Chromatin folding is a key step to pack DNA molecules 10,000-fold into a germ cell, but exactly how the meiotic chromatin is folded and how spatiotemporal folding impacts transcription, chromosome pairing, and recombination remains largely mysterious. Homologous chromosome pairing and recombination are required for accurate chromosome segregation. Mis-segregation of homologous chromosomes is a major cause of miscarriage and birth defects (e.g., Down Syndrome). Three papers have recently reported that high-order chromatin organizations/domains dynamically shape recombination landscape and germline transcriptomes. These dramatic chromatin reorganizations depend on chromatin axes because the domain boundary proteins (e.g., cohesins) are located in the axes. What remains unknown is 1) how meiotic chromosome axis contributes to meiotic gene transcription, homologous chromosome pairing, and chromosome stiffness via chromatin reorganization; and 2) whether the meiotic chromosome structure dynamics are sex- and stage- specific. Our long-term goal is to decipher the meiotic genome organization and its roles in transcription, homologous pairing, and recombination. The proposed work here will specifically test the overall hypothesis that meiotic chromosome axes regulate transcriptome and homologous pairing via controlling local and global chromatin folding in a stage- and sex- dependent manner. To test this hypothesis, we will pursue three specific aims: Determine whether meiotic chromosome axis regulates 1) transcription and homologous pairing via reorganizing local chromatin loops 2) chromosome stiffness and pairing by mediating global chromatin folding. 3) Uncover the temporal and sexual differences of meiotic transcriptomes and chromatin organization. Method:
In aim 1, local chromatin reorganization in spatial domains will be detected by chromosome conformation capture (Hi-C) contact map and the corresponding changes of transcriptional levels within these domains can be measured via RNAseq. Chromosome interactions will be examined locally by Hi- C analysis (aim 1) and stiffness will be measured globally by micromanipulation (aim 2). Fluorescent in situ hybridization will be used to verify the intra- and inter-chromosome interaction found in Hi-C map. Hi-C, single- cell RNAseq, and micromanipulation will be introduced to reveal the 4D meiotic genome reorganization and sexual dimorphism of transcriptome and chromatin folding in aim 3. The approach is innovative because multiple advanced methods including Hi-C, single-cell RNAseq, micromanipulation, nano-newton force measurement, and quantitative immunocytology will be integrated to generate a more complete picture of meiotic chromosomes. The proposed research is significant because it is expected to provide a deeper understanding of chromosome structure and how the structure varies in different stages and genders. Ultimately, insights from these studies will help us develop diagnosis and treatment for infertility, miscarriage, and birth defects.
Aneuploidy, mainly due to the defects of homologous chromosome segregation, is a common cause of human diseases, including infertility, miscarriage, and birth defects. The research proposed here will elucidate the 4D genome organization of meiotic chromosomes for both males and females, which provides insight information of meiotic gene regulation and homologous chromosome pairing and segregation. A better understanding of meiotic chromosome structure will help us elucidate the abnormal chromosome behaviors and shed light on the diagnose and treatment of infertility and aneuploidy-induced diseases.