The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation

Abstract

Many steps in gene expression and mRNA biosynthesis are coupled to transcription elongation and organized through the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). We showed recently that Spt6, a transcription elongation factor and histone H3 chaperone, binds to the Ser2P CTD and recruits Iws1 and the REF1/Aly mRNA export adaptor to facilitate mRNA export. Here we show that Iws1 also recruits the HYPB/Setd2 histone methyltransferase to the RNAPII elongation complex and is required for H3K36 trimethylation (H3K36me3) across the transcribed region of the c-Myc, HIV-1, and PABPC1 genes in vivo. Interestingly, knockdown of either Iws1 or HYPB/Setd2 also enhanced H3K27me3 at the 5' end of the PABPC1 gene, and depletion of Iws1, but not HYPB/Setd2, increased histone acetylation across the coding regions at the HIV-1 and PABPC1 genes in vivo. Knockdown of HYPB/Setd2, like Iws1, induced bulk HeLa poly(A)+ mRNAs to accumulate in the nucleus. In vitro, recombinant Spt6 binds selectively to a stretch of uninterrupted consensus repeats located in the N-terminal half of the CTD and recruits Iws1. Thus Iws1 connects two distinct CTD-binding proteins, Spt6 and HYPB/Setd2, in a megacomplex that affects mRNA export as well as the histone modification state of active genes.

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…In this regard it is known that IWS1 interacts with the CTD of the large subunit of RNAPII complex through Spt6 (ref. 43). During the reviewing process of this manuscript, Gérard et al published an independent study, which confirmed the existence of a LEDGF/p75-IWS1-Spt6 triple complex that was suggested to have an important function during HIV latency 46 .…”

“…This complex recruits HYPB/Setd2 histone methyltransferase, which controls H3K36me3 methylation, affecting co-transcriptional pre-mRNA splicing and export 42,43 . Because LEDGF is linked to both alternative splicing and interaction with H3K36me3, we chose to experimentally investigate the interaction between LEDGF/p75 and IWS1.…”

“…IWS1 forms a stable complex with SPT6 and the CTD of the large subunit of RNAPII 42 . In addition, IWS1 has been shown to recruit HYPB/Setd2, the only histone methyltransferase known to create H3K36me3 marks 43 . The unstructured character of IWS1 was experimentally verified via comprehensive biophysical characterization.…”

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

“…In particular, a C-terminal fragment of murine Spt6 (residues 1162-1726) that contains the SH2 domain did not bind to a Ser2-phosphorylated tandemrepeat CTD peptide, but bound to the N-terminal half of the CTD, which contains a stretch of 15 consensus repeats. 66 Thus, interactions that are not predicted by our modeling (Fig. 4) may occur between the SH2 domain and a longer CTD peptide.…”

Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling

“…Considering that these HMTs have different domain architectures and possibly different expression patterns, it is reasonable to postulate that the different H3K36 HMTs play distinct roles and cooperate to subtly regulate histone methylation and gene expression, thus controlling complicated developmental processes. Although H3K36 methylation has been implicated in multiple molecular functions including transcriptional elongation, suppression of cryptic transcription and mRNA export (33)(34)(35)(36), it remains unclear whether H3K36 methylation contributes to any specific developmental processes. In this study, we identified the genes altered by the Hypb disruption, some of which might be directly or indirectly related to the loss of H3K36me3, thus allowing study of potential functions of Hypb and H3K36 methylation on different genes.…”