Gene knockdown suggests a new paradigm for transcription factor TFIIB functionality

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data, TFIIB knockdown in primary and transformed cell lines, and in vitro transcription experiments, we report that TFIIB is dispensable for transcription of most human promoters, but is essential for HSV-1 gene transcription and replication. We report a novel cell cycle TFIIB regulation and involvement of the acetylated TFIIB variant in chromosomal condensation. Taken together, these results establish a new paradigm for TFIIB functionality as a determinant of the human transcriptome, which when downregulated has potent anti-viral effects. HeLa cells are electroporated with TFIIB specific siRNA and control siRNA to identify if all human genes require TFIIB for transcription. Sixty hours post electroporation with siRNA, when the cellular TFIIB protein content was nearly undetectable by anti- human TFIIB in western blotting and immunofluorescence cells were harvested and total RNA was isolated. The gene expression profile of cells that received TFIIBsiRNA was compared to the gene expression profile of cells that received control siRNA by applying Agilent Whole Human Genome Oligo Microarray. Some data were confirmed by RT-PCR. RNAs for which the array data and the RT-PCR data differ have been excluded. The microarray experiments were performed in duplicates, as two hybridizations were carried out for the TFIIB -suppressing cell clone against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.