Genetic Variation in Enhancers Modifies Cardiomyopathy Gene Expression and Progression

Abstract

Background: Inherited cardiomyopathy associates with a range of phenotypes, mediated by genetic and nongenetic factors. Noninherited cardiomyopathy also displays varying progression and outcomes. Expression of cardiomyopathy genes is under the regulatory control of promoters and enhancers, and human genetic variation in promoters and enhancers may contribute to this variability.

Methods: We superimposed epigenomic profiling from hearts and cardiomyocytes, including promoter-capture chromatin conformation information, to identify enhancers for 2 cardiomyopathy genes, MYH7 and LMNA. Enhancer function was validated in human cardiomyocytes derived from induced pluripotent stem cells. We also conducted a genome-wide search to ascertain genomic variation in enhancers positioned to alter cardiac expression and correlated one of these variants to cardiomyopathy progression using biobank data.

Results: Multiple enhancers were identified and validated for LMNA and MYH7, including a key enhancer that regulates the switch from MYH6 expression to MYH7 expression. Deletion of this enhancer resulted in a dose-dependent increase in MYH6 and faster contractile rate in engineered heart tissues. We searched for genomic variation in enhancer sequences across the genome, with a focus on nucleotide changes that create or interrupt transcription factor binding sites. The sequence variant, rs875908, disrupts a T-Box Transcription Factor 5 binding motif and maps to an enhancer region 2 kilobases from the transcriptional start site of MYH7. Gene editing to remove the enhancer that harbors this variant markedly reduced MYH7 expression in human cardiomyocytes. Using biobank-derived data, rs875908 associated with longitudinal echocardiographic features of cardiomyopathy.

Conclusions: Enhancers regulate cardiomyopathy gene expression, and genomic variation within these enhancer regions associates with cardiomyopathic progression over time. This integrated approach identified noncoding modifiers of cardiomyopathy and is applicable to other cardiac genes.

Keywords: enhancer, polymorphisms; epigenomics; genes, modifier; lamin A/C; myosin heavy chain; tissue engineering.

Figure 1.. Integrated epigenomic analysis identifies candidate enhancer regions for MYH7 and LMNA.

MYH7 encodes β-myosin heavy chain (MHC), the major contractile protein in the human left ventricle; mutations in MYH7 are a leading cause of inherited cardiomyopathy. Mutations in LMNA, which encodes lamin A/C also contribute to inherited cardiomyopathies. We intersected enhancer data from multiple sources to identify regulatory regions around these genes. Candidate enhancer regions are shown in yellow. A. The MYH6/7 genes are in near two clusters of candidate enhancers, highlighted in yellow boxes. B. Integrated epigenomic analysis identified three candidate enhancer clusters at the LMNA locus. The labels on the left indicate the data and cell/tissue source (full source listing is found in Table I in the Supplement). pcHi-C, promoter capture Hi-C. LV, left ventricle. IPSC-CMs, IPSC-derived cardiomyocytes.

Figure 2.. Enhancer activity in IPSC-Derived Cardiomyocytes (IPSC-CMs).

A luciferase reporter assay was used to test for enhancer activity in IPSC-CMs. The position of candidate enhancers is shown along the top in colored boxes. The clusters in Figure 1 were evaluated as smaller regions. A. Regions from 4 of 5 candidate enhancer regions demonstrated activity in IPSC-CMs, with the highest activity for MYH7 C3. B. Five of six candidate enhancer regions for LMNA showed activity in IPSC-CMs, with the highest being LMNA-C5. These data indicate the candidate MYH7 and LMNA enhancers have activity in human cardiomyocyte-like cells. Data is displayed as fold change to a negative control 500bp genomic desert region with mean ±SD. Expression was measured in an average of 32 assays per construct over four independent differentiations. Significance vs negative control determined by nonparametric one-way ANOVA with Dunn’s multiple comparisons correction. *<0.03, **<0.0021, ****<0.0001.

Figure 3.. Deletion of the MYH7-C3 enhancer increases MYH6 and reduces MYH7 mRNA and protein and produces faster contraction and relaxation in engineered heart tissues.

A. Gene editing was used to delete the MYH7-C3 enhancer heterozygously (^+/−) or homozygously (^−/−). MYH6 and MYH7 mRNA expression was assayed by qPCR and showed a dose-dependent increase in MYH6 expression and reduction in MYH7 expression. Therefore, the MYH7-C3 enhancer is required for MYH7 expression (unedited n=16, MYH7 C3^+/− n=8, MYH7 C3^−/− n=9.) B. Deletion of the MYH7-C4 enhancer had little effect, demonstrating a specificity of these findings to MYH7-C3 (unedited n=16, MYH7 C4^+/− n=9, MYH7 C4^−/− n=10.) C. α-MHC and β-MHC protein ratios were quantified using SDS-PAGE. D. Quantification of α-MHC/β-MHC protein ratios demonstrating correlation with the differences seen at the RNA level. E. Representative images of engineered heart tissues (EHTs) containing unedited or MYH7-C3 homozygous deleted IPSC-CMs. F. Average time to peak (top) and average relaxation times (bottom) measurements of EHT contractions containing unedited or MYH7-C3 deleted cells showed a decrease in time to peak contraction and relaxation in MYH7-C3 deleted EHTs, consistent with the shift from MYH7/β-MHC to MYH6/α-MHC and the known faster ATPase cycle for α-MHC. Each point represents the average time to peak measurement of a single EHT across multiple contractions (unedited n=14, MYH7 C3^+/− n=3, MYH7 C3^−/− n=7.) All data shown as mean ±SD. * determined by one-way ANOVA with Dunnett’s multiple comparisons correction. *<0.03, **<0.0021, ***<0.0002.

Figure 4.. Genomic variation in MYH7 enhancer regions.

A. We queried the human genetic variation database gnomAD for naturally occurring human sequence variants in MYH7 enhancers. We selected variants overlapping cardiac transcription factor binding motifs, and/or correlating with MYH7 expression in the GTEx eQTL dataset. rs7403916 and rs373958405 fall within MYH7 C2 and disrupt NKX2.5 motifs. These variants were evaluated for reporter activity in IPSC-CMs and rs373958405 demonstrated reduced activity compared to the reference allele, indicating this variant may reduce expression by disrupting the enhancer activity of MYH7-C2. B. MYH7-C3 contains rs7149564 and chr14_23912371_C. rs7149564 disrupts an NKX2.5 motif and produced a trending reduction in activity in IPSC-CMs. chr14_23912371_C generates a TCF21 motif and produced an increased IPSC-CM luciferase signal. MYH7-C4 contains rs116554832 and rs10873105. rs116554832 disrupts a TBX5 motif and reduced activity in IPSC-CMs. rs10873105 is correlated with MYH7 expression in GTEx skeletal muscle data and creates a Hox10 motif. This variant generated increased activity in IPSC-CMs. These data indicate that sequence variants in transcription factor binding sites within enhancer regions can alter enhancer function and potentially affect MYH7 gene expression. All data shown as mean ± SD. Data was derived from an average of 30 different assays per condition, from across least 3 separate differentiations. Significance determined by unpaired t-test. ****<0.0001.

Figure 5.. Computational pipeline to identify enhancer modifying variants.

A. Schematic showing the pipeline filtering steps used to identify enhancer modifying variants (EMVs) genome-wide. B. This strategy disproportionately identified significant GTEx eQTLs from heart tissues versus non-heart tissues, and disproportionately identified rare alleles, indicating both tissue specificity and sequence conservation. C. Significance determined in B & C by Fisher’s exact test. D. Candidate enhancers for five genes were selected for testing in the luciferase reporter assay in IPSC-CMs. Four of five of these candidate regions validated as functional enhancer regions with signal greater than the negative control region (as tested in an average of 32 assays using four separate differentiations). Significance vs negative control was determined by nonparametric one-way ANOVA with Dunn’s multiple comparisons correction. E. We then tested the reference and alternate alleles to assess whether the alternative variant shifted activity. The enhancer modifying variant in MYH6/7 and in GATA4 significantly changed expression (highlighted in yellow) (data derived from an average of 27 assays per condition, from four separate differentiations). Significance determined by unpaired t-test. All data shown as mean ± SD. *<0.03, ***<0.0002, ****<0.0001.

Figure 6.. Deletion of the C6 enhancer region alters MYH6/7 expression.

A. Schematic demonstrating the location of the MYH6/7-C6 enhancer region; the position of rs875908 is indicated. B. IPSC-CM MYH6 and MYH7 expression levels in cells deleted heterozygously or homozygously for the C6 enhancer region containing rs875908. MYH6/7 levels were assayed by qPCR, and showed a dose-dependent reduction in MYH7 expression. Therefore, rs875908, is within an enhancer region required for strong MYH7 expression in human cardiomyocyte like cells (unedited n=16, MYH7-C6^+/− n=11, MYH7-C6^−/− n=6). C. SDS-PAGE analysis of myosin heavy chain protein (MHC) isoforms in MYH7-C6 ^+/− and ^−/− cells. D. Quantitation of α-MHC/β-MHC ratios indicates that RNA differences correlate with changes in protein level. Significance determined by one-way ANOVA with Dunnett’s multiple comparisons correction. *<0.03, ****<0.0001.

Figure 7.. Correlation of MYH6/7 rs875908 EMV with MYH7 mRNA cardiac expression and longitudinal shift in left ventricular dimensions over time in human cardiomyopathy patients.

A. The location of the MYH6/7 regulatory variant is shown; it is bound by both GATA4 and TBX5 signals. The variant disrupts a site within the TBX5 transcription factor motif. B. eQTL data from the GTEx project shows that the variant genotype correlates with MYH7 expression across multiple striated muscle samples; the larger number of skeletal muscle samples yields genome wide significance in expression levels. C. Association of variant status with LVIDd/BSA over time in cardiomyopathy patients whose data was derived from the electronic data warehouse from Northwestern Medicine. D. Association of variant genotype with LVPWd/BSA over time in in cardiomyopathy cases. These data indicate that rs875908 associated with changes of left ventricular morphology including a more dilated left ventricle and thinner posterior wall. Significance determined using a linear regression model corrected for genetic ancestry and sex. LVIDd/BSA, left ventricular internal diameter during diastole corrected for body surface area. LVPWd/BSA, left ventricular posterior wall thickness during diastole corrected for body surface area.

Figure 8.. Schematic diagram of the MYH6/7 locus during cardiac development.

At the uninduced locus, the MYH7 and MYH6 promoter regions form a complex with a super-enhancer containing the MYH7-C3 enhancer. The effect of the super-enhancer is depicted as the yellow hue. In early development, this super-enhancer is primarily in contact with the MYH6 promoter, which recruits cardiac transcription factors, chromatin remodelers, and transcription machinery to drive MYH6 expression. During development, activation of the MYH7-C3 region reorganizes the complex and the MYH7 promoter preferentially contacts the regulatory regions. Through competition for transcriptional machinery or through an independent separate mechanism, the MYH6 gene is downregulated. Cells lacking the MYH7-C3 region can established the chromatin structure of the locus but are unable to switch interactions to the MYH7 promoter, causing an upregulation of MYH6 and a downregulation of MYH7. This model highlights the importance of an MYH7 enhancer which is an attractive therapeutic target. TFs, transcription factors.