Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown.Here, we define transcriptional consequences of reduced dosage of the CHD-linked transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5dependent pathways-including lineage decisions and genes associated with cardiomyocyte function and CHD genetics-in discrete subpopulations of cardiomyocytes. GRN analysis identified vulnerable nodes enriched for CHD genes, indicating that cardiac network stability is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c was validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite sensitivity to TBX5 dosage by diverse transcriptional responses in heterogeneous subsets of iPSC-derived cardiomyocytes. This predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

30Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD) 1 . This observation predicts gene regulatory network (GRN) imbalances 2 , but the nature 32 of dosage-vulnerable GRNs and their contribution to human cardiogenesis and CHDs are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD 34 transcription factor TBX5 during human cardiac differentiation from induced pluripotent stem (iPS) cells. Single cell RNAseq revealed that transcriptional responses to reduced TBX5 levels 36 are not homogeneous, and instead, discrete sub-populations of cardiomyocytes exhibit dysregulation of distinct TBX5 dose-sensitive genes related to cellular phenotypes and CHD-38 associated genetics. Cellular trajectory inference revealed TBX5 dosage-dependent differentiation paths, with implications for cardiac developmental identity. GRN analysis of the 40 single cell RNAseq data identified vulnerable nodes enriched for CHD genes, implicating a critical sensitivity to TBX5 dosage in cardiac network stability. A novel GRN-predicted genetic 42 interaction between TBX5 and MEF2C was validated in mouse, revealing a highly dosagesensitive pathway for CHD. Our results reveal unforeseen complexity and exquisite sensitivity to 44 TBX5 dosage in discrete sub-populations of iPSC-derived cardiomyocytes, providing mechanistic insights into human CHDs and quantitative transcriptional regulation in disease. linked to CHD predict reduction in dosage of transcriptional regulators, including transcription factors (TFs) and chromatin-modifying genes 1 . Despite advances in tracing the roles of 52 individual factors, how altered dosage of transcriptional regulators translates to altered transcriptional activity is not known, nor is it known how these altered GRNs disrupt heart 54 development, resulting in CHD.Heterozygous mutations in the T-box TF gene TBX5 cause Holt-Oram syndrome (HOS) 56 5,6 , which presents with upper limb defects, CHDs, diastolic dysfunction, or arrhythmias. In humans, homozygous TBX5 loss of function is presumed to cause fetal demise. Altered 58 expression of select genes in mice in vivo demonstrate a stepwise sensitivity to reductions in Tbx5 dosage 7,8 . These findings suggest that a reduction in TBX5 dosage perturbs downstream 60 gene expression, but the overall disrupted networks and mechanisms are not understood.Mechanistic investigation of human CHD has been hampered by a lack of relevant and 62 tractable models. Human heart tissue is largely inaccessible for molecular analysis, and pathological or surgical specimens are limited. However, induced pluripotent stem (iPS) cells, 64 genome editing, directed differentiation, and single cell RNAseq together provide a human cellular platform for gene-centered cardiac disease modeling at single cell resolution. In 66 considering how transcriptional regulator haploinsufficiency might cause CHD, at least two scenarios are possible: 1. That reduced dosage affects genes only in specific anatomical 68 locations, such as the ...

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. For instance, in pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. Conversely, H3K9 methylation represents an epigenetic barrier for reprogramming somatic cells back to pluripotency. How global H3K9 methylation levels are coupled with the acquisition and loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of an uncharacterized antisense long non-coding RNA to Suv39h1, which we name Suv39h1as. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, leading to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the global levels of H3K9 methylation to pluripotency in mouse ES cells.

The exogenous application of small peptides can beneficially affect clinical skin appearance (wrinkles) and architecture (collagen and elastic fibre deposition and epidermal thickness). However, the discovery of new bioactive peptides has not been underpinned by any guiding hypothesis. As endogenous extracellular matrix (ECM)-derived peptides produced during tissue remodelling can act as molecular signals influencing cell metabolism, we hypothesised that protease cleavage site prediction could identify putative novel matrikines with beneficial activities. Here, we present an in silico to in vivo discovery pipeline, which enables the prediction and characterisation of peptide matrikines which differentially influence cellular metabolism in vitro. We use this pipeline to further characterise a combination of two novel ECM peptide mimics (GPKG and LSVD) which act in vitro to enhance the transcription of ECM organisation and cell proliferation genes and in vivo to promote epithelial and dermal remodelling. This pipeline approach can both identify new matrikines and provide insights into the mechanisms underpinning tissue homeostasis and repair.

The extracellular matrices (ECMs) of mammalian tissues play important roles in mediating and maintaining tissue function. However, aberrant and progressive remodelling of ECM components is a key feature in the pathology and ageing of many organs, including skin. Crucially, these degradative processes not only impair function, but may also release peptide fragments, known as matrikines, with cell signalling capabilities. In addition to endogenously produced matrikines, many exogenously applied bioactive peptides, with proven activity in skin, are homologous to naturally occurring amino acid sequences within ECM proteins. Despite the ability of some peptides to induce clinical benefits in aged skin, there has been no published conceptual framework to guide the prediction of new therapeutic matrikines. Here, we tested the hypothesis that small bioactive peptides (matrikines) can be predicted by the in silico digestion of dermal proteins by ECM proteases. We first identified a target cohort of 27 ECM proteins that were abundant in the dermis and/or were reported to undergo age-related remodelling. Utilizing an established machine-learning tool (PROSPER) we developed a PYTHON algorithm that could predict peptides liberated by in silico cleavage with eight skin-active enzymes [matrix metalloproteinase (MMP)-2, MMP-3, MMP-7 and MMP-9, cathepsin G and cathepsin K, granzyme B and elastase-2]. For the 27 target proteins this approach predicted the identity of 453 tetrapeptides. These peptides were predicted to be liberated predominantly from collagens (I, III, IV, VI and VII), some elastic fibre-associated proteins (EMILIN1 and fibulin-1) and the adhesive glycoprotein fibronectin. Eight peptides were selected for synthesis and biological activity testing based on their predicted protein sources, high solubility and likelihood of successful manufacture. The ability of these peptides to promote ECM synthesis (procollagen I, fibronectin, decorin, collagen IV, hyaluronic acid and fibrillin-1) in cultured human dermal fibroblasts (HDFs) was assessed by enzyme-linked immunosorbent assay or immunofluorescence (fibrillin-1 only). With the exception of hyaluronic acid, all of the peptides enhanced the synthesis of at least some ECM markers, including procollagen-I and decorin. Deposition of fibrillin-1, which is a sensitive marker of both skin ageing and repair, was significantly enhanced by three peptides. The ability of the peptides to influence the wider HDF secretome was assessed by liquid chromatography tandem mass spectrometry proteomics. Four of the peptides modulated synthesis of proteins in a wide range of functional classes, including enzyme inhibitors, ECM-affiliated proteins, cell adhesion molecules and basement membrane components, while the remaining four exhibited more targeted activities. We conclude that bioinformatic prediction of cleaved peptide fragments from ECM proteins can identify multiple small peptides with potentially beneficial activities for skin. This approach can both identify new matrikines and provide insights into the mechanisms underpinning tissue repair. Funding sources: this study was funded by the No. 7 Beauty Company.