Trometry. Bands identified are indicated by arrowheads with human orthologs in parentheses. B and C, HEK293T cells were transfected with the indicated vectors or pcDNA3 control vector. Whole cell extracts were used for immunoprecipitation using a nonspecific antibody and anti-FLAG antibody or FLAG resin that pulls down NELF. Immunoprecipitates were immunoblotted (IB) with anti-HA antibody that detects HA-HDAC3 and HA-GPS2. Data represent three or more independent experiments.ical pathway. Activating NELF- and/or Pcf11-deficient cells through CD3 plus CD28 led to an increase in HIV transcription that was comparable with siControl-treated cells, suggesting that both these proteins function to regulate basal proviral transcription and that their repressive activities are overcome by T cell activation (Fig. 2F). To explore NELF-Pcf11 functional interactions, we transiently expressed NELF-B in HEK293T cells. NELF-B was sufficient to inhibit HIV transcription (Fig. 3A) and facilitate the recruitment of other NELF factors as well as Pcf11 to the HIV LTR without a concomitant increase in RNAP II (Fig. 3B). These data suggest that NELF and Pcf11 repress HIV transcription by interacting with each other. To examine whether NELF and Pcf11 physically interact in the context of a T cell, Jurkat T cells were lysed, and Pcf11 and associated proteins were immunoprecipitated with a Pcf11specific antibody. As shown in Fig. 3C, NELF-D coimmunoprecipitated with Pcf11. This interaction was validated by immunoprecipitating NELF-D to pull down Pcf11. Collectively, these data suggest that NELF recruits Pcf11 to the paused RNAP II to prematurely terminate transcription, thus reinforcing repression of HIV transcription. NELF Interacts with the NCoR1-Gps2-HDAC3 Complex– The ability of NELF to interact with Pcf11 raises the possibility that NELF may recruit additional transcriptional repressors to the HIV LTR. Mass spectrometric analysis was used to identify potential factors that interact with NELF and contribute to HIV transcriptional repression.U0126 We took advantage of previously described transgenic Drosophila lines that expressed FLAGSEPTEMBER 6, 2013 VOLUME 288 NUMBERtagged NELF subunits (34), assuming that key proteins that regulate RNAP II processivity are functionally and structurally conserved in flies and humans. Nuclear extracts from Drosophila embryos were immunoprecipitated using the epitope tag to enrich for NELF complexes (Fig.Amantadine hydrochloride 4A).PMID:23672196 The immunoprecipitations from the different transgenic Drosophila lines yielded similar protein, as assessed by SDS-PAGE electrophoresis and Coomassie Blue staining (34). Furthermore, NELF subunits were efficiently coimmunoprecipitated with the FLAG antibody. For example, as shown in Fig. 4A, NELF-A, NELF-B, and NELF-E were all immunoprecipitated by FLAG-NELF-D, verifying that subunits known to be associated with the NELF complex were pulled down. Because the FLAG-NELF-D immunoprecipitations provided consistent protein yields and pulled down the other NELF subunits in proper stoichiometry, we used these extracts for the mass spectroscopy analysis. We were particularly interested in potential corepressors that interact with NELF and contribute to the maintenance of a repressed HIV transcriptional state. Potential transcriptional repressors that were identified included Smrter, CG17002, and HDAC3. The respective human orthologs of these proteins, NCoR1, GPS2, and HDAC3 have been demonstrated to form a corepressor complex (24.
