H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora

Heterochromatin is a key feature of eukaryotic genomes. In the filamentous fungus Neurospora crassa, the mechanism leading to heterochromatin initiation is unknown. Here, we show that casein kinase II (CKII) are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA co-localizes with phosphorylated H3T11 (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3, and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation. Overall design: Bisulfite-sequencing sample preparation was performed using the MGIEasy WGBS Library Prep Kit (1000005251;MGI) for DNBSEQ™ platform (MGI) and the EZ DNA Methylation-Gold Kit (Zymo Research). Raw reads that pass quality control were performed with Bismark software (version 0.23.1) for alignment and quantification. Clean reads were mapped to N. crassa OR74A (NC10) genome using Bowtie2 (version 2.4.4). Only unique mappers were retained for downstream analyses. The position and density of 5mC was evaluated by Bismark software with command bismark methylation extractor and visualized with the Integrative Genomics Viewer (IGV). The pipeline for WGBS data processing, quantification and visualization are available on GitHub.