Manjinder Nanrey 12855110
To release RNAPII from pausing from the promoter proximal hairpin loop requires the recruitment of proteins such at TAT and P-TEFb. Currently supporting, but oversimplified, models tell us that Tat bind to the hairpin loop created by the Tar sequence of the HIV LTR, which then recruits P-TEFb. Through the use of CDK9, p-TEFb then phosphorylates Ser2 of the CTD, and allow for elongation. Nevertheless, it has been found that another protein called TCERG1 is involved in Ser 2 phosphorylation. This protein, originally called CA150 (2), has been characterized in the past, but there hasn’t been an appropriate model available that describes how TCERG1 works in vivo with regard to HIV-1 transcriptional elongation. In our current paper we have evaluated the role of TCERG1 and hypothesis that TCERG1 is a cofactor for HIV transcriptional elongation. We looked at the effects of TCERG1 knockdown by shRNA in PBL and Jerkat cells transfected with the HIV-1 LTR with the Luciferase reporter fused on the env gene, and found that both basal and Tat-activated transcription were diminished. This suggested TCERG1 served as a positive regulator of transcription. To test TCERG1’s effect on elongation we measured the amount of transcript at the distal regions of the LTR in HEK293T cells with a deletion of the Reverse transcriptase of HIV-1 transiently transfected plasmid. We found that TCERG1 knock down decreased transcript production by 30-40%. To directly measure the transcription rate we measured the level of transcript from the using 2 lines, one of which did not contain the shRNA for TCERG1 (control). We treated both cell lines with DRM, which block gene transcription in vivo, and measured the recovery of the distal transcript by RT-PCR at different intervals after wash. We found that recovery of the distal region for the knockdown cells were significantly slower than in the control. This shows that TCERG1 can alter the rate of transcription of HIV-1 in vivo as well as modify the level pre-mRNA containing the distal region. The mechanism that we were able to derive from our results showed that TCERG1 works by increasing the phosphorylation of Ser2 on the CTD, which then helps increase the level of HIV-1 transcription. To further this mechanism and to demonstrate how TCERG1 interacts with p-TEFb, we propose that TCERG1 interacts with P-TEFb through the interaction of its WW binding domains with ELL 2. We have shown by Tandem affinity purification that ELL2 binds with TCERG1, but did not include the data in the published paper. Experiments of others have shown that Tat and P-TEFb work with a complex of proteins called the SEC (super elongation complex) (1), and found ELL2 in the complex by affinity chromatography using HA-tagged Tat. In the analysis of ELL2 they found that it had, by shRNA knockdown, a significant effect on the basal and Tat mediated transcription as well. Other works have shown that TCERG1’s 3 WW domain interacts with...
