Integrating leiomyoma genetics, epigenomics, and single-cell transcriptomics reveals causal genetic variants, genes, and cell types.

Uterine fibroids (UF), that can disrupt normal uterine function and cause significant physical and psychological health problems, are observed in nearly 70% of women of reproductive age. Although heritable genetics is a significant risk factor, specific genetic variations and gene targets causally associated with UF are poorly understood. Here, we performed a meta-analysis on existing fibroid genome-wide association studies (GWAS) and integrated the identified risk loci and potentially causal single nucleotide polymorphisms (SNPs) with epigenomics, transcriptomics, 3D chromatin organization from diverse cell types as well as primary UF patient's samples. This integrative analysis identifies 24 UF-associated risk loci that potentially target 394 genes, of which 168 are differentially expressed in UF tumors. Critically, integrating this data with single-cell gene expression data from UF patients reveales the causal cell types with aberrant expression of these target genes. Lastly, CRISPR-based epigenetic repression (dCas9-KRAB) or activation (dCas9-p300) in a UF disease-relevant cell type further refines and narrows down the potential gene targets. Our findings and the methodological approach indicate the effectiveness of integrating multi-omics data with locus-specific epigenetic editing approaches for identifying gene- and celt type-targets of disease-relevant risk loci.

Engineered MED12 mutations drive leiomyoma-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.

Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the MED12 gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.

Increased Ca2+-dependent intrinsic tone and arterial stiffness in mesenteric microvessels of hypertensive pregnant rats.

Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanisms. We have shown increased vascular reactivity to extrinsic vasoconstrictors in HTN-Preg rats. Here, we test whether microvascular intrinsic tone and arterial stiffness could contribute to HTN-Preg, and examined the underlying cellular mechanisms. On gestational day 19, BP was recorded in normal pregnant (Preg) rats and Preg rats with reduced uterine perfusion pressure (RUPP), and mesenteric microvessels were mounted on a pressure myograph for measurement of intrinsic tone, simultaneous changes in [Ca2+]i (fura-2 340/380 ratio), and arterial stiffness. Arteries were incubated in Ca2+-containing and 0 Ca2+ (2 mM EGTA) Krebs, pressurized at 10 to 110 mmHg in 10 mmHg increments, and the % change in vessel diameter from initial diameter at 10 mmHg was analyzed for measurement of total (active + passive) intrinsic tone and passive intrinsic response, respectively. The passive response was then subtracted from the total intrinsic tone to determine the active myogenic tone. The strain-stress relationship was also constructed as a measure of arterial stiffness. BP was higher in RUPP vs Preg rats. In Ca2+-containing Krebs, increases in intraluminal pressure caused smaller increases in diameter and greater increases in [Ca2+]i in microvessels of RUPP vs Preg rats, suggesting increased Ca2+-dependent myogenic tone. In 0 Ca2+ Krebs, increases in pressure also caused less increases in diameter in microvessels of RUPP vs Preg rats, but with no changes in [Ca2+]i, suggesting changes in the structure and mechanics of the arterial wall. The total and passive strain-stress relationship was shifted to the left in microvessels of RUPP vs Preg rats, suggesting increased arterial wall stiffness. Histology and immunohistochemistry showed greater vascular wall thickness and collagen-I staining in RUPP vs Preg rats, supporting changes in the wall architecture and structural proteins. The increased active myogenic tone and underlying increases in Ca2+ signaling as well as the increased passive intrinsic response, arterial stiffness and collagen-I in the mesenteric microvessels could play a role in the regulation of blood flow to the splanchnic region and the increased vascular resistance and BP in HTN-Preg.