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Brian Krueger

Mentor: David Price, Ph.D.
Lab Room: 3120 MERF
Lab Phone: 319-335-6742

The elongation phase of RNA polymerase II (RNAPII) transcription is a critical control point of gene expression. After transcription initiation, RNAPII is controlled by both negative (N-TEF) and positive transcription elongation factors (P-TEF). The concerted efforts of the N-TEFs, DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF), are responsible for promoter proximal pausing of newly-initiated polymerases. However, the block placed on transcription elongation by DSIF and NELF can be reversed by the actions of positive transcription elongation factor b (P-TEFb). P-TEFb is essential for RNAPII elongation in higher eukaryotes and is composed of one of two isoforms of Cdk9 and one of three cyclins, T1, T2, or K in humans. P-TEFb is present in two forms within the nucleus. The large inactive form of P-TEFb is comprised of P-TEFb, HEXIM, and the small nuclear RNA 7SK. It is believed that HEXIM and 7SK form an inhibitory complex with P-TEFb to prevent aberrant activation of RNAPII transcription and to serve as an easily accessible pool that can be activated during times of stress.

The goal of my thesis research is to elucidate the mechanisms involved in releasing P-TEFb from this large inactive complex. One of our collaborators has recently discovered 3 new proteins that may be involved in regulating the binding and release of P-TEFb from HEXIM and 7SK. All three of these proteins could potentially modify the regulatory RNA 7SK. /In vitro/ and /in vivo/ experiments are currently being planned to determine how these proteins affect 7SK’s and HEXIM’s ability to inhibit P-TEFb.

The structure and sequence of 7SK may also be extremely important for P-TEFb regulation. We know that, based on unpublished data, recombinant HEXIM can bind double stranded RNA and inhibit P-TEFb /in vitro/. This result is relatively curious in light of recent results found using 7SK mutants. These results suggest that binding of HEXIM to 7SK is dependent on certain structural features and sequences /in vivo/. Taking both of these results into consideration, it is imperative that we determine the specific structure of 7SK that is required for P-TEFb inhibition /in vivo/ and also the modifications of HEXIM that prevent it from inhibiting P-TEFb while bound to nuclear double stranded RNAs other than 7SK. Understanding how P-TEFb is regulated is broadly important for understanding the progression of HIV and cardiac hypertrophy. Cardiac hypertrophy is a compensatory enlargement of the heart in response to stresses such as increased work load or impaired heart function.

Application of the lessons learned by studying P-TEFb regulation /in vitro/ and in cell culture will help in determining how release and hyperactivation of P-TEFb occurs in cardiac hypertrophy.

In addition to understanding the functional role of the “classical” activities of P-TEFb, I am also studying novel roles for P-TEFb in other processes. Recent data has implicated P-TEFb as one of the kinases that can modify and stabilize the G1 DNA damage check-point protein p53. /In vitro /experiments have shown that P-TEFb can phosphorylate p53 on Ser 33, 315, and 392 and activate p53’s ability to tetramerize and bind DNA. I am currently in the process of repeating these experiments to confirm P-TEFb’s involvement in this process.

Nguyen D, Krueger BJ, Sedore SC, Brogie JE, Rogers JT, Rajendra TK, Saunders A, Matera AG, Lis JT, Uguen P, Price DH. The Drosophila 7SK snRNP and the essential role of dHEXIM in development. Nucleic Acids Res. 2012 Jul;40(12):5283-97. Epub 2012 Feb 29. PubMed PMID: 22379134; PubMed Central PMCID: PMC3384314.

Krueger BJ, Varzavand K, Cooper JJ, Price DH. The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK. PLoS One. 2010 Aug 23;5(8):e12335. PubMed PMID:20808803; PubMed Central PMCID: PMC2925947.

Krueger BJ, Jeronimo C, Roy BB, Bouchard A, Barrandon C, Byers SA, Searcey CE, Cooper JJ, Bensaude O, Cohen EA, Coulombe B, Price DH. LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res. 2008 Apr;36(7):2219-29. Epub 2008 Feb 16. PubMed PMID: 18281698; PubMed Central PMCID: PMC2367717.