Akiko Furuya

NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

Identification of NAP1, a Regulatory Subunit of IκB Kinase-Related Kinases That Potentiates NF-κB Signaling

ABSTRACT The IκB kinase (IKK)-related kinase NAK (also known as TBK or T2K) contributes to the activation of NF-κB-dependent gene expression. Here we identify NAP1 (for NAK-associated protein 1), a protein that interacts with NAK and its relative IKKε (also known as IKKi). NAP1 activates NAK and facilitates its oligomerization. Interestingly, the NAK-NAP1 complex itself effectively phosphorylated serine 536 of the p65/RelA subunit of NF-κB, and this activity was stimulated by tumor necrosis factor alpha (TNF-α). Overexpression of NAP1 specifically enhanced cytokine induction of an NF-κB-dependent, but not an AP-1-dependent, reporter. Depletion of NAP1 reduced NF-κB-dependent reporter gene expression and sensitized cells to TNF-α-induced apoptosis. These results define NAP1 as an activator of IKK-related kinases and suggest that the NAK-NAP1 complex may protect cells from TNF-α-induced apoptosis by promoting NF-κB activation.

Human Spt6 Stimulates Transcription Elongation by RNA Polymerase II In Vitro

ABSTRACT Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

Structure–function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex

Background: The human Spt4/Spt5 complex, termed DRB‐sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription‐coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro.