Negative elongation factor NELF controls transcription of immediate early genes in a stimulus-specific manner

Abstract

The transcription rate of immediate early genes (IEGs) is controlled directly by transcription elongation factors at the transcription elongation step. Negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) stall RNA polymerase II (pol II) soon after transcription initiation. Upon induction of IEG transcription, DSIF is converted into an accelerator for pol II elongation. To address whether and how NELF as well as DSIF controls overall IEG transcription, its expression was reduced using stable RNA interference in GH4C1 cells. NELF knock-down reduced thyrotropin-releasing hormone (TRH)-induced transcription of the IEGs c-fos, MKP-1, and junB. In contrast, epidermal growth factor (EGF)-induced transcription of these IEGs was unaltered or even slightly increased by NELF knock-down. Thus, stable knock-down of NELF affects IEG transcription stimulation-specifically. Conversely, DSIF knock-down reduced both TRH- and EGF-induced transcription of the three IEGs. Interestingly, TRH-induced activation of the MAP kinase pathway, a pathway essential for transcription of the three IEGs, was down-regulated by NELF knock-down. Thus, stable knock-down of NELF, by modulating intracellular signaling pathways, caused stimulation-specific loss of IEG transcription. These observations indicate that NELF controls overall IEG transcription via multiple mechanisms both directly and indirectly.

Introduction

Extra-cellular stimuli induce the expression of immediate early genes (IEGs), which will orchestrate the expression of further genes needed to evoke cellular responses (growth, differentiation, apoptosis etc.). Transcription of some IEGs (c-fos, c-myc, junB, MKP-1, heat shock genes, etc.) is tuned by the regulation of transcription elongation [1], [2], [3], [4], [5]. For such IEGs, promoters are constitutively activated and promote RNA polymerase II (pol II) initiation even in resting cells. However, without further stimulation, the transcription is paused in the proximity of the promoters [4], [6] and possibly also further downstream [1], [7]. Upon stimulation, the transcription elongation gradually resumes. In this manner, external stimuli continuously control transcription rates and mRNA steady state levels for several IEGs [7].

What are the mechanisms and molecular elements involved in regulating the elongation phase of transcription? Longstanding evidence highlights the importance of the C-terminal domain (CTD) of a large subunit of pol II. Its selective phosphorylation on the serine residues in position 2 and 5 in the YSPTSPS repeat (CTD Ser-2 and CTD Ser-5, respectively) is related to transition of the pre-initiation complex to transcription initiation (CTD Ser-5) and to elongation (CTD Ser-2) [8], [9]. One of the key players in the transition to productive elongation is positive transcription elongation factor b (P-TEFb), which is composed of cyclin T and cyclin-dependent kinase 9 (CDK9) and phosphorylates CTD Ser-2. Further transcription elongation factors also participate in the regulation of transcription elongation: the transcription elongation factors which stall pol II elongation, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), have been studied intensively [10], [11], [12], [13], [14], [15]. In vitro, DSIF and NELF collaborate to stall pol II soon after initiation; When P-TEFb is recruited to the pol II complex and subsequently phosphorylates both Spt5 a subunit of DSIF and CTD Ser-2 of pol II, the transcription elongation resumes, producing the full-length transcripts. Recent in vivo observations combined with chromatin immuno-precipitation (ChIP) or immuno-histochemistry support these in vitro findings [6], [7], [16], [17], [18], [19], [20], [21]. DSIF is likely to act both as a negative or positive elongation factor depending upon the phosphorylation state of Spt5 [21]. Extra-cellular stimuli will favor the transition of the inhibitory DSIF which blocks elongation to its stimulatory form which associates with elongating pol II [6], [21].

Recent evidence suggests that a role of the promoter-proximal pol II pausing including DSIF, NELF and P-TEFb is a “checkpoint” to ensure capping of transcripts [9]. More recently, it has been shown that the pol II pausing blocks promoter-proximal nucleosome assembly to enhance gene expression [22]. Interestingly, this interaction with chromatin occurs in a gene-specific manner. In several model systems including Drosophila, zebrafish, nematode, and mouse, DSIF, NELF and P-TEFb control various physiological processes such as cell survival, neuronal development, ontogeny, and cardiac hypertrophy [23], [24], [25], [26], suggesting that these transcription elongation factors are essential regulators genetically and also physiologically.

Aida et al. reported that transient knock-down of NELF in HeLa cells up-regulated transcription of certain IEGs not only in cellular resting conditions but also after interleukin-6 (IL-6) stimulation [27], supporting the in vitro observation that NELF causes the pausing of pol II during elongation. The abolishment of the blockade is likely to accelerate pol II elongation and its new recruitment [22]. Using the same approach of transient knock-down, Narita et al. demonstrated that NELF associates with the nuclear cap binding complex (CBC) and the histone stem-loop binding protein (SLBP) to facilitate 3′ end processing of the transcripts at the 3′ regions of replication-dependent histone genes [28]. These latter findings suggest that NELF plays also a positive role for the transcription.

Aiyar et al. reported global changes of gene transcription after stable knock-down of NELF in the T47D cancer cell line grown in serum-containing standard medium [29]: 84 genes were up-regulated whereas 50 other genes were down-regulated. This suggests that NELF contributes both positively and negatively to gene transcription in a gene-specific manner. On the other hand, it remains unclear whether the up- or down-regulation caused by stable but not transient knock-down of NELF reflected only the direct effects of NELF on transcription elongation and RNA processing or were in fact due to comprehensive (direct and indirect) effects of NELF. Stable knock-down can induce the secondary responses (indirect effects) during its maintenance. Moreover, the indirect effects of NELF have not been intensively examined. The elucidation of comprehensive effects of NELF on gene transcription is useful to understand physiological relevance of the factor.

In the present study, we address this question by considering the roles of the negative elongation factors NELF as well as DSIF for the transcription of the IEGs c-fos, MKP-1, and junB in neuroendocrine GH4C1 cells. By using stable RNA interference (RNAi) of NELF and DSIF, we found that DSIF functions negatively in resting cells but positively after transcription elongation is stimulated. Findings with NELF stable knock-down were more complex. Interestingly, dependent upon stimuli, knock-down of NELF resulted in reduced or enhanced transcription of the IEGs. Furthermore, we found that decrease in activation of the ERK1/2 MAP kinase pathway was followed by the reduction of the IEG transcription. Thus, stable knock-down of NELF modulated IEG transcription stimulation-specifically and indirectly. NELF would control IEG transcription overall via multiple mechanisms both directly and indirectly.