Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription.
The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.
Cojocaru
Marilena
M
Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, QC, Canada H2W 1R7.
Jeronimo
Célia
C
Forget
Diane
D
Bouchard
Annie
A
Bergeron
Dominique
D
Côte
Pierre
P
Poirier
Guy G
GG
Greenblatt
Jack
J
Coulombe
Benoit
B
D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation; D023281:Genomics; D006801:Humans; D010455:Peptides / Q000737:chemistry; D012319:RNA Polymerase II / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D010455:Peptides; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D004247:DNA; C477608:POLR2G protein, human; D012319:RNA Polymerase II; C056604:transcription factor TFIIF