Heart Enhancers: Development and Disease Control at a Distance

Abstract

Bound by lineage-determining transcription factors and signaling effectors, enhancers play essential roles in controlling spatiotemporal gene expression profiles during development, homeostasis and disease. Recent synergistic advances in functional genomic technologies, combined with the developmental biology toolbox, have resulted in unprecedented genome-wide annotation of heart enhancers and their target genes. Starting with early studies of vertebrate heart enhancers and ending with state-of-the-art genome-wide enhancer discovery and testing, we will review how studying heart enhancers in metazoan species has helped inform our understanding of cardiac development and disease.

Keywords: cardiac gene expression; comparative genomics; enhancer; epigenomics and epigenetics; gene regulation; transcription factor (TF).

FIGURE 1

Early examples of validated of cardiac TF-enhancer interactions. The first exons of the cardiac genes are shown in dark blue. Enhancer elements are shown as gray boxes. The AR1 enhancer of mouse Nkx2.5 contains a repressive element in the middle, which is shown in black. Direct activators are listed above the enhancer elements while repressors are shown below. Upstream factors without direct binding evidence are indicated with dotted lines. E1: exon 1. These schematics are generated based on data from these publications: mouse Nkx2.5 (Searcy et al., 1998; Lien et al., 1999, 2002; Liberatore et al., 2002; Brown et al., 2004; Chi et al., 2005; Takeuchi et al., 2005; Chen and Cao, 2009; Clark et al., 2013; Doppler et al., 2014; Quinodoz et al., 2018); Chicken NKX2.5 (Lee et al., 2004); Mouse Gata4 (Rojas et al., 2005; Schachterle et al., 2012); zebrafish gata4 (Heicklen-Klein and Evans, 2004); mouse Gata6 (Molkentin et al., 2000); Chicken GATA6 (He and Burch, 1997; Davis et al., 2001; Adamo et al., 2004); mouse Mef2c (Dodou et al., 2004; Takeuchi et al., 2005; Pane et al., 2018); mouse Hand2 (McFadden et al., 2000).

FIGURE 2

Discovering conserved heart enhancers during early heart development: a case study. (A) Enhancers that are active at different stages of heart development show different evolutionary constraints. In mouse, enhancers that are active in mesoderm progenitors show higher sequence conservation than enhancers active in ESC and E11.5 embryonic hearts. But conservation levels of enhancers that active during the transition of mesoderm progenitors to cardiac progenitors and cardiac progenitors to cardiomyocytes remain less characterized. aCNEs, the accessible chromatin shared between zebrafish and human (or zebrafish and mouse) were identified within the mesoderm to cardiac progenitor transition (Yuan et al., 2018). Schematics generated based on Figure 5 (Nord et al., 2013). (B) Schematics showing sequence homology and shared enhancer signatures for aCNE1 locus across multiple species. aCNE1 was first discovered as an accessible chromatin region specific for an early cardiac progenitor-enriched population in zebrafish. Gray lines indicate the existence of orthologous sequences to aCNE1 in the given species (based on CNEs identified in Hiller et al., 2013). In mouse, aCNE1 regions are co-occupied by multiple cardiac TFs in cardiac cells (based on data from Luna-Zurita et al., 2016; Laurent et al., 2017). Human aCNE1 region shows chromatin accessibility in cardiac progenitor cells (based on data from Paige et al., 2012). The stickleback and the frog icons were created by Milton Tan and Soledad Miranda-Rottmann, respectively, and shared through (http://phylopic.org/) under the following license (https://creativecommons.org/licenses/by/3.0/). (C) Genome browser view of aCNE1 in zebrafish (ZaCNE1) and human (HaCNE1) genome. aCNE1 is located 108 kb upstream of hand2 in the zebrafish genome and 406 kb upstream of HAND2 in the human genome. Yellow boxes highlight the genes flanking aCNE1, indicating the conserved synteny that aCNE1 resides in. ATAC-seq data from Yuan et al. (2018) is plotted for ZaCNE1 and promoter capture Hi-C data from Montefiori et al. (2018) is plotted for HaCNE1. Note that aCNE1 display conserved cardiac-specific activity in both zebrafish (accessibility) and human (interacting with cardiac gene HAND2). ZaCNE1 and HaCNE1 shares an aligned GATA motif, the mutation of which can be used to determine if the activity of aCNE1 depends on this GATA motif. (D) Functional enhancer assays of WT and GATA motif mutated zebrafish and human aCNE1 sequence in zebrafish embryos. Candidate sequences are cloned into an enhancer vector to drive GFP expression. The whole cassette will be chromatinized after injecting into zebrafish embryos. For both ZaCNE1 and HaCNE1, GATA motif mutation leads to decreased enhancer activity compared to the respective WT sequences. This example illustrates that human and zebrafish aCNE1 share conserved activity and regulation despite less than 60% sequence identity. Schematics generated based on data from Yuan et al. (2018). Parts of this figure were created with BioRender.com.