Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expression.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator involved in the pathogenesis of rheumatoid arthritis. This study was undertaken to identify the MIF promoter elements responsible for regulating gene expression. METHODS: Luciferase reporter gene assays were used to identify the MIF promoter sequence responsible for basal activity. Bioinformatic analysis was used to predict transcription factor binding sites, and electrophoretic mobility shift assay (EMSA) was used to demonstrate transcription factor binding. Chromatin immunoprecipitation (ChIP) was used to demonstrate transcription factor loading on the MIF promoter. RESULTS: We identified the minimal promoter sequence required for basal MIF promoter activity that was also capable of conferring glucocorticoid-dependent inhibition in a T lymphocyte model cell line. Deletion studies and EMSA revealed 2 elements in the MIF promoter that were responsible for basal promoter activity. The 5' element binds CREB/activating transcription factor 1, and the 3' element is a functional hypoxia-responsive element binding hypoxia-inducible factor 1alpha. Further studies demonstrated that the cis elements are both required for glucocorticoid-dependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor alpha to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3. CONCLUSION: Our findings indicate that hypoxia and glucocorticoid signaling converge on a single element regulating MIF; this regulatory unit is a potential interacting node for microenvironment sensing of oxygen tension and glucocorticoid action in foci of inflammation.

Interaction of the TFIIB zinc ribbon with RNA polymerase II.

Transcription by RNA polymerase II requires the assembly of the general transcription factors at the promoter to form a pre-initiation complex. The general transcription factor TF (transcription factor) IIB plays a central role in the assembly of the pre-initiation complex, providing a bridge between promoter-bound TFIID and RNA polymerase II/TFIIF. We have characterized a series of TFIIB mutants in their ability to support transcription and recruit RNA polymerase II to the promoter. Our analyses identify several residues within the TFIIB zinc ribbon that are required for RNA polymerase II assembly. Using the structural models of TFIIB, we describe the interface between the TFIIB zinc ribbon region and RNA polymerase II.

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on the basis of their ability to compete with wild-type TFIIB in vivo. Moreover, analysis of the TFIIB mutant derivatives by chromatin immunoprecipitation showed that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II. Together, our results support a mode of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo.

FRET evidence for a conformational change in TFIIB upon TBP-DNA binding.

As a critical step of the preinitiation complex assembly in transcription, the general transcription factor TFIIB forms a complex with the TATA-box binding protein (TBP) bound to a promoter element. Transcriptional activators such as the herpes simplex virus VP16 facilitate this complex formation through conformational activation of TFIIB, a focal molecule of transcriptional initiation and activation. Here, we used fluorescence resonance energy transfer to investigate conformational states of human TFIIB fused to enhanced cyan fluorescent protein and enhanced yellow fluorescent protein at its N- and C-terminus, respectively. A significant reduction in fluorescence resonance energy transfer ratio was observed when this fusion protein, hereafter named CYIIB, was mixed with promoter-loaded TBP. The rate for the TFIIB-TBP-DNA complex formation is accelerated drastically by GAL4-VP16 and is also dependent on the type of promoter sequences. These results provide compelling evidence for a 'closed-to-open' conformational change of TFIIB upon binding to the TBP-DNA complex, which probably involves alternation of the spatial orientation between the N-terminal zinc ribbon domain and the C-terminal conserved core domain responsible for direct interactions with TBP and a DNA element.

Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.