CA150, a Nuclear Protein Associated with the RNA Polymerase II Holoenzyme, Is Involved in Tat-Activated Human Immunodeficiency Virus Type 1 Transcription

Abstract

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine-and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

Transcription Elongation Regulator 1 Is a Co-integrator of the Cell Fate Determination Factor Dachshund Homolog 1

“…Proteomic analysis identified the co-integrator protein TCERG1 as a candidate mediator of DACH1 transcriptional repression. TCERG1 is a promoter-specific transcription co-regulator of HIV Tat gene (26,27). TCERG1 expression enhanced DACH1-mediated trans-repression.…”

“…TCERG1 is a promoter-specific transcriptional co-regulator of human immunodeficiency virus (HIV) Tat gene (26,27). We therefore sought to determine whether TCERG1 functions as a co-regulator of DACH1.…”

Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

“…The CA150 antibody used in this study was generated in rabbits (Pineda Antikörper Service, Germany) by use of a truncated glutathione Stransferase (GST) fusion CA150 protein spanning amino acids 590 to 1098 as described previously (67). For immunofluorescence analysis, immunoglobulins (Igs) were purified from antiserum on protein A-Sepharose columns (Pharmacia) by following standard procedures.…”

“…This repression is promoter specific, as other viral promoters are not affected, and it is also dependent on a specific functional TATA box element (66). The primary sequence of CA150 contains notable motifs and domains, many of which have been also found in other regulators of gene expression, including three WW and six FF domains (1,3,67). CA150 interacts directly with the phosphorylated CTD of RNAPII via its FF domains (6), and it also interacts with the splicing factor SF1 through the WW domains (18).…”

“…CA150 (also named TCERG1, for transcription elongation regulator 1; HUGO Gene Nomenclature Committee) was initially purified from HeLa cell nuclear extract as a transcriptional cofactor that regulates human immunodeficiency virus type 1 (HIV-1) Tat gene activation (67). Transient overexpression of CA150 reduces both basal and Tat-activated transcription from the HIV-1 promoter by inhibiting elongation efficiency (66).…”