The Mechanism Study Of EAF1 In Tat-dependent HIV-1 Transcription

In eukaryotic cells,the transcripton of protein-coding genes is conducted by RNA polymerase Ⅱ.After the identification of positive-transcription elongation factor b(P-TEFb)and characterization of its function in transcription elongation state,accumulating evidence has revealed that the transcription elongation state is also a highly ordered and exceedingly precision process,which is ignored previously.As development study of P-TEFb,it is found that P-TEFb is binding to Brd4,and existing in two complexes,named 7SK snRNP and Super Elongation Complex(SEC)respectively.The activity of P-TEFb is dependent on hyperphosphorylating the Cterminal domain(CTD)of the largest subunit of Pol Ⅱ,which can antagonize the inhibiton of PoⅢ and stimulate transcription elongation activity of PoⅢ,to make sure the full-length pre-mRNA is synthesized.P-TEFb also participates in HIV-1 reactivation,recruited by Tat,encoded by HIV-1,to bind to TAR RNA.EAF1,also called ELL-associated Factor 1,is a novel protein,detected through yeast two hybrid in 2001.The following studies reveal that EAF1 is a transcriptional activator,containing a transcriptional activation domain in its C terminal.We wonder how EAF1 effects HIV-1 transcription.In this study,to studing the role of EAF1 in HIV-1 transcription,we choosed F1C2 cell line,a HeLa-based cell line,to construct a stable knockout EAF1 cell line,and used HIV-1 promter coupling Luciferase to do some relate experiments.The luciferase assay shows that after knockout EAF1,Tat-dependent transcription rises,indicating EAF1 inhibits HIV-1 promoter transcription.When knockout EAF1,the stability of ELL 1 or ELL2 has little change compered with ctrl group.The mechanism is:EAF1 competes with AFF1/4 to bind to ELL2,and that is why EAF1 inhibits Tatdependent HIV-LTR transcription.These experiments prove EAF1 is not a component of SEC,which associated with SEC because of competing.