Katsumi Kitagawa, Pharm.D., PhD, to Receive NIH Grant to study “The Role of EWSR1 at the Centromere.”

The centromere is a unique chromosomal region that is essential for high-fidelity chromosome transmission during cell division. In mitosis, microtubules attached to the kinetochore help ensure the faithful segregation of sister chromatids into daughter cells. Our goal is to understand the mechanisms that govern centromere function. Centromere identity relies on the deposition of the centromere-specific histone H3 variant, CENP-A but not on specific DNA sequences. After DNA replication, “old” centromeric nucleosomes are transferred onto the newly replicated chromatids. In mammals, deposition of CENP-A occurs in the G1 phase of the cell cycle. The mechanisms of CENP-A deposition and maintenance are crucial for proper centromere inheritance and function but remain to be delineated. We recently found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition and maintenance at the centromere. We show that EWSR1 and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma) bind to CENP-A through the SYGQ2 region within its prion-like domain known to be important for phase separation. We found that EWSR1 is required for CENP-A deposition at the centromere. CENP-A is associated with centromeric RNA, which is important for centromere function. We demonstrate that EWSR1 plays a key role in promoting this association. We also have biochemical evidence that EWSR1 binds R-loops, a three-stranded RNA-DNA hybrid, through its RNA-recognition motif. Based on these findings, we hypothesize that EWSR1 plays crucial roles in maintaining centromere structure and function by binding RNA and anchoring CENP-A in centromeric chromatin. We will test this hypothesis. Aim 1: To determine the function of EWSR1 and RNA/R-loops in centromere maintenance. We generated EWSR1 conditional KO cells using an AID (auxin-inducible degron) system, which allows us to remove EWSR1 upon auxin addition. We will test if auxin-induced degradation of EWSR1 abolishes CENP-A signals at the centromere at different cell cycle stages. We will induce the expression of SETX, which clears RNA-DNA hybrids, at different cell cycle stages to ask whether centromeric RNA/R-loops are required for CENP-A maintenance at the centromere. Aim 2: To investigate the function of EWSR1-CENP-A interaction. We will generate EWSR1 mutants defective in binding to CENP-A by introducing mutations into the SYGQ2 region of EWSR1. We will examine the phenotypes of the EWSR1 KO cells expressing CENP-A binding deficient mutants. We will also identify the EWSR1-binding domain of CENP-A. We will then isolate CENP-A mutants impaired for EWSR1 binding. We will express Flag-tagged mutants in CENP-A KO cells to examine the localization of CENP-A and the phenotypes of the mutant cells. We will investigate if EWSR1 association with CENP-A is unique for interphase. We will investigate the mechanism that regulates cell cycle specificity of EWSR1-CENP-A interaction. Aim 3: To test if EWSR1 anchors CENP-A at the centromere via RNA binding. To test this model, we will directly examine this model by constructing and testing CENP-A and EWSR1 variants with engineered functional domains.

Project Narrative/Relevance Statement Our studies will make important contributions to help clarify the mechanisms of centromere maintenance and specify the role of EWSR1 in centromere function and chromosome segregation, which will lead to insights relevant to health and disease, including cancer, birth defects, and developmental disorders such as Down syndrome.