Bromodomain-containing protein 4–independent transcriptional activation by autoimmune regulator (AIRE) and NF-κB

Fang Huang,Wei Shao,Koh Fujinaga,B Matija Peterlin

doi:10.1074/jbc.ra117.001518

Abstract

Autoimmune regulator (AIRE) and nuclear factor-κB (NF-κB) are transcription factors (TFs) that direct the expression of individual genes and gene clusters. Bromodomain-containing protein 4 (BRD4) is an epigenetic regulator that recognizes and binds to acetylated histones. BRD4 also has been reported to promote interactions between the positive transcription elongation factor b (P-TEFb) and AIRE or P-TEFb and NF-κB subunit p65. Here, we report that AIRE and p65 bind to P-TEFb independently of BRD4. JQ1, a compound that disrupts interactions between BRD4 and acetylated proteins, does not decrease transcriptional activities of AIRE or p65. Moreover, siRNA-mediated inactivation of BRD4 alone or in combination with JQ1 had no effects on AIRE- and NF-κB-targeted genes on plasmids and in chromatin and on interactions between P-TEFb and AIRE or NF-κB. Finally, ChIP experiments revealed that recruitment of P-TEFb to AIRE or p65 to transcription complexes was independent of BRD4. We conclude that direct interactions between AIRE, NF-κB, and P-TEFb result in efficient transcription of their target genes.

Highlights

Transcription is the fundamental step for gene expression
Mammalian RNA polymerase II (RNAP II)2 is engaged at most promoters, and promoter clearance occurs when the C-terminal domain of RNAP II is phosphorylated on serines at position 5 (Ser-5) in the heptapeptide repeats by the cyclin-dependent kinase 7 (CDK7) from the general transcription factor TFIIH [1, 2]
The removal and depletion of Bromodomain-containing protein 4 (BRD4) did not decrease the ability of autoimmune regulator (AIRE) to activate its responsive promoters on plasmids and in chromatin

Summary

To whom correspondence should be addressed

Elongation, the positive transcription elongation factor b (P-TEFb) is required. P-TEFb consists of cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CycT1) or CycT2 [5]. The autoimmune regulator (AIRE) plays a critical role in regulating central tolerance in the thymus It directs the expression of otherwise tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs) [8]. AIRE is recruited to the stalled RNAP II on TSA promoters by binding to unmodified histone H3 Lys-4 and DNA-dependent protein kinase [10, 11]. AIRE binds to P-TEFb. Phosphorylation and ubiquitylation of AIRE can increase its transcriptional activities [12]. After binding to the indicated promoters, p65 recruits P-TEFb to activate the transcription of its target genes [18]. It has been suggested that the transcriptional activation by AIRE or p65 is dependent on BRD4, which bridges interactions between P-TEFb and acetylated AIRE or p65 proteins [26, 27]. Transcriptional activities by AIRE and p65 on promoters are independent of BRD4

Results

Discussion

Experimental procedures