Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription

Abstract

Transcription factor (TF)IID is a central player in activated transcription initiation. Recent evidence suggests that the role and composition of TFIID are more diverse than previously understood. To investigate the effects of changing the composition of TFIID in a simple system, we depleted TATA box-binding protein-associated factor (TAF)1 from Drosophila cells and determined the consequences on metal-induced transcription at an inducible gene, metallothionein B. We observe a marked increase in the levels of both the mature message and pre-mRNA in TAF1-depleted cells. Under conditions of continued metal exposure, we show that TAF1 depletion increases the magnitude of the initial transcription burst but has no effect on the timing of that burst. We also show that TAF1 depletion causes delay in the shutoff of transcription upon removal of the stimulus. Thus, TAFs are involved in both establishing an upper limit of transcription during induction and efficiently turning the gene off once the inducer is removed. Using genome-wide nascent sequencing, we identify hundreds of genes that are controlled in a similar manner, indicating that the findings at this inducible gene are likely generalizable to a large set of promoters. There is a long-standing appreciation for the importance of the spatial and temporal control of transcription. Here we uncover an important third dimension of control: the magnitude of the response. Our results show that the magnitude of the transcriptional response to the same signaling event, even at the same promoter, can vary greatly depending on the composition of the TFIID complex in the cell.

Keywords: MtnA; RNA polymerase; coactivator; heavy metal.

Fig. 1.

D. melanogaster metallothionein A and B expression following depletion of TBP and TAF subunits. S2 cells treated with dsRNA targeting TFIID or mock-treated were induced with Cu or Cd and mRNA levels were measured by RT-qPCR. MtnA (A and C) and MtnB (B and D) expression was determined by RT-qPCR and normalized to the ribosomal protein (RP)49 transcript. Values shown are fractions of the mock-treated samples. Error bars represent SE of qPCR triplicates. Experiments were repeated in at least two independent biological replicates, and data are consistent with those shown. (E) RNAP II occupancy at MtnA in untreated S2 cells. (Upper) Data signal enrichment in immunoprecipitation versus input in our S2 cells. (Lower) Analysis of publicly available modENCODE data for comparison. (F) RNAP II occupancy at MtnB in untreated S2 cells as in E.

Fig. 2.

Induction of MtnB following addition of Cu. S2 cells treated with TAF1 dsRNA or mock-treated were induced with Cu for the indicated times and RNA levels were measured by RT-qPCR. (A) MtnB mature mRNA normalized to RP49. (B) MtnB intron-containing pre-mRNA normalized to RP49 intron-containing pre-mRNA. Primer binding sites are indicated by the arrows in the gene diagrams above the graphs. (C) MtnB pre-mRNA levels plotted as a fraction of the 1-h time point. Error bars are SEMs of qPCR. (D) ChIP of TBP or RNA polymerase II at the MtnB promoter at 80-min treatment with copper. Data are plotted as the precipitation relative to the housekeeping gene RPL6. All experiments were repeated in two independent biological replicates, and data are consistent with those shown.

Fig. 3.

Shutoff of MtnB pre-mRNA expression following removal of stimulus. (A) S2 cells treated with TAF1 dsRNA or mock-treated were induced with Cu for 1.5 h. Cu was then chelated with BCS. Cells were washed twice with media containing BCS and then incubated in complete media for the indicated times. (B) MtnB pre-mRNA expression normalized to RP49 pre-mRNA levels. (C) Normalized MtnB pre-mRNA levels plotted as a fraction of the 1.5-h time point for both washout conditions. Error bars represent SEMs. Two independent biological replicates were performed and results were comparable. Arrows indicate time of washout.

Fig. 4.

Genome-wide analysis of nascent RNA in TAF1-depleted cells. (A) RT-qPCR validation of 13 genes identified in the initial nascent-seq dataset. (B) Motifs identified as overrepresented in the promoters of genes showing either increased transcription or decreased transcription by nascent-seq.