Biology subjects

Kitzman, J. O.

Publications and source records attributed to Kitzman, J. O..

14 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↗

bioRxiv2026

High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

The tumor suppressor RAD51D is essential for homologous recombination (HR). Pathogenic variants in RAD51D are associated with breast and ovarian cancers. However, most clinical missense variants are of unknown significance. We performed a multiplex assay of variant effect to test 6,888 RAD51D coding variants for loss-of-function. The resulting variant-to-function map perfectly separates known pathogenic and benign variants and is validated by orthogonal HR and biochemical assays across 70 clinical variants. Our screen shows that variants in the DNA-binding or ATPase core most severely compromise HR, and we identify the RAD51D-RAD51C interface within the BCDX2 complex as essential for regulating its ATPase activity. We hypothesize that, paradoxically, the primary function of RAD51D is to slow the ATPase activity of BCDX2, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover the mechanisms by which variants compromise its biochemical functions. HighlightsO_LIUsed a multiplexed assay of functional effect (MAVE) to assess the functionality via olaparib sensitivity of 6,888 RAD51D coding variants, which can be used for variant classification C_LIO_LIProvided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants C_LIO_LIIdentified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes C_LIO_LIDetermined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity C_LI

cancer biology↗

bioRxiv2025

Massively parallel reporter assay reveals promoter-, position-, and strand-specific effects in transcription start sites

Massively parallel reporter assays (MPRA) are a high-throughput method of assessing the activity of candidate cis-regulatory sequences, and can be used to detect allelic differences at disease-associated variants. Previous MPRA studies have screened thousands of functional SNPs associated with various complex traits and conditions. Most MPRA libraries utilize a single plasmid configuration, a single minimal promoter to drive expression, and a single-strand orientation, which may fail to capture the context-dependent activity of disease-associated cis-regulatory elements. We interrogate the potential regulatory differences introduced by variable MPRA plasmid promoters and positions. We used an MPRA library to quantify the activity of 1,305 pancreatic islet-derived transcription start sites generated from CAP analysis of gene expression profiling. We cloned fragments upstream or downstream of a reporter gene along with either the human insulin (INS) promoter or a synthetic housekeeping promoter (SCP1). We used elastic net regression to predict position-specific fragment activity based on enrichment of transcription factor binding site motifs, and generalized linear models to predict position-specific fragment activity from tissue-specific chromatin state regulatory annotations. Our results support the use of MPRA strategies that account for context-dependent factors when assaying candidate regulatory elements in pursuit of understanding complex genetic diseases.

genomics↗

bioRxiv2025

Saturation mapping of MUTYH variant effects using DNA repair reporters

Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying the pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder alleles, and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,032 existing missense VUS and 90 variants with conflicting interpretations in MUTYH, and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.

genomics↗

bioRxiv2024

Functional dissection of metabolic trait-associated gene regulation in steady state and stimulated human skeletal muscle cells

Type 2 diabetes (T2D) is a common metabolic disorder characterized by dysregulation of glucose metabolism. Genome-wide association studies have defined hundreds of signals associated with T2D and related metabolic traits, predominantly in noncoding regions. While pancreatic islets have been a focal point given their central role in insulin production and glucose homeostasis, other metabolic tissues, including liver, adipose, and skeletal muscle, also contribute to T2D pathogenesis and risk. Here, we examined context-specific genetic regulation under basal and stimulated states. Using LHCN-M2 human skeletal muscle cells, we generated transcriptomic profiles and characterized regulatory activity of 327 metabolic trait-associated variants via a massively parallel reporter assay (MPRA). To identify condition-specific effects, we compared four different conditions: (1) undifferentiated, or (2) differentiated with basal media, (3) media supplemented with the AMP analog AICAR (to simulate exercise) or (4) media containing sodium palmitate (to induce insulin resistance). RNA-seq revealed these treatments extensively perturbed transcriptional regulation, with 498-3,686 genes showing significant differential expression between pairs of conditions. Among differentially expressed genes, we observed enrichment of relevant biological pathways including muscle differentiation (undifferentiated vs. differentiated), oxidoreductase activity (differentiated vs. AICAR), and glycogen binding (differentiated vs. palmitate). The results of our MPRA found broadly different levels of activity between all conditions. Our MPRA screen revealed a shared set of 7 variants with significant allelic activity across all conditions, along with a proportional number of variants showing condition-specific allelic bias and the total number of active oligos per condition. We found that a lead variant for serum triglyceride levels, rs490972, overlaps SP transcription factor motifs and has differential regulatory activity between conditions. Comparison of MPRA activity with paired gene expression data allowed us to predict that regulatory activity at this locus is mediated by SP1 transcription factor binding. While several of the MPRA variants have been previously characterized in other metabolic tissues, none have been studied in these stimulated states. Together, this work uncovers context-dependent transcriptomic and regulatory dynamics of T2D- and metabolic trait-associated variants in skeletal muscle cells, offering new insights into their functional roles in metabolic processes.

genomics↗

bioRxiv2024

Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing messenger RNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINE RNAs use strategies to hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.

molecular biology↗

bioRxiv2023

Population-scale skeletal muscle single-nucleus multi-omic profiling reveals extensive context specific genetic regulation

Skeletal muscle, the largest human organ by weight, is relevant in several polygenic metabolic traits and diseases including type 2 diabetes (T2D). Identifying genetic mechanisms underlying these traits requires pinpointing cell types, regulatory elements, target genes, and causal variants. Here, we use genetic multiplexing to generate population-scale single nucleus (sn) chromatin accessibility (snATAC-seq) and transcriptome (snRNA-seq) maps across 287 frozen human skeletal muscle biopsies representing nearly half a million nuclei. We identify 13 cell types and integrate genetic variation to discover >7,000 expression quantitative trait loci (eQTL) and >100,000 chromatin accessibility QTLs (caQTL) across cell types. Learning patterns of e/caQTL sharing across cell types increased precision of effect estimates. We identify high-resolution cell-states and context-specific e/caQTL with significant genotype by context interaction. We identify nearly 2,000 eGenes colocalized with caQTL and construct causal directional maps for chromatin accessibility and gene expression. Almost 3,500 genome-wide association study (GWAS) signals across 38 relevant traits colocalize with sn-e/caQTL, most in a cell-specific manner. These signals typically colocalize with caQTL and not eQTL, highlighting the importance of population-scale chromatin profiling for GWAS functional studies. Finally, our GWAS-caQTL colocalization data reveal distinct cell-specific regulatory paradigms. Our results illuminate the genetic regulatory architecture of human skeletal muscle at high resolution epigenomic, transcriptomic, and cell-state scales and serve as a template for population-scale multi-omic mapping in complex tissues and traits.

genomics↗

bioRxiv2023

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Most genome-wide association signals for complex disease reside in the noncoding genome, where defining function is nontrivial. MPRAs (massively parallel reporter assays) offer a scalable means to identify functional regulatory elements, but are typically conducted without regard to cell type, pairing cloned fragments with a generic housekeeping promoter. To explore the context-sensitivity of MPRAs, we screened enhancer activity across a panel of nearly 12,000 198-bp fragments spanning over 300 type 2 diabetes- and metabolic trait-associated regions in the 832/13 rat insulinoma beta cell line, a relevant model of pancreatic beta cells. We explored these fragments context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin (INS) gene. We identified clear effects of MPRA construct design on enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. Promoter choice also influenced MPRA activity (n = 698/11,656), mostly biased towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory regions. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

genomics↗

bioRxiv2023

Benchmarking splice variant prediction algorithms using massively parallel splicing assays

BackgroundVariants that disrupt mRNA splicing account for a sizable fraction of the pathogenic burden in many genetic disorders, but identifying splice-disruptive variants (SDVs) beyond the essential splice site dinucleotides remains difficult. Computational predictors are often discordant, compounding the challenge of variant interpretation. Because they are primarily validated using clinical variant sets heavily biased to known canonical splice site mutations, it remains unclear how well their performance generalizes. ResultsWe benchmarked eight widely used splicing effect prediction algorithms, leveraging massively parallel splicing assays (MPSAs) as a source of experimentally determined ground-truth. MPSAs simultaneously assay many variants to nominate candidate SDVs. We compared experimentally measured splicing outcomes with bioinformatic predictions for 3,616 variants in five genes. Algorithms concordance with MPSA measurements, and with each other, was lower for exonic than intronic variants, underscoring the difficulty of identifying missense or synonymous SDVs. Deep learning-based predictors trained on gene model annotations achieved the best overall performance at distinguishing disruptive and neutral variants. Controlling for overall call rate genome-wide, SpliceAI and Pangolin also showed superior overall sensitivity for identifying SDVs. Finally, our results highlight two practical considerations when scoring variants genome-wide: finding an optimal score cutoff, and the substantial variability introduced by differences in gene model annotation, and we suggest strategies for optimal splice effect prediction in the face of these issues. ConclusionSpliceAI and Pangolin showed the best overall performance among predictors tested, however, improvements in splice effect prediction are still needed especially within exons.

bioinformatics↗

bioRxiv2022

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived PKR antagonist RhTRS1 in place of its native PKR antagonists; E3L and K3L (VACV{Delta}E{Delta}K+RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a "molecular foothold" to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACV{Delta}E{Delta}K+RhTRS1 replication in human cells, mediated by both PKR and RNase L. We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage nine. Using our Illumina-based pipeline, we found that some SNPs which had evolved during the prior AGM adaptation were rapidly lost, while 13 singlebase substitutions and short indels increased over time, including two SNPs unique to HFF adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an "intermediate species" and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

evolutionary biology↗

bioRxiv2020

Massively parallel functional testing of MSH2 missense variants conferring Lynch Syndrome risk

The lack of functional evidence for the majority of missense variants limits their clinical interpretability, and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed "variants of uncertain significance" (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the [~]1,300 extant missense VUSs in MSH2, and may facilitate the prospective classification of newly discovered variants in the clinic.

genetics↗

bioRxiv2019

Integrating enhancer RNA signatures with diverse omics data identifies characteristics of transcription initiation in pancreatic islets

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ~20% of which are islet-specific and occur mostly distal to known gene TSSs. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validate transcriptional enhancer activity (5% FDR) for 2,279 of 3,378 (~68%) tested islet CAGE elements. TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting functional enhancers at GWAS loci.

genomics↗

bioRxiv2019

Chromatin information content landscapes inform transcription factor and DNA interactions

Interactions between transcription factors (TFs) and chromatin are fundamental to genome organization and regulation and, ultimately, cell state. Here, we use information theory to measure signatures of TF-chromatin interactions encoded in the patterns of the accessible genome, which we call chromatin information enrichment (CIE). We calculate CIE for hundreds of TF motifs across human tissues and identify two classes: low and high CIE. The 10-20% of TF motifs with high CIE associate with higher protein-DNA residence time, including different binding sites subclasses of the same TF, increased nucleosome phasing, specific protein domains, and the genetic control of both gene expression and chromatin accessibility. These results show that variations in the information content of chromatin architecture reflect functional biological variation, with implications for cell state dynamics and memory.

genomics↗