A Tandem SH2 Domain in Transcription Elongation Factor Spt6 Binds the Phosphorylated RNA Polymerase II C-terminal Repeat Domain (CTD)

Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 285(53):41597-603. DOI: 10.1074/jbc.M110.144568
Source: PubMed


Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA polymerase II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo as its deletion in cells is lethal in the presence of 6-azauracil.

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    • "Spt6, along with Spt4 and Spt5 (mammalian DSIF), were discovered in yeast as canonical transcription factors that suppressed Ty element insertions in promoters of genes (30,70–72). Spt6, the only protein in the yeast genome with a tSH2 domain (73), binds to the Ser2/Ser5 and Tyr1-phosphorylated CTD forms of RNAPII in vitro (34,38) and may regulate and maintain chromatin structure by way of its histone chaperone activities (32,33,74). While much has been learned regarding the role of Spt6 in transcription elongation (75), an unresolved mystery has been how this protein contributes to H3K36 methylation in yeast and mammals. "
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    ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II is sequentially modified for recruitment of numerous accessory factors during transcription. One such factor is Spt6, which couples transcription elongation with histone chaperone activity and the regulation of H3 lysine 36 methylation. Here, we show that CTD association of Spt6 is required for Ser2 CTD phosphorylation and for the protein stability of Ctk1 (the major Ser2 CTD kinase). We also find that Spt6 associates with Ctk1, and, unexpectedly, Ctk1 and Ser2 CTD phosphorylation are required for the stability of Spt6-thus revealing a Spt6-Ctk1 feed-forward loop that robustly maintains Ser2 phosphorylation during transcription. In addition, we find that the BUR kinase and the polymerase associated factor transcription complex function upstream of the Spt6-Ctk1 loop, most likely by recruiting Spt6 to the CTD at the onset of transcription. Consistent with requirement of Spt6 in histone gene expression and nucleosome deposition, mutation or deletion of members of the Spt6-Ctk1 loop leads to global loss of histone H3 and sensitivity to hydroxyurea. In sum, these results elucidate a new control mechanism for the regulation of RNAPII CTD phosphorylation during transcription elongation that is likely to be highly conserved.
    Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt1003 · 9.11 Impact Factor
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    • "Indeed, the recently solved structures of the C-terminal region of Spt6 revealed that the region actually contains two SH2 domains in tandem that are intimately associated with each other (Figure 1C) [48-51]. The phospho-binding pocket of the N-terminal SH2 domain, which contributes to CTD phosphopeptide binding, contains an arginine that is invariant among eukaryotic SH2 domains [49,50,52]. In contrast, the corresponding pocket in the C-terminal SH2 domain lacks an arginine, and NMR titration studies suggest that this pocket is not used for peptide binding. "
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    ABSTRACT: SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKCdelta and PKCtheta C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases.
    Cell Communication and Signaling 11/2012; 10(1):32. DOI:10.1186/1478-811X-10-32 · 3.38 Impact Factor
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    • "Set1A/1B (histone methylase) Ser5-P Lee and Skalnik 2008 MLL1/2 (histone methylase) Ser5-P Hughes et al. 2004; Milne et al. 2005 Set2 (histone methylase) Ser2/5-P Li et al. 2003; Xiao et al. 2003; Kizer et al. 2005 HYPB (histone methylase) Ser2/5-P Li et al. 2005; Sun et al. 2005 Rpd3S (histone deacetylase) P-CTD Drouin et al. 2010; Govind et al. 2010 Spt6 Ser2-P Yoh et al. 2007; Sun et al. 2010 Guanylyltransferase (capping) Ser5-P Ho and Shuman 1999; Fabrega et al. 2003; Ghosh et al. 2011; Schwer and Shuman 2011 Prp40 (U1 snRNP) P-CTD Morris and Greenleaf 2000 PSF/p54 (multifunctional) CTD, P-CTD Emili et al. 2002; Rosonina et al. 2005 U2AF65 (U2 snRNP) P-CTD David et al. 2011 CstF50 (CstF) CTD, P-CTD Fong and Bentley 2001 Yhh1 (CPSF) P-CTD Dichtl et al. 2002b Ssu72 Ser5-P Ganem et al. 2003; Krishnamurthy et al. 2004; Xiang et al. 2010; Werner-Allen et al. 2011 Ess1/Pin1 Ser5-P Yaffe et al. 1997; Verdecia et al. 2000 Pcf11 (CF II) Ser2-P Barilla et al. 2001; Licatalosi et al. 2002; Meinhart and Cramer 2004 Rtt103 (termination factor) Ser2-P Kim et al. 2004b; Lunde et al. 2010 Sen1 (termination factor) Ser2-P Ursic et al. 2004; Chinchilla et al. 2012 Nrd1 (termination factor) Ser5-P Conrad et al. 2000; Steinmetz et al. 2001; Vasiljeva et al. 2008 The CTD-binding preference of factors involved in transcription elongation, RNA processing, and termination. The multifunctional protein complex PSF/p54 and CstF50 can bind to either unphosphorylated CTD or phosphorylated CTD (P-CTD). "
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    ABSTRACT: The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity.
    Genes & development 10/2012; 26(19):2119-37. DOI:10.1101/gad.200303.112 · 10.80 Impact Factor
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