A Novel Domain in Set2 Mediates RNA Polymerase II Interaction and Couples Histone H3 K36 Methylation with Transcript Elongation

Abstract

Histone methylation and the enzymes that mediate it are important regulators of chromatin structure and gene transcription. In particular, the histone H3 lysine 36 (K36) methyltransferase Set2 has recently been shown to associate with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), implying that this enzyme has an important role in the transcription elongation process. Here we show that a novel domain in the C terminus of Set2 is responsible for interaction between Set2 and RNAPII. This domain, termed the Set2 Rpb1 interacting (SRI) domain, is encompassed by amino acid residues 619 to 718 in Set2 and is found to occur in a number of putative Set2 homologs from Schizosaccharomyces pombe to humans. Unexpectedly, BIACORE analysis reveals that the SRI domain binds specifically, and with high affinity, to CTD repeats that are doubly modified (serine 2 and serine 5 phosphorylated), indicating that Set2 association across the body of genes requires a specific pattern of phosphorylated RNAPII. Deletion of the SRI domain not only abolishes Set2-RNAPII interaction but also abolishes K36 methylation in vivo, indicating that this interaction is required for establishing K36 methylation on chromatin. Using 6-azauracil (6AU) as an indicator of transcription elongation defects, we found that deletion of the SRI domain conferred a strong resistance to this compound, which was identical to that observed with set2 deletion mutants. Furthermore, yeast strains carrying set2 alleles that are catalytically inactive or yeast strains bearing point mutations at K36 were also found to be resistant to 6AU. These data suggest that it is the methylation by Set2 that affects transcription elongation. In agreement with this, we have determined that deletion of SET2, its SRI domain, or amino acid substitutions at K36 result in an alteration of RNAPII occupancy levels over transcribing genes. Taken together, these data indicate K36 methylation, established by the SRI domain-mediated association of Set2 with RNAPII, plays an important role in the transcription elongation process.Successful synthesis of mRNA by RNA polymerase II (RNAPII) requires tight regulation of the initiation, elongation, and termination processes of transcription. The process of transcription elongation is affected in part by the binding of regulatory factors to the phosphorylated C-terminal domain (CTD) of the RNAPII subunit Rpb1. Recent studies have highlighted an important role for histone methylation in the process of transcription elongation. In particular, studies have shown that the Set1 and Set2 methyltransferases, which catalyze methylation of histone H3 lysines 4 (K4) and 36 (K36), respectively, are associated with RNAPII at different stages of the transcription elongation cycle. While Set1 associates with RNAPII via the Paf1 transcription elongation complex in a manner that is dependent on the serine 5 (Ser5)-phosphorylated CTD, Set2 is recruited to RNAPII in a manner that is dependent on the CTD and the Ctk1 kinase (CTDK-I) that effects...

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…Second, Figure 5B confirms that the WW domain-containing construct of Set2 can indeed bind directly to the PCTD. There are thus at least two independent phosphoCTD binding domains in Set2, one encompassed by amino acids 425-551 and the other by amino acids 619-733 (the SRI region; see ref 65). Third, Figure 5C shows that the N-terminal 160 amino acids in Ssd1 are sufficient for phosphoCTD binding.…”

“…Recent results lend credence to this hypothesis. For example, as mentioned earlier, deletion of either CTDK-I catalytic activity or the SRI region of Set2 leads to a specific loss of H3 K36 methylation, suggesting that binding of Set2 to the PCTD made by CTDK-I is central to its activity in vivo (37,38,65 (68), and splicing of newly synthesized transcripts can be assayed. In contrast to wild type, in which unspliced precursor is never observed (e.g., Figure 9B, lanes 2 and 3), ctk1Δ cells consistently contain significant amounts of the unspliced RPS17A transcript (lanes 5 and 6).…”

Transcription termination by nuclear RNA polymerases

“…For instance, in yeast, Ser2 phosphorylation of the CTD potentiates interaction with the essential polyadenylation factor Pcf11 (de Vries et al 2000;Barilla et al 2001;Licatalosi et al 2002;Ahn et al 2004). The H3-K36 methyltransferase Set2 binds elongating RNAPII, recognizing the doubly phosphorylated CTD (Ser5/Ser2) (Hampsey and Reinberg 2003;Kizer et al 2005). Interestingly, a genomic analysis employing tilling arrays in mammalian cells showed that H3-K36 methylation often decreases near the poly(A) site during or prior to RNAPII release, leading to the intriguing possibility that H3-K36 methylation plays a role in transcription termination (Lian et al 2008).…”

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

“…Set2 interacts with the repeating C-terminal domain (CTD) of RNA polymerase II (RNAPII) (12,(21)(22)(23)(24)(25). In yeast, the CTD of RNAPII consists of 26 repeats of a heptapeptide repeat sequence (YSPTSPS) in which all three serine residues can be phosphorylated (26 -28).…”