expression of Hoxa1 in vivo (Dupe et al. 1997), required for the activation of the 3′ Hox genes are still suggesting the requirement of additional cis-regulatory se- unidentified. quences. To identify novel regulatory elements of HoxA Epigenetic mechanisms are associated with establish- genes, we performed ChIP-seq (chromatin immunoprecip- ing Hox gene expression patterns during embryonic itation [ChIP] combined with high-throughput sequenc- development and stem cell differentiation (Soshnikova ing) of the enhancer-associated histone marks H3K27ac and Duboule 2009). The antagonism between trithorax and H3K4me1 and the active promoter mark H3K4me3 group proteins (TrxG) and polycomb group (PcG) pro- in ESCs treated with RA. In addition to proximal enhanc- teins is known to enforce changes in H3K4me3 and ers of Hoxa1, two putative enhancer regions (referred to H3K27me3 levels at Hox clusters, resulting in activation here as E1 and E2), located in the gene desert between or repression, respectively, during development (Ring- Skap2 and Hoxa1, are enriched with H3K27ac upon RA rose and Paro 2004; Schuettengruber et al. 2007; Piunti treatment (Fig. 1B). These regions are marked initially and Shilatifard 2016). In mammals, the COMPASS (com- by H3K4me1 in undifferentiated ESCs, and, upon RA-in- plex of proteins associated with Set1) family of six pro- duced differentiation, this histone mark spreads to form tein complexes is responsible for the implementation of a broad domain covering the majority of the intergenic re- the vast majority of H3K4 methylation. The MLL1 gion (Fig. 1B). Additionally, moderate levels of H3K4me3 branch of the COMPASS family, which is related to Dro- were found at E1 upon differentiation (Fig. 1B). The E1 re- sophila Trx, is required for proper expression of many gion overlaps with the Halr1 gene; however, unlike the genes within the HoxA and HoxC clusters in mouse em- H3K4me3 peak that mainly covers the transcription start bryonic fibroblasts (MEFs) (Wang et al. 2009). MLL2 site (TSS) region, the H3K27ac-coated E1 is located within depletion in ESCs leads to loss of H3K4me3 at promoters the Halr1 gene body (Supplemental Fig. S1A). These data of bivalently marked genes, which includes the Hox suggest that E1 and E2 may gain enhancer activity upon genes, and at numerous intergenic regions bearing signa- RA induction and can potentially impact transcriptional tures of transcriptional enhancers (Hu et al. 2013b, 2017). activation of HoxA genes. Nevertheless, none of the members of the COMPASS Looping between enhancers and promoters plays an in- family has been demonstrated to participate in the early structive role in transcriptional activation (Deng et al. activation of anterior Hox genes during ESC differentia- 2012, 2014). To identify regions that gain interaction tion to date. with the HoxA cluster during RA-induced differentiation, To understand the relative contributions of cis-regula- we generated chromatin interaction profiles of the Hoxa1 tory sequences and the COMPASS family, we individually promoter in ESCs using circularized chromosome confor- and combinatorially examined the contribution of two mation capture (4C) combined with high-throughput se- shadow enhancers, a long noncoding RNA (lncRNA), quencing (4C-seq). Silent Hoxa1 interacts with the and members of the COMPASS family to early activation previously identified RARE-containing proximal enhanc- of HoxA genes during