Solution structure of the Set2–Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1

Abstract

The phosphorylation state of the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II changes as polymerase transcribes a gene, and the distinct forms of the phospho-CTD (PCTD) recruit different nuclear factors to elongating polymerase. The Set2 histone methyltransferase from yeast was recently shown to bind the PCTD of elongating RNA polymerase II by means of a novel domain termed the Set2-Rpb1 interacting (SRI) domain. Here, we report the solution structure of the SRI domain in human Set2 (hSRI domain), which adopts a left-turned three-helix bundle distinctly different from other structurally characterized PCTDinteracting domains. NMR titration experiments mapped the binding surface of the hSRI domain to helices 1 and 2, and Biacore binding studies showed that the domain binds preferably to [Ser-2 ؉ Ser-5]-phosphorylated CTD peptides containing two or more heptad repeats. Point-mutagenesis studies identified five residues critical for PCTD binding. In view of the differential effects of these point mutations on binding to different CTD phosphopeptides, we propose a model for the hSRI domain interaction with the PCTD. RNA polymerase II carries an intrinsically unstructured, flexible domain at the C terminus of its largest subunit. The principal function of this C-terminal repeat domain (CTD), which comprises multiple repeats of a consensus heptamer Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is to serve as a binding scaffold for various nuclear factors (reviewed in refs. 1-3). The CTD of preinitiating RNA polymerase II is mostly unphosphorylated, whereas after initiation and during elongation it is hyperphosphorylated, principally on Ser-5 and Ser-2 residues of the repeats (4-6); we refer to this form as the phospho-CTD (PCTD). Attendant with changes in patterns of CTD phosphorylation, the ensemble of CTD-bound proteins changes as RNA polymerase II progresses through the transcription cycle (5). Although knowledge of the number and types of PCTD-associating proteins (PCAPs) has expanded rapidly (7, 8), information about the molecular nature of PCAP-PCTD interactions remains quite limited; detailed binding properties and͞or 3D structures are known for only a few PCTD-interacting domains (PCIDs) (9-14).Describing in molecular detail the interactions between the PCTD and its binding partners is an important step in advancing our understanding of the PCTD and its manifold functions. One recently identified binding partner of the PCTD is the Saccharomyces cerevisiae Set2 (yeast Set2; ySet2), a histone methyltransferase that modifies K36 of histone H3 in nucleosomes of transcribed genes (15)(16)(17)(18)(19). The identification of ySet2 as a PCAP together with studies of transcription in set2 mutant strains suggests a role for the PCTD in chromatin structure modulation during elongation (20). Deletion studies of ySet2 mapped a novel PCID, termed the Set2-Rpb1 interacting (SRI) domain by Kizer et al. (20), to the C-terminal segment of ySet2. The yeast SRI domain binds preferentially to PCTD peptides with both S...

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…In a similar line of argument, a universal RNAPII CTD cycle along genes has been proposed, which is orchestrated by complex interplays between kinases, phosphatases and proline isomerases 46 . The determination of the minimal distances required between the two general phosphorylation marks Ser5 and Ser2 and their possible combinations with other modifications such as pSer7 might therefore be a key step in the understanding of CTD interacting proteins that recognize different phospho-isoforms during the transcription cycle [47][48][49][50] . (1) (2) (3) (4) (5) (6) (7) w/o inhibitor (1) (2) (3) (4) (5) (6) (7) Variable reg.…”

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

“…The primary sequences of the SET and SRI domains of the enzyme responsible for H3K36 methylation from H. sapiens, S. cerevisiae, X. tropicalis, D. melanogaster, D. rerio, and M. musculus were aligned using ClustalOmega (56) and annotated with reported SETD2 mutations in ccRCC (1,4,11,12). The structures of the SETD2 SET domain (34), SETD2 SRI domain (44), and Set2 SRI domain (43) have been reported previously. To predict the structure of the yeast SET domain, the amino acid sequence (UniProtKB, P46995) was submitted to I-TASSER using the default parameters (35)(36)(37)(38).…”

“…The physical relationship of these helices appears conserved between Set2 and SETD2. We therefore selected the Arg-2510 residue for further study because this amino acid is recurrently mutated (R2510H and R2510L) in ccRCC (1,12) and is predicted to be important for SETD2-RNAPII interaction by in vitro peptide interaction assays (44).…”

Phosphorylation and functions of the RNA polymerase II CTD

“…Correspondingly, the PCTD-binding domain of Set2 (the SRI [Set2-Rpb1-interacting] domain) is essential for cotranscriptional methylation (Kizer et al 2005). The binding specificity of this ∼100-amino-acid domain was determined using a series of synthetic CTD peptides of varying length and phosphorylation patterns (Kizer et al 2005;M. Li et al 2005).…”