Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6_Chromatin occupancy of Rad6 and Swd2 in several strains.

Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2 and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. We investigated the genome-wide occupancy patterns of Rad6, Swd2 and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both 5’region and 3’region of genes, which are overlapped with its tightly bound two complexes, Set1 and CPF (Cleavage and Polyadenylation Factor), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5ʹ region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5’region was impared and rather increased in the 3’ region. This study highlights that catalytic activity of Rad6 is essential for all the ways of Swd2’s binding to the transcribed genes and Set1 redistributes the Swd2 to 5’region for accomplishments of H3K4me3 in the genome-wide level. Sample number (1) ~ (5): We performed a-HA ChIPseq with strains expressing Swd2-3HA at its chromosomal locus in wildtype or ∆rad6 background to identify the chromatin occupancy of Swd2-3HA by the presence or absence of Rad6. Experiments were performed in duplicates, and we performed a-HA ChIPseq with a yeast strain without any epitope tag ((5)Notag) to reveal the background signal of the experiment. Sample number (6) ~ (12): We performed a-Myc ChIPseq with strains expressing Rad6-9Myc at its chromosomal locus in wildtype to identify the chromatin occupancy of Rad6-9Myc. Also, we performed a-Myc ChIPseq with yeast strains expressing Swd2-3HA and Rad6-9Myc (or Rad6-C88A-9Myc) at its chromosomal loci to identify the change of the chromatin occupancy of Rad6-9Myc by the catalytic activity of Rad6. Experiments were performed in duplicates, and we performed a-Myc ChIPseq with a yeast strain without any epitope tag ((12)Notag) to reveal the background signal of the experiment. Sample number (13) ~ (21): We performed a-HA ChIPseq with yeast strains expressing Swd2-3HA and Rad6-9Myc (or Rad6-C88A-9Myc) at its chromosomal loci to identify the change of the chromatin occupancy of Swd2-3HA by the catalytic activity of Rad6. Also, we performed a-HA ChIPseq with strains expressing Swd2-6HA at its chromosomal locus in wildtype or ∆set1 background to identify the change of chromatin occupancy of Swd2-6HA by the presence or absence of Set1. Experiments were performed in duplicates, and we performed a-HA ChIPseq with a yeast strain without any epitope tag ((21)Notag) to reveal the background signal of the experiment.