Linking Mitochondrial Calcium to Histone Methylation Underlying Lung Regeneration

PROJECT SUMMARY/ABSTRACT Chronic Respiratory Diseases (CRDs) are the third leading cause of death and disability in the United States, with a significant impact on the sociobiological and economic health of the country. Many patients with CRDs present with dysregulation of lung healing after injury. Therefore, there is an unmet need to better understand the molecular mechanisms underlying lung regeneration in order to develop novel treatment strategies for these patients. Most of the gas exchange region of the lung is lined by Alveolar Epithelial Type 1 (AT1) cells. When these cells are damaged, Alveolar Epithelial Type 2 (AT2) cells activate, proliferate, and differentiate into new AT1 cells to replace them. Failure of AT2-to-AT1 differentiation contributes to the pathogenesis of CRDs. However, the mechanisms underlying this transition are not completely understood. Data suggest that expression of Mitochondrial Calcium Uptake 1 (MICU1) protein, as well as a reduction of trimethylated histone H3 residues lysine 9 (H3K9me3) and lysine 36 (H3K36me3), are both vital for AT2-to-AT1 differentiation. We hypothesize that alterations in MICU1-dependent mitochondrial calcium uptake drive AT2 differentiation by rewiring cellular metabolism to favor the accumulation of α-ketoglutarate at the expense of succinate, thereby increasing the activity of α-ketoglutarate-dependent histone demethylases and promoting an AT1 pattern of gene expression. Therefore, our specific aims are to: (1) Determine the distribution of H3K9me3 and H3K36me3 marks across the genome, and their functional consequences, during AT2-to-AT1 cell differentiation in AT2 wildtype and MICU1 knockout cells, and (2) Determine whether increased H3K9me3 and H3K36me3 are responsible for the decreased AT2-to-AT1 differentiation capacity of AT2 MICU1 knockout cells. Aim 1 will utilize CUT&RUN, ATAC-Seq, and RNA-Seq, while Aim 2 use CRISPR/Cas9 gene editing both in vitro and in vivo. The proposed experiments will take place in the Center for Translational Medicine in the Lewis Katz School of Medicine at Temple University, an institution with a track record of excellence in both clinical medicine and scientific research. The training plan, developed by Co-Sponsors Drs. Tian and Elrod, will be tailored to ensure success of the applicant, Morgan Pantuck, as she progresses through Temple's MD/PhD program. Overall, this grant will provide an excellent training vehicle for the applicant while also increasing our understanding of the molecular mechanisms underlying lung regeneration, supporting development of novel treatments for CRDs.