A Conserved Protein Motif Is Required for Full Modulatory Activity of Negative Elongation Factor Subunits NELF-A and NELF-B in Modifying Glucocorticoid Receptor-regulated Gene Induction Properties

Luo, Min; Lu, Xiaoyong; Zhu, Rong; Zhang, Zhenhuan; Chow, Carson C.; Li, Rong; Simons, S. Stoney

doi:10.1074/jbc.m113.512426

Cited by 12 publications

(57 citation statements)

References 61 publications

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“…7) was unexpected. Earlier results for two other components of the NELF complex (NELF-A and NELF-B) showed that both act as a competitive decelerator at two sites before or at the CLS (20). The results of Fig.…”

Section: Discussionsupporting

confidence: 56%

“…The present results with NELF-E would initially appear to contradict our earlier report that NELF-E decreases the A max of GR transactivation (20). However, this discrepancy was found to be due mostly to differences in the nature of the vectors employed.…”

Section: Nelf-e Is An Accelerator In Competition Assays Withcontrasting

confidence: 57%

“…They have also been shown to act at various steps in GR transactivation (Figs. 1-4) (16,20). We therefore asked whether BRD4 binds to any of the NELF components directly.…”

Section: Brd4 Binds To Nelf-e-cdk9 Brd4mentioning

confidence: 99%

“…Information about each factor is then obtained from a series of graphs, similar to Lineweaver-Burk graphs in biochemistry, that plot combinations of the A max and EC 50 values from the competition assays. Specifically, the graphs reveal the number of sites at which each factor acts, their kinetically defined mechanism of action, and their relative order of action in the overall sequence leading to changes in gene expression (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). This approach has been extensively validated, and the derived information is novel and not available from other methodologies.…”

mentioning

confidence: 99%

“…In addition, several well known transcription factors have been found to modulate GRregulated gene induction, such as components of the transcription negative elongation factor (NELF) complex (20) and CDK9 (16), which is the kinase subunit of P-TEFb. The NELF complex is a negative regulator of RNA polymerase II (Pol II), enforcing a pause in early elongation about 100 bp after transcription initiation.…”

mentioning

confidence: 99%

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Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

Pradhan¹,

Blackford²,

Devaiah³

et al. 2016

Journal of Biological Chemistry

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Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (A max ) and the steroid concentration required for half-maximal induction (EC 50 ) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 < induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.Steroid hormone action has been studied intensively since the discovery of steroid hormone receptors almost 50 years ago (1) for three reasons. First, the receptors are obligatory mediators for the biological responses of the five classes of endogenous steroid hormones. Second, steroids and their receptors provide an ideal system in which to define the molecular mechanisms that regulate the controlled induction of gene transcription. Such studies have resulted in the identification of many species or factors that are required for transcription initiation, elongation, and termination (2-4). Third, the clinical benefits of steroid hormones are often severely limited by undesired side effects (5-8). A better molecular understanding of steroid action should lead to fewer side effects, which would significantly expand the clinical applications of steroids in treating human pathologies.The majority of factors identified to date for steroid-regulated gene induction participate in the early steps of transcription initiation (e.g. chromatin remodeling, assembly of the transcription preinitiation complex, and synthesis of the first oligonucleotides) (9). Two interesting classes of factors for steroid-regulated gene transcription are coactivators and corepressors (2-4). One of the earliest and most widely studied of these factors is TIF2/GRIP1, which is a member of the p160 family of coactivators with molecular masses of about 160 kDa (10, 11). Despite the detailed understanding of many of the factors involv...

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Section: Discussionsupporting

confidence: 56%

Section: Nelf-e Is An Accelerator In Competition Assays Withcontrasting

confidence: 57%

“…They have also been shown to act at various steps in GR transactivation (Figs. 1-4) (16,20). We therefore asked whether BRD4 binds to any of the NELF components directly.…”

Section: Brd4 Binds To Nelf-e-cdk9 Brd4mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

Pradhan¹,

Blackford²,

Devaiah³

et al. 2016

Journal of Biological Chemistry

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Pause Patrol: Negative Elongation Factor’s Role in Promoter-Proximal Pausing and Beyond

Diao,

Su,

Vos

2025

Journal of Molecular Biology

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A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

Zhu

Pradhan

et al. 2014

Biochemistry

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A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.

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A Conserved Protein Motif Is Required for Full Modulatory Activity of Negative Elongation Factor Subunits NELF-A and NELF-B in Modifying Glucocorticoid Receptor-regulated Gene Induction Properties

Cited by 12 publications

References 61 publications

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

Pause Patrol: Negative Elongation Factor’s Role in Promoter-Proximal Pausing and Beyond

A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

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