Abstract Loss of function in the chromatin remodeler CHD7 causes CHARGE syndrome, characterized by variable penetrance and diverse abnormalities. However, establishing genotype-phenotype correlations has been challenging, as most CHD7 inactivating mutations are null alleles. Through CHD7 missense variant analysis at potential phosphorylation sites, we identified T730 (T720 in mice) as a critical residue associated with pathogenesis. Using a CHD7 T730 missense variant (Chd7^T720A) and a frameshift null allele (Chd7^fs) in a mouse model, we found that Chd7^fs/fs mice were non-viable, while Chd7^fs/+ mice exhibited haploinsufficiency-related circling behavior. Notably, Chd7^fs/T720A mice died before postnatal day 2, indicating the Chd7^T720A allele is hypomorphic. Micro-CT analysis at E18.5 revealed that heterozygous mice primarily exhibited hypertrophic cardiomyopathy (HCM), while homozygous mice developed both HCM and dilated cardiomyopathy (DCM). RNA-seq analysis of neonatal Chd7^T720A/T720A hearts revealed a disrupted transcriptome, which in males and females was characterized by downregulation of mitochondrial energy metabolism genes and enrichment of ETS family transcription factor targets. We further identified GSK3β, GSK3α, HIPK1, and DYRK2 as candidate kinases for this site, suggesting a regulatory role in CHD7. This missense variant causing developmental heart abnormalities establishes the first genotype-phenotype correlation for CHD7, and offers new insights into CHARGE syndrome pathogenesis. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-00606-1. Keywords: CHARGE syndrome, CHD7, T730 phosphorylation, Cardiomyopathy, GSK3 Subject terms: Genetics, Diseases Introduction Modulation of chromatin structure by ATP-dependent remodeling factors is central to the regulation of transcription and, consequently, key biological processes. These enzymes alter chromatin organization, in part, by moving or evicting nucleosomal histones that considerably restrict DNA accessibility^[42]1. There are 4 major families of chromatin remodeling factors: the Switch/Sucrose Non-Fermenting (SWI/SNF), Imitation Switch (ISWI), Inositol 80 - Swi2/Snf2-Related 1 (INO80-SWR1) and Chromodomain Helicase DNA-binding (CHD) families. This classification is based on protein domains associated with their central catalytic domain, which is homologous to the yeast SNF2 enzyme^[43]2. For instance, SWI/SNF-family ATPases harbor bromodomains while CHD-family enzymes are characterized by their chromodomains. CHD proteins form the largest family of remodeling enzymes and all 9 members are involved in vertebrate development^[44]3. CHD7 (Chromodomain Helicase DNA-binding Protein 7) is a key chromatin remodeler involved in developmental gene expression, and its loss-of-function mutations cause CHARGE syndrome, a multisystem disorder characterized by Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth, Genital and Ear abnormalities^[45]4. Despite extensive research on CHD7 mutations, most studies have focused on null or frameshift mutations that lead to complete CHD7 function loss^[46]5–[47]8. While sequencing of CHD7 is used as a molecular diagnostic for CHARGE^[48]9, how different pathogenic variants affect CHD7 function is largely unknown. For instance, truncating/stop codon variants in CHD7 are typically found in individuals with CHARGE, while individuals with IHH predominantly have missense variants^[49]10. CHD7 variants that do not generate null alleles may even be classified as ‘benign’ or ‘variants of uncertain significance’ despite producing amino acid substitutions at conserved CHD7 residues and the presence of CHARGE phenotypes. The inability to ascertain whether CHD7 missense mutations are benign or pathogenic is further compounded by their distribution along the entire gene, making it difficult to determine whether they affect a specific or multiple CHD7 biochemical functions^[50]9. To investigate genotype-phenotype relationships for CHD7, we focused on a highly conserved phosphorylatable residue, threonine 730 (T730; T720 in mice), and generated a missense variant allele (Chd7^T720A) to examine its role in heart development. Notably, mice carrying the Chd7^T720A allele had distinct cardiomyopathies, linking this missense variant to defects in heart development. Furthermore, in vitro kinase assays identified GSK3β, GSK3α, HIPK1, and DYRK2 as kinases for T730 suggesting this may be a phosphorylation site on CHD7, and RNA-seq revealed transcriptional changes in the heart of animals carrying the T720A missense variant linked to mitochondrial dysfunction and ETS family transcription factor dysregulation. These in vivo and in vitro findings establish the first genotype-phenotype correlation for a CHD7 missense variant, and lay the groundwork for exploring upstream regulatory pathways governing CHD7 function, with potential implications for CHARGE syndrome and related cardiomyopathies. Results CHD7 threonine 730 (T730): a conserved and clinically relevant site on CHD7 To explore genotype-phenotype correlations and dissect how loss of specific CHD7 functions contribute to disease pathogenesis, we focused on potential phosphorylation sites, reasoning that these could potentially regulate specific CHD7 functions. We conducted an extensive review of previously identified phosphorylation sites on CHD7 involving collation of all documented phosphorylation sites from the PhosphoSitePlus database ([51]https://www.phosphosite.org/)^[52]11. These phosphorylation sites were then correlated with clinical data for missense variants listed in the single nucleotide polymorphism database (dbSNP) at NextProt ([53]https://www.nextprot.org/entry/NX_Q9P2D1/sequence)^[54]12. A total of eighteen clinically significant missense variants were identified at phosphorylatable serine (S), threonine (T) or tyrosine (Y) residues: S734P, T730I, T2472A, S2229P, S2395P or Y, T2952S, T298A, Y313C, S316I, S559L or W, S612N or T, T689M, S782C, T786I, Y1217D, S2110G, S2231C or F, T2532M. Clinical relevance of these variants ranged from associations with CHARGE syndrome to manifestations in hypogonadotropic hypogonadism, neurodevelopmental disorders, and various neoplasms, as detailed in Supplementary Table [55]1. Among these sites, T730 emerged as a key site of interest, as a T730 missense variant (T730I c.2189 C > T), cataloged as rs552946889 in reference SNP reports with an allele frequency of 0.00476% (38 cases in 797927 samples), was associated with both Kallmann syndrome and CHARGE syndrome (ClinVar; [VCV000363451.13]). In gnomAD, CHD7-T730A and CHD7-T730I have been reported with allele counts of 1 and 57, respectively. While CHD7-T730I has been observed in individuals with hypospadias and cryptorchidism, its clinical significance remains uncertain, as it has been classified as ‘uncertain significance’ or ‘likely benign’ in ClinVar (Variation ID: 363451). Given the variable penetrance observed in CHARGE syndrome, changes in cell signaling, perhaps in response to environmental variation, could impact CHD7 activity during key periods of development and contribute to disease pathogenesis. Given that cardiac defects are a major feature of CHARGE syndrome, understanding how phosphorylation affects CHD7 activity provides valuable insights into the molecular mechanisms underlying CHD7-related congenital heart disease. T730 is evolutionarily conserved from Drosophila(Kismet) to humans, underscoring its biological significance. Phosphorylation at this site (pT730) has been observed in cell lines^[56]13–[57]15 and mouse tissues (Guo A, CST Curation Set 2923 and 2717; Year 2007, [58]https://www.phosphosite.org/curatedInfoAction.action?record=3366534 ). Consequently, we focused on T730 for further exploration into regulatory pathways that could link specific genetic alterations to the multifaceted phenotypes of CHARGE syndrome and, more broadly, CHD7-related disorders. Table 1. Genotype-Phenotype Correlations. Chd7 ^+/+ Chd7 ^fs/+ Chd7 ^T720A/+ Chd7 ^T720A/T720A Chd7 ^fs/T720A Total number 139 15 176 128 6 (All animals died before P2) Sex ratio 76 M, 63 F 10 M, 5 F 93 M, 81 F(2 ND) 65 M, 63 F 5 M, 1 F Circling 0 N = 14/14 (9 M, 5 F): 100%. 1 M was dead after birth. 0 0 Animals did not survive to be evaluated. Round head 0 N = 1/15: 7% 0 0 N = 1/1 (1 M): 100%, 5 ND Abnormal eye 0 N = 13/14 (8 M, 5 F): 93%. Unilateral microphthalmia 0 0 N = 5/5 (4 M, 1 F): 100% had unilateral microphthalmia. N = 2/5 (2 M): 40% had anophthalmia. 1 M was indeterminant. [59]Open in a new tab M, male; F, female; ND, not determined; P2, postnatal day 2. Pathogenic effects of a CHD7 T730 missense variant in a murine model To establish the functional importance of T730, we generated mice carrying a murine mT720A (hT730A equivalent) Chd7 allele variant (Chd7^T720A) using a Chd7 cDNA ([60]NM_001277149.1) and CRISPR-Cas9 (Fig. [61]1a and Supplementary Fig. [62]1a). T730I has been identified as a clinically relevant CHD7 variant, but its functional consequences remain uncharacterized. Our goal is to investigate the broader functional effects of phosphorylation loss at this site, rather than solely replicating the human variant’s effects. T730 in CHD7 is a conserved threonine residue, and a predicted phosphorylation site, although a functional role for this residue is unknown. Indeed, T730 has been identified as clinically relevant, as a T730I mutation has been linked to CHD7 (ClinVar, Variation ID: 363451). To better understand the potential impact of loss of phosphorylation loss at this site, we generated variants substituting threonine with a non-phosphorylatable alanine (T730A) residue. Concurrently, we generated a null allele carrying a frameshift deletion (ΔC2718) that introduces a premature stop codon upstream of the CHD7 chromodomains, subsequently referred to as Chd7^fs (Fig. [63]1b). Given that approximately 78% of individuals with CHARGE syndrome have either nonsense or frameshift mutations (44% and 34%, respectively)^[64]8, the Chd7^fs allele was also characterized as a potential new murine CHARGE model. Fig. 1. [65]Fig. 1 [66]Open in a new tab Murine T730 missense and frameshift-null alleles. (a) Alignment of mouse and human CHD7 proteins showing conservation between the human T730 and mouse T720 phosphorylation sites. (b) Schematic representation of the T720A point mutation and the frameshift mutation generated by CRISPR/Cas9-mediated technology. The frameshift null allele (Chd7^fs) terminates at the N-terminal region due to the deletion of a cytosine at position 2718 in the Chd7 cDNA ([67]NM_001277149.1). (c) Offspring produced by mating mice with Chd7^T720A and/or Chd7^fs alleles. The number of pups is shown along with the expected and observed percentages. Chd7^T720A/fs mice died by postnatal day 2, and all pups from Chd7^fs/+ mothers died after delivery. Subsequent breeding experiments among the various genotypes carrying these alleles were conducted to evaluate offspring viability. While Chd7^fs/+ mice with a wild-type (WT) allele were viable, no Chd7^fs/fs homozygotes were obtained from crosses between heterozygous Chd7^fs/+ x Chd7^fs/+ mice, as expected for null alleles; four separate matings between heterozygous Chd7^fs/+ mice, including one that was left undisturbed for four months, yielded no homozygous Chd7^fs/fs offspring. Chd7^fs/+ mice carrying one null allele exhibited characteristic circling behavior indicative of inner ear defects due to Chd7 haploinsufficiency^[68]16, as well as small eyes (microphthalmia) and, in one case, a round head consistent with hydrocephalus (Table 1). In contrast, crosses between heterozygous Chd7^T720A/+ mice produced offspring in Mendelian ratios (Fig. [69]1c), including Chd7^T720A/T720A homozygotes. These mice did not display circling behavior, nor grossly observable eye defects, suggesting phosphorylation at T720 is not linked to pathogenesis of these defects. Strikingly, however, Chd7^fs/T720A progeny from Chd7^T720A/T720A females crossed with Chd7^fs/+ males all displayed either prenatal or immediate postnatal lethality, with none surviving beyond postnatal day 2 (Fig. [70]1c; Table 1), indicating the Chd7^T720A allele was not able to fully substitute for WT Chd7. Of the live-born Chd7^fs/T720A pups that survived long enough to be genotyped (but later died), 5 were males and only 1 was female. Although this observation suggests females may be more adversely affected than males, Fisher’s exact test showed that the deviation from the expected 1:1 ratio was not statistically significant (p = 0.55). While both Chd7^fs/+ and Chd7^fs/T720A mice exhibited eye defects such as microphthalmia, Chd7 mice displayed more severe phenotypes, including anophthalmia (Table [71]1 and Supplementary Fig. [72]1b). Pathogenicity of theChd7^T720Aallele and its impact on cardiac development To further investigate these findings, we conducted a phenotypic analysis using micro-computed tomography (micro-CT) of E18.5 mouse embryos, allowing us to examine Chd7^T720A/+, Chd7^T720A/T720A, Chd7^fs/+, and Chd7^fs/T720A mice before viability loss. This analysis included assessments of gross morphological abnormalities, such as dysmorphisms, growth delays, edema, and craniofacial anomalies, as well as anatomical defects in major organs. Notably, the most pronounced and consistently observed abnormalities were found in the brain, eyes, and heart (Fig. [73]2a and b). We observed highly penetrant eye defects, including unfused eyelids and microphthalmia, in Chd7^fs/+ and Chd7 embryos, suggesting a strong association with the Chd7^fs allele. In contrast, all Chd7^T720A/+ and Chd7^T720A/T720A embryos had normal eyes. In the brain, reduced lateral ventricle size was observed across most mutant genotypes. These findings corroborate previous research on CHD7 haploinsufficiency, with the Chd7^fs allele phenotypes closely aligning with the Chd7^COA1/+mouse model carrying the K719X nonsense mutation^[74]17. Fig. 2. [75]Fig. 2 [76]Open in a new tab Gross morphology and 2D digital section phenotyping with iodine-contrasted micro-CT. (a) Table summarizing the occurrence of organ abnormalities in Chd7 mutants across different genotypes: WT (Chd7^+/+), T720A/+ (Chd7), T720A/T720A (Chd7^T720A/T720A), frameshift null fs/+ (Chd7^fs/+), and compound heterozygous fs/T720A (Chd7^fs/T720A). The analysis was performed on mouse embryos at embryonic day 18.5 (E18.5) using micro-CT. Shaded areas in the table indicate the presence of abnormalities in various organs including the brain, eye, heart, and kidney. (b) Representative micro-CT images showing the phenotypic abnormalities observed in Chd7 mutants. The abnormalities are indicated by red arrows and are organized by organ: Brain (reduced lateral ventricular size in Chd7^T720A/+ and Chd7^fs/T720A mutants), eye (microphthalmia observed in two Chd7^fs/+ mutants, heart (enlarged ventricular chamber, thinning of the ventricular wall, and ventricular septal defect in Chd7^fs/+ mutant; or reduced ventricular chamber and thickened ventricular wall in Chd7^fs/T720A mutant), and kidney (enlarged kidney in Chd7^T720A/T720A mutant; or hydronephrosis in Chd7^fs/T720A mutant). Scale bars for reference are included in the images (2.5 mm, 5 mm, and 2 mm). We also observed highly penetrant heart abnormalities in both male and female mice carrying Chd7^fs and Chd7^T720A alleles (Fig. [77]2b). Micro-CT section analysis revealed embryos carrying these alleles exhibited both enlarged and reduced ventricular chambers compared to WT embryos. Embryos with enlarged ventricular chambers displayed thinning of the ventricular walls, characteristic of DCM. In contrast, embryos with reduced ventricular chambers showed thickened ventricular walls and septa, consistent with HCM. Additionally, two of the Chd7^fs/+embryos (one male and one female) exhibited a ventricular septal defect (VSD), a hallmark in CHARGE syndrome and its mouse models^[78]16,[79]18, characterized by a discontinuous gap in the ventricular septum. Interestingly, these embryos also showed enlarged ventricular chambers, consistent with a DCM-like phenotype. Given that CHD7 functions as a chromatin remodeler involved in cardiac development, its haploinsufficiency may disrupt transcriptional programs required for myocardial homeostasis. This could contribute to chamber dilation through mechanisms involving cardiomyocyte survival, extracellular matrix remodeling, or altered contractility. In more severe cases, progressive myocardial thinning may further impair ventricular septation, ultimately leading to VSD. The presence of VSD in some, but not all, Chd7^fs/+ embryos suggests that while these defects may arise through overlapping mechanisms, they are not necessarily linked in all cases. Further studies will be necessary to elucidate the precise relationship between CHD7 function, myocardial development, and ventricular septation. Notably, this ventricular septal defect was not observed in mice carrying the Chd7^T720A allele. Quantitative 3D analysis of cardiac morphology To quantify the impact of the Chd7^fs and Chd7^T720A alleles on heart morphology, we developed a 3D segmentation method to measure the volumes of the left and right ventricular chambers and ventricular walls using micro-CT datasets (Fig. [80]3a). The volume of the ventricular chambers (left and right), normalized by the total ventricular volume (chambers + walls), was defined as the Volume of Chambers (VoC). A Z-score was computed for each sample, comparing it to WT average. A Z-score < −2 (2 standard deviations below the mean) with a Bonferroni-corrected p-value < 0.05 indicated a significantly smaller VoC than WT embryos, while a Z-score > 2 with p < 0.05 indicated a significantly larger VoC than WT embryos (Supplementary Table 2). Fig. 3. [81]Fig. 3 [82]Open in a new tab Comparative analysis and quantitation of cardiomyopathy in E18.5 mouse embryos. (a) Cardiac imaging and 3D reconstruction. Cross-sectional views of hearts are shown on the left, with 3D reconstructions highlighting the left ventricular wall (yellow) and right ventricular chamber (green) in the middle panel. The right panel shows the ventricular wall and septum. Three phenotypes are shown: normal, enlarged ventricular chamber (DCM), and reduced ventricular chamber (HCM). (b-c) Quantitative analysis of cardiac morphology. Ratios of the volumes of the left and right ventricular chamber were normalized to total ventricle volumes by measuring individual volumes in cubic millimeters (mm³). Comparisons were made among Chd7^+/+ (WT), Chd7^fs/+, Chd7^T720A/+, Chd7^T720A/T720A, and Chd7^fs/T720A mice. Z scores were calculated using the average and standard deviation of WT, with significant thresholds marked at Z = ± 2. DCM and HCM phenotypes are color-coded in pink and blue, respectively. This 3D quantification method refined our cardiac phenotype characterization, differentiating between ventricular chamber volume changes and ventricular wall thickness variations. Four out of five Chd7^fs/+ embryos exhibited altered VoC, with three showing enlarged VoC (DCM-like phenotype) and one showing reduced VoC (HCM-like phenotype) (Fig. [83]3, Supplementary Tables 2, and Supplementary Fig. [84]2). All Chd7^T720A/+ embryos (n = 8) had significantly reduced VoC, consistent with an HCM-like phenotype. Among Chd7^T720A/T720A embryos (n = 7), three exhibited enlarged VoC (DCM-like), three exhibited reduced VoC (HCM-like), and one was not significantly different from WT embryos. In Chd7^fs/T720A embryos (n = 6), five showed reduced VoC (HCM-like phenotype), while one was not significantly different from WT embryos. These quantitative data in Fig. [85]3 directly support and validate the qualitative observations shown in Fig. [86]2, reinforcing the reliability of our phenotype assessment. Thus, 3D quantification confirmed the cardiomyopathy phenotypes identified in 2D sections. Impact of theChd7^T720Aallele on the transcriptome in heart To investigate the functional differences between WT and Chd7^T720A alleles, we evaluated how the T730 missense variant affects CHD7’s activity as a transcriptional co-activator/co-repressor using RNA-seq at postnatal day 0 (P0). In Chd7^T720A/T720A hearts, a total of 2012 differentially expressed genes (DEGs) (FDR < 0.01, ≥ 1.5-fold change) were identified in males (1264 up-regulated, 748 down-regulated) and 581 in females (489 up-regulated, 92 down-regulated) relative to age- and sex-matched controls. These changes were visulaized through heat maps, principal component analysis (PCA), and volcano plots (Fig. [87]4a and b, and Supplementary Fig. [88]3a). Interestingly, there was no overlap between DEGs that were down-regulated in male and female Chd7^T720A/T720A hearts, while 179 DEGs were up-regulated in both sexes. This finding highlights an unexpected sex-specific difference, particularly given the young age of these animals and that both males and females presented with cardiomyopathy (Supplementary Fig. [89]3b). Fig. 4. [90]Fig. 4 [91]Open in a new tab Transcriptional profiling of neonatal hearts from Chd7^T720A/T720A mice. (a-b) Heat maps (a) and PCA plots (b) showing DEGs in Chd7^T720A/T720A males (left) and females (right) neonatal hearts compared to Chd7^+/+ (WT) controls (N = 3 per group for both WT and mutant). (c) Volcano plots highlighting the most significantly enriched pathways for DEGs determined by ORA of GO-BP gene sets in male and female Chd7^T720A/T720A hearts. Key pathways such as cytoskeleton organization, lipid metabolic process, cell adhesion, and cell cycle are shown. (d) GSEA enrichment plots showing downregulation of the mitochondrial protein containing complex gene sets in homozygous Chd7^T720A/T720A mouse neonatal hearts compared to those of wild type controls. The NES values are displayed for both males (NES = −3.72) and females (NES = −8.12). (e) Bar chart showing NES from GSEA of commonly disrupted pathways in male and female Chd7^T720A/T720A neonatal hearts. Pathways include mitochondrial components, the inner mitochondrial membrane, and the tricarboxylic acid cycle enzyme complex. To investigate the functional significance of these gene expression changes, we performed over-representation analysis (ORA) using the Gene Ontology: Biological Processes (GO: BP) compendium to identify pathways enriched in the disrupted transcriptomes of Chd7^T720A/T720A mice. In males, the most significantly enriched pathways were related to cytoskeletal organization, cell adhesion, lipid metabolic process, and cell cycle (Fig. [92]4c and Supplementary Fig. [93]3c). In female Chd7^T720A/T720A hearts, up-regulated DEGs were primarily associated with immune cell activation, while cell adhesion pathways were enriched in both sexes (Fig. [94]4c and Supplementary Fig. [95]3c). Figure [96]4c presents volcano plots highlighting the most significantly up- and down-regulated DEGs in males, as well as up-regulated DEGs in females within these pathways. Given that many cytoskeletal elements are involved in both cell division and cell adhesion, we examined the extent of overlap between these GO terms. As shown in Supplementary Fig. [97]3 d, only a quarter of the identified genes were shared across two or more of these pathways, suggesting that they reflect distinct gene sets involved in cell cycle, cytoskeletal organization, and cellular adhesion. To identify common transcriptional changes in the neonatal hearts of both male and female Chd7^A/T720A mice, we performed gene set enrichment analysis (GSEA) on the entire transcriptome of P0 hearts. As shown in the enrichment plots and bar charts in Fig. [98]4d and e, neonatal Chd7^T720A/T720A hearts from both sexes exhibited negative enrichment for genes involved in mitochondrial respiration, indicating downregulation of this pathway. This finding suggests that CHD7 regulates the expression of oxidative phosphorylation genes which is impaired in Chd7^T720A/T720A hearts of both sexes. While Chd7^T720A/T720A homozygotes exhibited widespread transcriptional dysregulation affecting multiple biological processes, in the presence of a WT allele (Chd7^T720A/+), heterozygote hearts showed more limited pathway-level disruption despite a considerable number of DEGs (Supplementary Fig. [99]4a and 4b). Unlike the strong down-regulation observed in homozygotes, Chd7^T720A/+ hearts exhibited positive enrichment of genes involved in mitochondrial respiration, indicating a possible compensatory response of the WT allele to maintain cellular energy homeostasis (Supplementary Fig. [100]4c). CHD7 T730 missense variant alters ETS family transcription factor target gene expression The role of CHD7 as a transcriptional co-regulator is well established^[101]19–[102]24, and previous studies have shown that CHD7 co-localizes on chromatin with the ETS family transcription factor RUNX1^[103]25. To further investigate this interaction, we performed an in silico comparison of CHD7 and ETS family transcription factor target genes. Using the Harmonizome ENCODE Transcription Factor Target gene set for CHD7 and the MSigDB Transcription Factor Target gene sets for the ETS family transcription factors^[104]26,[105]27, we found a significant overlap of 57–73% (Fig. [106]5a). Next, we asked if DEGs in neonatal hearts of Chd7^T720A/T720A homozygotes were significantly enriched for ETS recognition motifs. As shown in Fig. [107]5b, up-regulated DEGs in both male and female Chd7^T720A/T720A hearts were significantly enriched for targets of ETS1, ETS2, RUNX1, PU1, PEA3, ELF1 and GABPβ, with -log p-values ranging from p ≤ 10^−7 to 10^−36, depending on sex and the specific ETS family transcription factor (notably, no down-regulated DEGs were shared between males and females, Supplementary Fig. [108]3b). Additionally, RUNX1 target gene enrichment was observed using GSEA against the ENRICHR RUNX1 LINCS L1000 CRISPR KO Sigs gene set^[109]28, as shown in the enrichment plots in Fig. [110]5c. These findings suggest that T720 palys a key role in CHD7 function as a co-regulator of ETS family transcription factors, potentially influencing RUNX1-mediated transcriptional programs. Given that RUNX1 activation has been linked to pathological myocardial remodeling and heart failure via TGF-β signaling, these findings suggest that CHD7 loss-of-function may contribute to DCM through transcriptional dysregulation. While the exact mechanism remains unclear, CHD7 may regulate RUNX1 directly or through broader chromatin remodeling effects that influence cardiac transcriptional networks. Further studies will be necessary to determine the precise role of CHD7 in myocardial remodeling and disease progression. Fig. 5. [111]Fig. 5 [112]Open in a new tab Altered ETS family transcription factor target gene expression in the hearts of CHD7 T730 missense mutant mice. (a) Overlap between CHD7 and ETS family transcription factor targets as reported in the Harmonizome database. The table shows the number of target genes for each transcription factor, the overlap with CHD7 targets, and the percentage of overlap. (b) Bar chart depicting enrichment scores for ETS family transcription factors targets among DEGs from homozygous Chd7^T720A/T720A male and female neonatal hearts. (c) GSEA enrichment plots for RUNX1 upregulated target genes in the Chd7^T720A/T720A transcriptome of male and female neonatal hearts. The NES values are displayed for male (NES = 1.35) and female (NES = 1.18). Multiple kinases phosphorylate CHD7 at T730 and the adjacent S734 residue Because T730 can potentially be phosphorylated, with regulatory consequences for CHD7 activity, we analyzed data from Kinase Library^[113]29, accessible through PhosphoSitePlus to ascertain if T730 was a potential kinase target. The Kinase Library algorithm predicts kinase activity by integrating phosphorylated residues in proximal sequences (phospho-priming) with empirical data. This in silico analysis identified two isoforms of glycogen synthase kinase-3 (GSK3) as the primary candidates for T730 phosphorylation, with a confidence score of 100.0% for GSK3β and 99.996% for GSK3α (Supplementary Fig. [114]5a) when adjacent residues at S725 (pS725) or S734 (pS734) were phosphorylated (primed). Interestingly, a CHD7 serine-to-proline mutation (S734P) was identified in a patient with a neurodevelopmental disorder and classified as a variant of unknown significance (Supplementary Table 1). Notably, both pT730 and pS734 phosphorylation events have been observed in human cell lines, mouse heart and brain tissues^[115]13,[116]15, and mouse NIH3T3 cells^[117]14. These findings suggest that phosphorylation of these CHD7 residues may be part of a yet-to-be-characterized but functionally important kinase signaling pathway. A preliminary round of in vitro kinase assays was performed using a peptide containing T730 (Fig. [118]6a and b) and 20 kinases with aligned motifs to identify potential kinases that phosphorylate this site. The results showed that this peptide was phosphorylated by GSK3β, and even more robustly DYRK2 and HIPK1 (Supplementary Fig. [119]5b). However, this assay could not distinguish between the two potential phosphorylation sites, T730 and S734, nor determine the impact of S734 priming on GSK3β activity. To address this, we designed peptides in which either T730 or the potential priming site S734 was substituted with non-phosphorylatable alanine (T730 A or S734A, respectively), as well as a “pre-primed” peptide containing pS734 (Fig. [120]6b). These substitutions allowed us to determine whether specific kinases target T730 or S734 and how phosphorylation at S734 influences T730 phosphorylation efficiency. S734A was chosen as a non-phosphorylatable control to determine the role of priming phosphorylation at this site without introducing structural perturbations. While potentially clinically relevant, proline substitution (S734P) was not used in this context, as this amino acid induces local conformational rigidity, which could independently alter kinase recognition and activity. Instead, our kinase assays were specifically designed to assess phosphorylation site preference, ensuring that observed changes reflect phosphorylation loss rather than structural effects. Using these peptides, in vitro kinase assays revealed distinct phosphorylation preferences among kinases. DYRK2 preferentially phosphorylated S734, as