We know that defects in recombination and synapsis between homologous chromosomes during meiotic prophase will result in infertility, pregnancy loss or genetic defects. However, we know very little about how these events are coordinated with meiotic cell cycle progression. Our preliminary data implies that the PLKs (Polo-like kinases), PLK1 and PLK4, ensure that these meiotic events are coordinated with chromosome segregation. We have generated a Plk1 conditional knockout (CKO) mouse to delineate PLK1 functions during meiosis. To assess temporally distinct roles, we will delete Plk1 at three different stages prior to the prophase to metaphase I (G2/MI) transition of meiosis (Aim 1a). Based on our preliminary data we hypothesize that PLK1 ensures timely resolution of homologous recombination events, proficient desynapsis between homologs and protection of sister chromatid cohesion during meiosis I. The Plk1 CKO models will be used to determine the substrates that are phosphorylated by PLK1 to ensure accurate meiotic progression (Aim 1b). Our preliminary data shows that PLK1 phosphorylates two proteins required for homolog synapsis, and these modifications are required to stimulate desynapsis during the G2/MI transition. During mammalian meiosis, the X-Y chromosomes pair and are transcriptionally silenced via a specialized DNA damage response known as Meiotic Sex Chromosome Inactivation (MSCI). In addition, regions of homologous chromosomes that fail to synapse are also subject to this DDR, in a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC). Errors in MSCI and MSUC result in meiotic failure and chromosome missegregation. PLK4 colocalizes with these DDR proteins on the X-Y and unsynapsed chromosomes. Using a Plk4 CKO mouse and a dominant negative allele of Plk4, we will assess the meiotic defects that ensue (Aim 2a). We hypothesize that PLK4 is required for signaling aberrant homolog synapsis and ensuring robust MSCI and MSUC signaling. We have determined that PP4R3A (Protein Phosphatase 4 Regulatory Subunit 3A) is phosphorylated by PLK4. PP4R3A is a component of the protein phosphatase, PP4, which is required to dephosphorylate histone H2AFX following DNA damage repair. However, H2AFX must remain phosphorylated on the X-Y chromosomes during MSCI. Using biochemical assays we will test whether phosphorylation of PP4R3A by PLK4 inhibits PP4-mediated H2AFX dephosphorylation (Aim 2b). By delineating the novel roles of PLK1 and PLK4 during meiosis, we will develop new concepts of how meiotic processes are regulated and coordinated. Our proposed research will contribute to the diagnosis and prevention of genetic abnormalities that cause birth defects, affect physical and mental development, and increase the risk of cancer and infertility.
During the formation of sperm and eggs, chromosomes must segregate accurately. However, approximately 10% of clinically reported pregnancies are chromosomally abnormal, resulting in pregnancy loss or genetic defects such as Down syndrome. Very little is known about the genetic failures that cause these chromosomal abnormalities in sex cells; however, we will significantly enhance our knowledge of this phenomenon by achieving the goals set within this proposal.
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