Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

Abstract

During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.

Introduction

The gene encoding Spt6 was originally identified in a genetic screen in yeast as a suppressor of transposon insertion in the promoter region of a reporter gene.¹ Spt6 was later described as an essential nuclear protein.2, 3 Like Spt4 and Spt5, Spt6 is required for transcription elongation,4, 5, 31, 70 and co-localizes with Spt5 and RNA polymerase (Pol) II at transcriptionally active loci on Drosophila polytene chromosomes.6, 7 Spt6 interacts with the structured part of histone H3 and can deposit nucleosomes on DNA in a supercoiling assay.⁸ Loss of normal Spt6 function leads to transcription from cryptic promoters within the coding region of genes,⁹ suggesting that Spt6 is required to re-establish correct chromatin structure after Pol II passage. Spt6 also has a role in the re-assembly of nucleosomes in promoter regions, and is required for repression of transcription reinitiation.¹⁰ Mutations in Spt6 influence mRNA 3′-end formation,¹¹ splicing, and mRNA export.¹²

Spt6 is associated with the Pol II machinery,6, 7, and its SH2 domain is apparently important for this. Spt6 binds to Spt54, 5, and occupies the 5′-region of the uninduced heat shock gene hsp70.⁶ In vivo, Spt6 and Spt5 co-localize with actively transcribing Pol II that is phosphorylated at Ser2 of the C-terminal repeat domain (CTD) of the Pol II largest subunit.⁷ In vitro, the C-terminal region of mouse Spt6, which includes the SH2 domain, binds the Ser2-phosphorylated CTD, and this interaction is required for normal mRNA processing and exit from the nucleus.¹²

The Spt6 SH2 domain is of special interest because it apparently has phosphoserine-binding specificity, whereas SH2 domains generally bind to phosphotyrosine-containing peptides. Further, it is the only SH2 domain encoded in the yeast genome¹³ and thus may represent an ancestor of the large variety of SH2 domains encoded in the genome of higher eukaryotes. SH2 domains are highly abundant in animal cells as adaptor modules that connect proteins in tyrosine kinase pathways, and are important mediators in cellular signaling.14, 15

Structural information is available for SH2 domains and their complexes with phosphopeptides, including that of the Src SH2 domain–phosphopeptide complex.¹⁶ SH2 domains can be divided into two structural subfamilies: the Src-type and the STAT-type subfamilies.¹⁷ Whereas both families of domains share a central fold, the Src-type domains contain an extra β-sheet (βE-βF motif) that participates in the read-out of residues C-terminal to the phosphotyrosine,¹⁶ and the STAT-type domains show an extended helical structure at their C-terminus (αB' and αB). Phosphorylated peptides bind two pockets on the SH2 domain surface, one that recognizes the phosphorylated tyrosine side chain, and one that contacts residues C-terminal of the phosphotyrosine, generally at register +3. Binding of the peptide to the second pocket is a major determinant of the interaction specificity.¹⁸ Although in some cases binding of phosphoserine- and phosphothreonine-containing peptides to SH2 domains has been reported,19, 20 there is no structural information on the interaction of a SH2 domain with peptides other than those containing phosphotyrosine.

Here, we report the high-resolution structure of the SH2 domain of Spt6, suggest a model for how this domain binds to the phosphorylated Pol II CTD, and show that the domain is required for normal expression of a subset of yeast genes. Comparison of known SH2 domain structures reveals that the Spt6 SH2 domain structure is intermediate between SH2 domains of the two subfamilies in higher eukaryotes, reflecting the likely structure of the common ancestor of all SH2 domains.