PAF1 N-terminal extension-generated PAF1c-SKIP-CDKB module regulates plant-specific cell cycle in the plant kingdom

Through systematic biochemical analyses, we identified a novel protein-protein interaction between PAF1 and SKIP in Chlamydomonas reinhardtii. To investigate the functional significance of this interaction, we generated targeted mutants using CRISPR-Cas9 genome editing technology. Phenotypic characterization revealed that both PAF1 and SKIP mutants exhibit coordinated regulation of the cell cycle progression. To elucidate the molecular mechanism underlying PAF1-SKIP mediated cell cycle regulation, we employed an integrated multi-omics approach. RNA sequencing (RNA-seq) analysis was performed to identify differentially expressed genes, while chromatin immunoprecipitation sequencing (ChIP-seq) was utilized to map the genomic loci directly bound by the PAF1-SKIP protein complex. This comprehensive strategy enabled us to delineate the transcriptional network governed by the PAF1-SKIP regulatory module. The Chip experiment was conducted using three materials: CrWT, CrPAF1-HA, and CrSKIP-HA. Chlamydomonas cells were cultured and harvested, then cross-linked with 1% formaldehyde. After terminating the cross-linking with Gly, the cells were sonicated in lysis buffer, followed by immunoprecipitation (IP) using HA antibody. The resulting products were subjected to DNA recovery and subsequent Chip-seq sequencing analysis.