Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.

RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster. OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo. METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa. CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

RATIONALE: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. OBJECTIVE: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. METHODS AND RESULTS: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. CONCLUSIONS: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes.

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor-ß (TGF-ß). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-ß receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-ß-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.

Conserved NPPB+ Border Zone Switches From MEF2- to AP-1-Driven Gene Program.

BACKGROUND: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb, encoding B-type natriuretic peptide, after infarction. METHODS: Transgenic reporter mice were used to identify the Nppb-positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. RESULTS: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB, ANKRD1, DES, UCHL1, JUN, and FOXP1. Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. CONCLUSIONS: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1-driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress.

The paralogous genes Nppa and Nppb are organized in an evolutionarily conserved cluster and provide a valuable model for studying co-regulation and regulatory landscape organization during heart development and disease. Here, we analyzed the chromatin conformation, epigenetic status and enhancer potential of sequences of the Nppa-Nppb cluster in vivo Our data indicate that the regulatory landscape of the cluster is present within a 60-kb domain centered around Nppb Both promoters and several potential regulatory elements interact with each other in a similar manner in different tissues and developmental stages. The distribution of H3K27ac and the association of Pol2 across the locus changed during cardiac hypertrophy, revealing their potential involvement in stress-mediated gene regulation. Functional analysis of double-reporter transgenic mice revealed that Nppa and Nppb share developmental, but not stress-response, enhancers, responsible for their co-regulation. Moreover, the Nppb promoter was required, but not sufficient, for hypertrophy-induced Nppa expression. In summary, the developmental regulation and stress response of the Nppa-Nppb cluster involve the concerted action of multiple enhancers and epigenetic changes distributed across a structurally rigid regulatory domain.

Epigenetic State Changes Underlie Metabolic Switch in Mouse Post-Infarction Border Zone Cardiomyocytes.

Myocardial infarction causes ventricular muscle loss and formation of scar tissue. The surviving myocardium in the border zone, located adjacent to the infarct, undergoes profound changes in function, structure and composition. How and to what extent these changes of border zone cardiomyocytes are regulated epigenetically is not fully understood. Here, we obtained transcriptomes of PCM-1-sorted mouse cardiomyocyte nuclei of healthy left ventricle and 7 days post myocardial infarction border zone tissue. We validated previously observed downregulation of genes involved in fatty acid metabolism, oxidative phosphorylation and mitochondrial function in border zone-derived cardiomyocytes, and observed a modest induction of genes involved in glycolysis, including Slc2a1 (Glut1) and Pfkp. To gain insight into the underlying epigenetic regulatory mechanisms, we performed H3K27ac profiling of healthy and border zone cardiomyocyte nuclei. We confirmed the switch from Mef2- to AP-1 chromatin association in border zone cardiomyocytes, and observed, in addition, an enrichment of PPAR/RXR binding motifs in the sites with reduced H3K27ac signal. We detected downregulation and accompanying epigenetic state changes at several key PPAR target genes including Ppargc1a (PGC-1α), Cpt2, Ech1, Fabpc3 and Vldrl in border zone cardiomyocytes. These data indicate that changes in epigenetic state and gene regulation underlie the maintained metabolic switch in border zone cardiomyocytes.

Keratosis Follicularis Spinulosa Decalvans is caused by mutations in MBTPS2.

Keratosis Follicularis Spinulosa Decalvans (KFSD) is a rare genetic disorder characterized by development of hyperkeratotic follicular papules on the scalp followed by progressive alopecia of the scalp, eyelashes, and eyebrows. Associated eye findings include photophobia in childhood and corneal dystrophy. Due to the genetic and clinical heterogeneity of similar disorders, a definitive diagnosis of KFSD is often challenging. Toward identification of the causative gene we reanalyzed a large Dutch KFSD family. SNP arrays (1 M) redefined the locus to a 2.9-Mb region at Xp22.12-Xp22.11. Screening of all 14 genes in the candidate region identified MBTPS2 as the candidate gene carrying a c.1523A>G (p.Asn508Ser) missense mutation. The variant was also identified in two unrelated X-linked KFSD families and cosegregated with KFSD in all families. In symptomatic female carriers, skewed X-inactivation of the normal allele matched with increased severity of symptoms. MBTPS2 is required for cleavage of sterol regulatory element-binding proteins (SREBPs). In vitro functional expression studies of the c.1523A>G mutation showed that sterol responsiveness was reduced by half. Other missense mutations in MBTPS2 have recently been identified in patients with IFAP syndrome. We postulate that both phenotypes are in the spectrum of one genetic disorder with a partially overlapping phenotype.

Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction.

Genome-wide association studies have implicated common genomic variants in the gene desert upstream of TBX3 in cardiac conduction velocity. Whether these noncoding variants affect expression of TBX3 or neighboring genes and how they affect cardiac conduction is not understood. Here, we use high-throughput STARR-seq to test the entire 1.3 Mb human and mouse TBX3 locus, including two cardiac conduction-associated variant regions, for regulatory function. We identified multiple accessible and functional regulatory DNA elements that harbor variants affecting their activity. Both variant regions drove gene expression in the cardiac conduction tissue in transgenic reporter mice. Genomic deletion from the mouse genome of one of the regions caused increased cardiac expression of only Tbx3, PR interval shortening and increased QRS duration. Combined, our findings address the mechanistic link between trait-associated variants in the gene desert, TBX3 regulation and cardiac conduction.

Identification and Characterization of a Transcribed Distal Enhancer Involved in Cardiac Kcnh2 Regulation.

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.

A transgenic mouse model for the simultaneous monitoring of ANF and BNP gene activity during heart development and disease.

AIM: The expression of Nppa (ANF) and Nppb (BNP) marks the chamber myocardium in the embryo, and both genes serve as early and accurate markers for hypertrophy and heart failure. Non-invasive visualization of Nppa-Nppb expression in living mice would enable to evaluate the disease state during the course of time in heart disease models. We sought to develop a method to assess the pattern and level of Nppa and Nppb expression within living mice. METHODS AND RESULTS: A modified bacterial artificial chromosome containing a genomic segment spanning the Nppa-Nppb locus was randomly integrated into the mouse genome. Firefly Luciferase was inserted into Nppa and the red fluorescent protein gene Katushka into Nppb. Both reporters precisely recapitulated the spatio-temporal patterns of Nppa and Nppb, respectively. In a hypertrophy model (transverse aortic constriction) and myocardial infarction model (left anterior descending coronary artery occlusion), the non-invasively measured bioluminescent signal from Luciferase correlated with Nppa expression, and the intensity of red fluorescence with levels of the expression of Katushka and Nppb. After myocardial infarction, the border zone of the infarct area was readily identified by an increased intensity of Katushka fluorescence. CONCLUSIONS: A genomic region sufficient to regulate the developmental pattern and stress response of Nppa and Nppb has been defined. The double reporter mice can be used for the functional imaging and investigation of cardiac hypertrophy and myocardial infarction in vivo.