Biology subjects

Renberg, A.

Publications and source records attributed to Renberg, A..

5 recordsLinked to original sources

bioRxiv2026

Mapping and rewiring the MYBPC3 promoter for rescue of haploinsufficiency driven hypertrophic cardiomyopathy

Autosomal dominant loss-of-function variants in the gene MYBPC3 are, collectively, the most common genetic cause of hypertrophic cardiomyopathy (HCM) and are a prototype of haploinsufficient human disease. Typical for haploinsufficiency-associated genes, hundreds of unique loss-of-function pathogenic variants have been reported for MYBPC3 - therapeutic gene editing to correct each of these variants poses major regulatory and logistical hurdles. Upregulating wild-type allele expression could offer a generalizable therapeutic strategy, but the capacity to modulate native MYBPC3 transcription is unknown. Here, we present a variant-agnostic approach to rescue haploinsufficiency by mapping and rationally redesigning the MYBPC3 promoter. Using massively parallel reporter assays (MPRAs) in human induced pluripotent stem cell-derived cardiomyocytes, we performed saturation mutagenesis of the MYBPC3 promoter at single base-pair resolution. This defined a new class of clinically relevant noncoding loss-of-function variants while revealing essential cis-regulatory grammar anchored by key transcription factor binding sites (TFBSs). Furthermore, by systematically screening thousands of variant combinations, modular promoter elements, and heterologous TFBS insertions, we identified synergistic sequence edits that drive robust increases in MYBPC3 expression. Together, our findings improve the clinical interpretation of noncoding variants and establish a scalable blueprint for promoter editing to treat MYBPC3-associated HCM and other haploinsufficient diseases.

molecular biology↗

bioRxiv2026

Genomic Engineering of Gene Dosage: A Generalizable Framework for Modeling Haploinsufficiency-Mediated Human Disorders through Splicing Modulation

Heterozygous loss-of-function variants causing gene dosage reduction underlie many human genetic disorders, yet preclinical mouse models frequently fail to recapitulate human disease phenotypes due to post-translational compensation. Here, we present a generalizable framework to modulate gene dosage by alternative splicing via genome editing. By shifting proportions of transcripts toward nonsense-mediated decay, this approach enables precise titration of functional protein levels. Applying this concept to Mybpc3, we combined hypomorphic splice-altering alleles to overwhelm post-translational buffering in mice, successfully reproducing hallmark structural and functional features of human hypertrophic cardiomyopathy. Extending this concept, we engineered a portable Modulated Alternative Splice Cassette (MASC) inserted into Dsp, yielding patient-level protein reductions and characteristic arrhythmogenic cardiomyopathy pathologies, including subepicardial cardiac fibrosis and immune cell infiltration. These novel haploinsufficient models of Mybpc3 hypertrophic cardiomyopathy and Dsp arrhythmogenic cardiomyopathy will critically enable mechanistic and therapeutic testing under protein stoichiometry conditions that closely replicate the patient disorders. Finally, systematic in silico predictive modeling across 645 human haploinsufficiency-associated genes demonstrated the generalizability of MASC insertion effects on splicing across diverse tissue types. Notably, the framework developed here leaves endogenous gene expression regulatory logic intact, allowing these models to be used to develop and validate therapeutic approaches that target transcription. Overall, this strategy provides a scalable roadmap for engineering high-fidelity animal models for human diseases caused by haploinsufficiency.

genetics↗

bioRxiv2025

Desmoplakin Haploinsufficiency Underlies Cell-Cell Adhesion Failure in DSP Cardiomyopathy and is Rescued by Transcriptional Activation

BackgroundTruncating variants in desmoplakin (DSPtv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear. MethodsWe analyzed explanted human heart tissue from patients with DSPtv (N=3), titin truncating variants (TTNtv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered DSPtv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue. ResultsCompared to both control and TTNtv hearts, DSPtv human hearts exhibited reduced DSP at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. DSPtv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, p<0.001). Adhesion failure was prevented by the myosin inhibitor, mavacamten. CRISPRi-mediated DSP knockdown replicated this susceptibility to adhesion failure. Conversely, CRISPRa robustly increased DSP expression and rescued cell-cell adhesion failure in DSPtv CMBs (9% failure post-CRISPRa, p<0.001 vs. un-treated). Rescue occurred even when only the DSPII isoform was upregulated in a model with biallelic DSP transcript 1 loss of function. ConclusionsDSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of DSP reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.

genetics↗

bioRxiv2022

Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues

Mitochondrial damage is a hallmark of metabolic diseases, including diabetes and metabolic dysfunction-associated steatotic liver disease, yet the consequences of impaired mitochondria in metabolic tissues are often unclear. Here, we report that dysfunctional mitochondrial quality control engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity across multiple metabolic tissues. Surprisingly, we demonstrate that defects in the mitochondrial quality control machinery, which we observe in pancreatic {beta} cells of humans with type 2 diabetes, cause reductions of {beta} cell mass due to dedifferentiation, rather than apoptosis. Utilizing transcriptomic profiling, lineage tracing, and assessments of chromatin accessibility, we find that targeted deficiency anywhere in the mitochondrial quality control pathway (e.g., genome integrity, dynamics, or turnover) activate the mitochondrial integrated stress response and promote cellular immaturity in {beta} cells, hepatocytes, and brown adipocytes. Intriguingly, pharmacologic blockade of mitochondrial retrograde signaling in vivo restores {beta} cell mass and identity to ameliorate hyperglycemia following mitochondrial damage. Thus, we observe that a shared mitochondrial retrograde response controls cellular identity across metabolic tissues and may be a promising target to treat or prevent metabolic disorders.

cell biology↗

bioRxiv2021

The human disease gene CLEC16A encodes an intrinsically disordered protein region required for mitochondrial quality control

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic {beta}-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.

molecular biology↗