Uterine leiomyoma (LM, fibroid) is the most common tumor in women. No long-term medical treatment is available. Each LM seems to originate from the clonal expansion of a single mutated LM stem cell (LSC) in the myometrium (MYO). LSC comprise 5% of tumor mass and differentiate into an intermediate cell population (LIC, 7%), which then become terminally differentiated cells (LDC) comprising 88% of the tumor bulk. Driver mutations of mediator complex subunit 12 (mut-MED12) occur in 70% of all LM. Progesterone (P4) and its receptor PR are essential for LM growth. PR-rich LIC transduce P4 signaling to PR-deficient LSC via paracrine factors to activate their proliferation. Ulipristal acetate (UPA), a PR-selective P4 antagonist, shrank LM and reduced its symptoms, but its use was halted because of risk of liver injury. Our overall goal is to define the role of genomewide P4 action in the etiology of mut-MED12‒associated LM tumorigenesis and identify novel therapeutic targets. We found that mut-MED12 physically interacts with PR and genomewide PR-chromatin interaction landscapes are dramatically dysregulated in LM expressing mut-MED12 vs. normal MYO tissue carrying wild type MED12. Mut-MED12 enhances PR recruitment to cis-regulatory elements of P4 target genes encoding paracrine growth factors, cytokines and extracellular matrix proteins critical for LSC proliferation and LM tumorigenesis. Furthermore, the uteri of mice with a human-equivalent gain-of-function mutation in Med12 develop LM in response to P4, whereas Med12 knockout blocks P4/PR signaling in mouse uterus. We hypothesize that mut-MED12 alters PR-chromatin interaction signatures and enhances P4 action in LIC, providing a support niche for LSC survival and proliferation. Using ChIP-seq, RNA-seq, STARR-seq, and CRISPR/Cas9-gene editing strategies, and in vivo PDX and mut-Med12 knock-in mouse models, we propose the following Aims: (1) Determine whether PR-chromatin interaction loci specifically associated with mut- MED12 stimulate P4 action in a distinct stem-support cell population (LIC), which then send tumorigenic paracrine signals to increase LSC activity and tumor growth. We will test the hypothesis that mut-MED12 interacts with PR and alters its interaction with chromatin, thereby enhancing P4 responsiveness of LICs to activate gene transcription and paracrine signaling that support the function of adjacent LSC. (2) Define whether tumorigenic activity of mut-Med12 is mediated via altering PR-chromatin interaction landscapes in a human-equivalent mut-Med12 mouse model. We will test the hypothesis that mut-Med12 disrupts chromatin features surrounding PR-binding sites, thereby supporting gene transcription and pathways critical for LM tumorigenesis, whereas UPA shrinks LM via altering the aberrant PR-chromatin-epigenomic interactions and reversing inappropriate expression of disease-associated genes. Deciphering the genomewide mechanisms at a defined cell population level will help us identify genotype-specific novel targets associated with mut-MED12 for pharmacogenomics and precision medicine in the treatment of LM.