Browsing by Subject "H2A.Z"

Uterine leiomyomas (ULs) are common benign tumors that originate from the smooth muscle cells of the uterine wall known as the myometrium. Around 70% of pre-menopausal women are affected by these tumors. The high prevalence of ULs is a significant public health issue and ULs are the leading cause for hysterectomy. Many tumors remain asymptomatic, but 15-30% of affected women develop symptoms ranging from pain and heavy menstrual bleeding to pregnancy complications and infertility. Despite their common occurrence, the underlying mechanisms of UL genesis are still largely unknown. Based on mutually exclusive recurring genetic alterations, ULs can be divided into molecular subclasses. Three main molecular subclasses have been established: MED12 mutated tumors, HMGA2 overexpressing tumors and tumors with biallelic FH inactivation. Combined, these three subclasses represent around 90% of ULs, indicating that additional smaller molecular subclasses also exist. Recently, novel mutations associated with ULs have been identified in genes encoding for subunits of the SRCAP chromatin remodeling complex that deposits histone variant H2A.Z onto chromatin. These included loss-of-function mutations in YEATS4, DMAP1 and ZNHIT1, and resulted in deficient H2A.Z loading in the tumors. The detailed functional consequences of these driver mutations need to be further investigated to fully understand their significance in UL genesis. This work aimed to elucidate the effects of YEATS4 mutations by characterizing previously established CRISPR-Cas9 edited immortalized human myometrial cell models carrying heterozygous mutations in YEATS4 using various molecular biology methods. Subcellular fractionation and western blot analysis was used to detect chromatin bound H2A.Z from cell lysates. Quantitative PCR was performed to determine relative YEATS4 expression levels in mutated and wild-type cells. No significant reduction of chromatin bound H2A.Z or YEATS4 expression was observed in the studied heterozygous mutants when compared to wild-type immortalized myometrial smooth muscle cells. Additional myometrial cell models were created by CRISPR-Cas9 gene editing. Objective was to achieve homozygous YEATS4 mutations to better reflect the changes previously reported in ULs. One homozygous YEATS4 mutant cell line was achieved. Understanding the detailed molecular mechanisms behind UL genesis will be instrumental for developing curative non-invasive therapies in the future. Insight into dysregulated pathways and identification of UL biomarkers could improve diagnostic accuracy and help design personalized targeted therapies effective for specific UL subclasses. Characterization of each molecular subclass offers a unique opportunity to understand UL genesis.