Control and function of epigenetic reconfiguration in pollen

Correct inheritance of epigenetic marks of the genome and small RNAs across the next generation is essential for the normal development of mammals and plants. A unique vital feature of gametogenesis is resetting the genetic and epigenetic plan for totipotency. Current evidence suggests that in the germ cell lineage of mammals and plants, epigenetic reprogramming occurs on a genome-wide scale, which profoundly reconfigures the landscape of DNA methylation and chromatin dynamics. The purpose of the proposed work is to investigate how these reprogramming events are controlled and play roles in the plant male gametophyte (pollen) using the model plant Arabidopsis thaliana. Specifically, this research program is designed to (1) elucidate roles for active DNA demethylation in pollen, which is a process that requires the DEMETER DNA glycosylase, and (2) elucidate mechanisms that regulate reconfigurations of chromatin dynamics in pollen. The genetic and functional genomic approaches in Part 1 of the research project will provide novel insights into the function of DNA demethylation in plant reproduction. The analysis of the heterochromatin decondensation defective 1 (hdd1) mutant and the isolation of hdd1 suppressors in Part 2 of the project will help to understand the control and function of the dynamic chromatin remodeling during gametogenesis.

Centromeres are the fundamental unit required for segregation of chromosomes during cell division, and they are defined by the centromere-specific histone H3 variant CenH3/CENP-A. Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. In contrast to the relatively well-known process of de novo assembly of CenH3 at centromeres, little is known about how CenH3 is actively removed, an essential biological process during the life of a cell to prevent cancer. We discovered the process of centromere disassembly, demonstrating that it occurs via an active, proteolytic mechanism directed by the evolutionarily conserved AAA-ATPase molecular chaperone CDC48A. The terminally differentiated pollen vegetative cell of the flowering plant Arabidopsis thaliana undergoes loss of CenH3; centromeric heterochromatin decondensation; and translocation of bulk ribosomal RNA (rRNA) gene (rDNA) repeats loci into the nucleolus, a sub-nuclear compartment, where rRNA gene transcription takes place. We found that mutations in the CDC48A gene block all of these dynamic chromosome processes. rDNA transcriptional activity and cell growth are coupled processes. The pollen vegetative cell form a tube by rapid extended tip growth to transport two sperm cells to the ovule over long distances for fertilization, whch demands high rDNA transcriptional activity. Consisent with this notion, cdc48a mutations also block the single nucleus-driven pollen tube growth, and cause male sterility. We propose that the CDC48A molecular chaperone actively removes CenH3 from centromeres and disrupts centromeric heterochromatin to transcribe whole rRNA gene repeats, which facilitates ribosome biogenesis and protein synthesis, and ultimately fuels pollen tube formation and is essential for plant reproduction.