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. 2017 Apr 15;31(8):787-801.
doi: 10.1101/gad.294744.116. Epub 2017 May 9.

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 , Ali Shilatifard  1   2
Affiliations

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression

Kaixiang Cao et al. Genes Dev. .

Abstract

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, 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 methyltransferases 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 independently 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: COMPASS; Hox genes; SET1A; chromatin structure; enhancer; epigenetic marks.

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Figures

Figure 1.
Figure 1.
Identification of distal cis-regulatory elements at the HoxA cluster. (A) University of California at Santa Cruz (UCSC) genome browser view of RNA-seq signals of undifferentiated and RA-treated ESCs at two different time points. The HoxA gene cluster is shown. Genes on the Watson strand are labeled in black, and those on the Crick strand are marked in blue. (CPM) Counts per million mapped reads. (B) UCSC genome browser view of H3K4me1, H3K27ac, and H3K4me3 ChIP-seq tracks at the HoxA cluster in undifferentiated and RA-treated ESCs. The centers of potential distal regulatory regions are marked with blue stripes. (E1) Putative enhancer 1; (E2) putative enhancer 2. (C) 4C-seq (circularized chromosome conformation capture [4C] combined with sequencing) analysis with the Hoxa1 promoter as the viewpoint (left) and E1 as the viewpoint (right) in undifferentiated and RA-treated ESCs. (Blue arrow) Increased contact between HoxA-proximal enhancers and the viewpoint. The median and 20th and 80th percentiles of a sliding 5-kb window determine the main trend line. The color-coded scale represents enrichment relative to the maximum median value at a resolution of 12 kb.
Figure 2.
Figure 2.
Redundancy of the putative enhancers on the activation of HoxA genes. (A) UCSC genome browser view of Hoxa1 expression levels in wild-type, E1 knockout, E2 knockout, and double-knockout ESCs treated with RA. The arrow indicates transcription direction. (B) UCSC genome browser view of Hoxa3–Hoxa7 expression levels in wild-type, E1 knockout, E2 knockout, and double-knockout ESCs treated with RA. (C) UCSC genome browser view of Cyp26a1 expression levels in wild-type, E1 knockout, E2 knockout, and double-knockout ESCs treated with RA. (D) Correlation analysis of gene expression levels between wild-type and E1 knockout (top), E2 knockout (middle), and double-knockout (bottom) ESCs treated with RA. Plots were generated based on two biological replicates of RNA-seq experiments from two independent cell clones for each genotype. The X-axis represents the expression level (log2 normalized CPM) of wild-type cells, and the Y-axis represents the expression level of knockout cells. Significantly down-regulated genes (compared with wild type) are labeled in green, and up-regulated ones are labeled in purple. HoxA genes that were changed significantly (adjusted P < 0.01) are marked with red dots, while unchanged ones are marked with black dots. Other unchanged genes are labeled with gray dots. (E) Heat map analysis comparing the expression fold changes of the 39 Hox genes and Halr1 in wild-type ESCs with that in E1 knockout, E2 knockout, and double-knockout cells. The heat map was generated based on four biological replicates of RNA-seq experiments from two independent cell clones for each genotype. All values were normalized to wild-type values at the indicated time points to derive the fold changes.
Figure 3.
Figure 3.
Effects of enhancer deletion on epigenetic marks and the three-dimensional (3D) chromatin architecture. (A) UCSC genome browser view of H3K4me1 ChIP-seq tracks at the HoxA gene cluster in wild-type and double-knockout ESCs. E1 and E2 are marked with blue stripes. (B) UCSC genome browser view of H3K27ac ChIP-seq tracks at the HoxA gene cluster in wild-type and double-knockout ESCs. E1 and E2 are marked with blue stripes. (C) 4C-seq analysis with the Hoxa1 promoter as the viewpoint in wild-type (left) and double-knockout (right) ESCs without (top) or with (bottom) RA treatment. The median and 20th and 80th percentiles of a sliding 5-kb window determine the main trend line. The color-coded scale represents enrichment relative to the maximum median value at a resolution of 12 kb.
Figure 4.
Figure 4.
Inactivation of MLL3 and MLL4 in ESCs has little effect on Hox gene activation. (A,B) UCSC genome browser view of RNA-seq signals and Sanger sequencing results showing successful deletion of the indicated exons (red arrows and rectangles) at Kmt2c (A) and Kmt2d (B) genes, respectively. Genome locations of gRNAs are labeled in blue, and PAM sequences are labeled in green. Red dashed lines represent deleted sequences. (C) Coimmunoprecipitation assay using Rbbp5 antibody and IgG with nuclear extract from wild-type and MLL3 knockout ESCs. Antibodies used for Western blotting are labeled beside each blot. (D) Western blotting assay of wild-type and MLL4ΔSET ESCs with antibodies against MLL4 and Hsp90. (E) Western blotting assay of wild-type, MLL3 knockout, and MLL4ΔSET cell lysates with H3K4me1, H3K4me3, and H3 antibodies. (F) UCSC genome browser view of RNA-seq signals at the Hoxa1 (top) and Hoxa3–a7 (bottom) genes of wild-type, MLL3 knockout, and MLL4ΔSET ESCs upon RA induction. (G) UCSC genome browser view of H3K4me1 ChIP-seq at the HoxA cluster in undifferentiated (top) and RA-treated (bottom) wild-type, MLL3 knockout, and MLL4ΔSET ESCs, respectively.
Figure 5.
Figure 5.
Depletion of SET1A leads to failures in Hox gene activation. (A) Western blotting assay of SET1A (left) and H3K4me3 (right) with control and SET1A-depleted cell lysates, respectively. HSP90 and H3 Western blots served as loading controls, respectively. (B) UCSC genome browser view of RNA-seq signals at the Hoxa1 (left) and Hoxa3–a7 (right) genes of RA-treated wild-type (blue) and double-knockout (pink) ESCs infected with respective nontargeting shRNA (nonTsh) and SET1A shRNA (SET1Ash). (C) Correlation analysis of gene expression levels in SET1Ash- and nonTsh-infected wild-type (top) and double-knockout (bottom) ESCs treated with RA. The X-axis represents the expression level (log2 normalized CPM) of nonTsh-infected cells, and the Y-axis represents the expression level of SET1Ash-infected cells. Significantly changed Hox genes are labeled with red dots. Significantly down-regulated genes (compared with nonTsh-infected cells) are labeled in green, and up-regulated ones are labeled in purple. The numbers of significantly changed genes are shown in each plot. (D–F) UCSC genome browser view of H3K4me3 (D), H3K27ac (E), and H3K4me1 (F) ChIP-seq at Hoxa3–a7 genes in undifferentiated or RA-treated wild-type (left) and double-knockout (right) ESCs infected with respective nonTsh and SET1Ash. Black arrowheads indicate peaks of histone marks altered in SET1A-depleted cells.
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