Repression of RNA Polymerase II Elongation In Vivo Is Critically Dependent on the C-Terminus of Spt5

Hui Chen,Hiroshi Handa,Su Guo,Xavier Contreras,B Matija Peterlin,Yuki Yamaguchi,Laszlo Tora

doi:10.1371/journal.pone.0006918

Abstract

The stalling of RNA polymerase II (RNAPII) at the promoters of many genes, including developmental regulators, stress-responsive genes, and HIVLTR, suggests transcription elongation as a critical regulatory step in addition to initiation. Spt5, the large subunit of the DRB sensitivity-inducing factor (DSIF), represses or activates RNAPII elongation in vitro. How RNAPII elongation is repressed in vivo is not well understood. Here we report that CTR1 and CTR2CT, the two repeat-containing regions constituting the C-terminus of Spt5, play a redundant role in repressing RNAPII elongation in vivo. First, mis-expression of Spt5 lacking CTR1 or CTR2CT is inconsequential, but mis-expression of Spt5 lacking the entire C-terminus (termed NSpt5) dominantly impairs embryogenesis in zebrafish. Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions. Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus. Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

Highlights

The production of mRNA is a multi-step process that involves transcription initiation, elongation, and termination [1,2,3]
The conversion of Spt5 from a repressor to an activator involves positive transcription elongation factor b (P-TEFb), which phsosphorylate RNA polymerase II (RNAPII) Cterminal domain (CTD) [20,37] as well as the C-terminal repeat 1 (CTR1) domain of Spt5 [15,16,17,18,19], leading to the hypothesis that CTR1 acts as a mini-CTD for assembling active elongation complexes [19]
We demonstrate that C-terminus of Spt5 (CSpt5) composed of partially redundant CTR1 and CTR2CT domains is a repressive module, by showing that N-terminus of Spt5 (NSpt5) lacking this module exerts a dominant negative effect on the repressive but not the stimulatory activity of the endogenous Spt5, while has no stimulatory activity on its own

Summary

Introduction

The production of mRNA is a multi-step process that involves transcription initiation, elongation, and termination [1,2,3]. Biochemical studies have identified over a dozen proteins and a small nuclear RNA, which regulate RNAPII elongation in vitro [2,10,11]. Among the identified elongation factors, the DRB-SensitivityInducing-Factor (DSIF) that is composed of Spt and Spt, can repress and activate RNAPII elongation on an artificial DNA template under different assay conditions in vitro [12,13]. The C-terminal repeat 1 (CTR1) of Spt5 [15,16,17,18,19], together with RNAPII Cterminal domain (CTD) [13,20] and Negative Elongation Factors (NELF) [21] are targets of phosphorylation by P-TEFb, a protein kinase composed of CDK9 and Cyclin T subunits, which reverses elongation repression and promotes positive elongation

Methods

Results

Conclusion