Endometriosis, adenomyosis, and uterine fibroids are prevalent gynecologic disorders causing pain, infertility, and reduced quality of life. Despite their impact, the molecular mechanisms underlying these conditions remain poorly understood, and robust genetic mouse models are lacking. We investigated whether the epigenetic enzyme SUV4-20H2, which deposits the histone modification H4K20me3 to compact chromatin and suppress cell proliferation, regulates uterine homeostasis and female fertility. Using Suv4-20h2 knockout mice, we performed fertility trials, histological analysis, immunohistochemistry, and RT-qPCR on uterine tissue. Fertility trials revealed a consistent pattern of delayed litter intervals in knockout females across multiple breeding cycles compared to wild-type controls. Histological analysis revealed fluid-filled uterine cysts and disrupted glandular architecture resembling adenomyosis. Immunohistochemistry demonstrated increased epithelial proliferation in knockout uteri. Gene expression analysis revealed elevated Wnt4, Muc1, and Ctnnb1, genes previously associated with endometriosis and aberrant uterine proliferation. These findings suggest that H4K20me3-mediated chromatin repression is required to maintain uterine epithelial quiescence, and that its loss drives proliferative, disease-like uterine phenotypes. Suv4-20h2 knockout mice may represent a novel genetically defined epigenetic model of uterine disease, with potential implications for identifying SUV4-20H2 and H4K20me3 as diagnostic markers or therape

Bohring-Opitz Syndrome (BOS) is a neurodevelopmental disease caused by de novo truncating mutations in ASXL1, an important chromatin regulator, and often presents with intellectual disability, distinctive musculoskeletal features, and seizures. There are currently no therapeutics to treat BOS, only to mitigate symptoms. Given this, functional assays are needed to determine how truncating mutations in ASXL1 affect protein-protein and protein-genome interactions compared to those of the wild-type ASXL1 protein. With no reliable anti-ASXL1 antibody, we developed a stable inducible ASXL1 overexpression system in induced pluripotent stem cells (iPSCs) to study the effect mutations in ASXL1 have on early neurodevelopment. After validating our system, we are optimizing Immunoprecipitation-Mass Spectrometry (IP-MS) and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) to identify the binding partners of ASXL1 at the protein and genomic level. We also noted increased stability of the truncated protein compared to wild type ASXL1 in iPSCs. Interestingly, many known/predicted ubiquitination sites are clustered at the C-term of the protein, and previously described BOS mutations result in the loss of many of these predicted ubiquitination sites. This may suggest a possible gain-of-function mechanism for BOS, and data from our IP-MS and ChIP-Seq analysis will shed light on novel interactions associated with truncated ASXL1 that are distinct from the normal function of the canonical protein.

Strongylocentrotus purpuratus may seem unlikely to learn about the human genus from; however, radial spoke head protein 9 homolog, a gene in this organism, also exists in humans, and its mutations are associated with the disease primary ciliary dyskinesia. In an interest to learn more about a gene indicated in human disease, this study’s goal was to identify and clone the coding region of a strongylocentrotus purpuratus gene for future use. Identification of the gene was performed using BLAST, Echinobase, and InterPro sequence analysis tools. For gene cloning, PCR, Gel electrophoresis, and DNA purification were performed, with band sizes from gel electrophoresis of purified and non-purified PCR products yielding percent errors between 3.34% - 16% from expected. Ligation reactions, bacterial competent cell preparation and transformation, colony PCR, and gel electrophoresis of colony PCR products were also performed, with band sizes from gel electrophoresis of colony PCR products yielding percent errors between 0.11% – 8.56% from expected. Bacterial inoculation, plasmid DNA isolation, restriction digests, and sanger sequencing commenced the cloning process, with sanger sequencing analysis yielding no statistically significant results regarding cloned gene identity. Lastly, phylogenetic analysis yielded a phylogenetic tree displaying evolutionary relationships that matched the expected. Thus, the study concluded sufficient evidence that the coding region of the gene was identified but insufficient evidence that it was cloned.

Gene regulation is mediated by cis-regulatory elements (CREs), which are bound by transcription factors (TFs) to drive differential expression across thousands of genes. The regulatory grammar of CREs involves the combinatorial arrangement of TF binding sites, which governs the cooperative interactions underlying transcriptional responses. The bone morphogenetic protein pathway activates SMAD proteins, which form heteromeric transcription factor complexes that bind low-affinity motifs to drive osteogenic differentiation. The master regulator Runx2 and the Yamanaka factor KLF4 have been shown to interact with SMAD to regulate gene expression. However, the rules of “AND” grammar (regulation via simultaneous binding) in CREs involving SMAD and these TFs remain unclear. Here, we investigate the regulatory grammar of SMAD with Runx2 or KLF4 by analyzing chromatin immunoprecipitation sequencing (ChIP-seq) datasets to identify co-occupied enhancers. I quantified the spacing between motifs and assessed the frequency of specific distances for statistical enrichment. SMAD-Runx2 binding site spacings were enriched at approximately 50 base pairs (bps), while SMAD-KLF4 binding site spacings were enriched at approximately 65 bps. These results suggest that the cooperative interactions of these TFs may depend on defined spatial structures within CREs. This work provides further insight into the structural basis of regulatory grammar and how TF organization controls gene expression.

Mitochondrial diseases are a heterogeneous group of conditions characterized by defects in oxidative phosphorylation. A hallmark of these diseases is the accumulation of dysfunctional mitochondria, which contribute to oxidative stress. Mitophagy, the selective autophagic degradation of damaged mitochondria, is critical for maintaining mitochondrial quality control, and is frequently impaired in mitochondrial diseases. During mitophagy, dysfunctional mitochondria are engulfed by an autophagosome and delivered to lysosomes, where their fusion forms mitolysosomes. Because mitophagy relies on the proper function of autophagy machinery, evaluating autophagic dynamics is essential to determine whether mitochondrial clearance defects arise from mitophagy-specific failures or broader autophagic disruption. We selected three patient-derived fibroblasts with mitochondrial disease that demonstrate mitophagy impairment, two with an increased number of mitolysosomes per cell and one with a decreased number, relative to control fibroblasts. To further explore autophagy dynamics in these cells, we monitored LC3 accumulation over time, a marker of autophagosome formation, to quantify autophagic flux. Together, these data will elucidate the differences in mitochondrial disease states relative to one another and control. Further, this work will determine whether impaired mitochondrial clearance results from broader autophagic failure or a defect specific to mitophagy, providing insights into strategies to restore organelle homeostasis.