Brd4 is an atypical kinase that regulates transcription

Division Of Basic Sciences - Nci

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Abstract

Despite the interest in BRD4, there is relatively little understanding of either its global functions or its structure. BRD4 is a protein of 1362 amino acids in humans and 1400 amino acids in mice. The N-terminal half of the molecule encodes two bromodomains that bind to acetylated lysines. One of the major targets for BRD4 binding is to acetylated histones which results in the recruitment of a variety of transcription factors to regions of active chromatin, including super-enhancers and typical enhancers. Thus, BRD4 plays a passive role in transcriptional regulation. BRD4 also plays a direct role in regulating transcription, mediating pause release through its atypical kinase activity which we discovered. As part of the transcription initiation complex at the promoter, BRD4 phosphorylates the Pol II carboxy-terminal domain at Ser-2 which is associated with pause release and with the activation of Topoismerase I that is required to relieve the torsional stress caused by Pol II synthesis and is required for pause release. In contrast to its functional domains, relatively little is known about the biophysical properties of BRD4. Structural analysis has only been achieved for the two bromodomains and ET domain. Computational modeling of BRD4's secondary structure predicts that the amino-terminal half of the molecule is highly ordered whereas the carboxy terminal half is extensively intrinsically disordered. The goal of our studies is to further characterize the mechanisms by which BRD4 regulates transcription through its biophysical, biochemical and structural characterization. To begin to characterize the biophysical properties of BRD4, we first analyzed its pattern of elution by size exclusion chromatography (SEC). With a molecular weight of 156 kD, BRD4 would be expected to elute from a Sepharose 6 column at 16.30 ml. Surprisingly, we repeatedly observed purified recombinant BRD4 to elute as in a peak corresponding to a molecular weight of 900kD. Similarly, aberrant migration of BRD4 was observed on native-PAGE gels. These findings were particularly surprising in light of our observation that BRD4 migrates as a single band with a molecular weight of 156kD in denaturing gels, as would be expected of a molecule of 1400 aa. To further characterize the conformation of BRD4 in solution, we performed sedimentation velocity experiments using analytical ultracentrifugation (AUC). Analysis of sedimentation velocity experiments resulted in a sedimentation coefficient distribution with a concentration-independent major peak at s20,w of 6.7 S and a best-fit frictional ratio of 1.87. This extended conformation would retard the elution of BRD4 in SEC and its migration in native gels. Therefore, considering the experimentally measured frictional ratio for BRD4 of 1.87 and the anomalous migration in SEC, the dimeric state is most consistent with our observations. As noted above, BRD4 phosphorylates the CTD of RNA polymerase II, which consists of 52 repeats of the heptad YSPTSPS. It plays a critical role in regulating transcription initiation and elongation through the differential phosphorylation of its serine, threonine, and tyrosine residues by multiple kinases, including BRD4. Although we had demonstrated the phosphorylation of Ser2 of the CTD, we have not previously shown a stable interaction between BRD4 and the CTD. Therefore, we undertook to determine by AUC the stoichiometry and affinity of interaction between BRD4 and purified CTD1-52 in solution. At a ratio of BRD4:CTD of 1:2, the sedimentation coefficient distribution in AUC clearly shows a range of BRD4/CTD complexes. Our results clearly document a direct interaction between BRD4 and CTD, albeit of low affinity and driven by concentration. We have previously described BRD4 as an atypical kinase. Rather, predicted kinase subdomains are distributed within the N-terminus, consistent with the features of atypical kinases. Mutation of one, or even a few, of the kinase domains does not completely inhibit its kinase activity. As noted above, BRD4 trans-phosphorylates the Pol II CTD, as well as a number of other substrates, including TAF7. To assess the kinetics of trans-phosphorylation, we examined two of BRD4's substrates, the CTD and TAF7. Phosphorylation of the Pol II CTD occurred with a Km of 259 nM. The observation of a low to intermediate affinity of interaction is consistent with the findings by AUC. Phosphorylation of TAF7 by BRD4 occurred with a significantly lower Km of 49.7 nM, indicative of a higher affinity of interaction. Both followed classical Michaelis-Menten kinetics. Similarly, BRD4 hydrolyzed ATP efficiently with a Km of 37.6 nM+6.8nM, as assessed by the extent of autophosphorylation and following classical enzyme kinetics. These results establish that despite the atypical patterns of kinase subdomains, BRD4 kinase activity follows classical Michaelis-Menten enzyme kinetics. To further characterize the BRD4 kinase activity and map a minimal segment required for activity, we generated a series of truncation and deletion mutants. Deletion of the N-terminal half of BRD4 (1-722) abrogated kinase activity whereas truncation of the C-terminal half (730-1400) retained kinase activity. In addition to two bromodomains, the salient domains within the 1-722 region are the A motif, B motif, BID domain, ET and SEED domains. Deletions of the ET and SEED domains retained kinase activity, as did a fragment extending from 1-600 aa. To further map the kinase activity, we generated a BRD4 mutant deleted of the segment 351-598 (d351-598), which is enriched in predicted kinase subdomains and spans bromodomain 2 and the B-BID domains (BD2-B-BID). Deletion of this region (d351-598) abrogated BRD4's auto and trans phosphorylation activities by 97.5%, indicating that BRD4's kinase domain is contained within the BD2-B-BID. Importantly, smaller deletions within this segment retained activity, consistent with the distributed kinase domains of an atypical kinase. To directly demonstrate that the BRD4 kinase domain is located in the 351-598aa segment, peptide fragments of BRD4 spanning the segments 275-730, 358-730 and 358-646 were generated and tested for their ability to trans-phosphorylate the CTD or TAF7. All three fragments were able to auto-phosphorylate, mapping both the kinase activity and the sites of phosphorylation within this segment. The patterns of trans-phosphorylation were markedly different when the CTD or TAF7 were used as substrates. Among the fragments, only the largest fragment 275-730 could trans-phosphorylate the CTD. Surprisingly, both 275-730 and 358-730 fragments could efficiently phosphorylate TAF7. Thus, the kinase activity largely resides within the 351-598 segment which spans the BD2, B and BID domains. The inability of the 358-730 fragment to phosphorylate the CTD, but not TAF7, suggests that the CTD binding site maps to the 275-358 region. In contrast, the TAF7 binding site maps to the region 358-730. The finding of different binding sites of the CTD and TAF7 on BRD4 is consistent with their observed differences in enzyme kinetics. Furthermore, peptides derived from the viral MLV integrase and cellular demethylase, NSD3, which have been shown to bind to the ET domain, are also phosphorylated by BRD4. Thus, BRD4 kinase activity maps to a region between 351 and 598 aa and is capable of both autophosphorylation and trans-phosphorylation of a variety of substrates binding to various sites on the molecule. A manuscript describing these findings is currently under revision.