TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription - PubMed (original) (raw)

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription

Jer-Yuan Hsu et al. Genes Dev. 2008.

Abstract

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways.

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Figures

Figure 1.

Figure 1.

Depletion of TBP reduces TATA-dependent but not DPE-dependent transcription. Drosophila S2 cells were depleted of the indicated factors by RNAi, and then transfected with TATA-dependent or DPE-dependent luciferase reporter genes. The activities of the RNAi-depleted extracts are reported as luminescent units per microgram of protein of RNAi-depleted extracts relative to the luminescent units per microgram of protein of mock RNAi-treated control extracts. The experimental scheme and reporter constructs are depicted at the bottom of the figure. “PC4-like” is Ssb-C31a, which is the Drosophila protein that is most closely related to mammalian PC4. The error bars represent the standard deviation.

Figure 2.

Figure 2.

Mot1 and NC2 act in opposition to TBP to promote DPE transcription relative to TATA transcription. (A) Mot1, NC2, and TBP are efficiently depleted by RNAi in S2 cells. (B) The ability of Mot1 and NC2 to affect DPE-dependent versus TATA-dependent transcription requires TBP. RNAi depletion analysis of the indicated factors was carried out as in Figure 1. (C) Overexpression of TBP has the opposite effect as overexpression of Mot1 or NC2 upon DPE-dependent versus TATA-dependent transcription. The indicated expression vectors were cotransfected with DPE-dependent or TATA-dependent luciferase reporter genes. In each series of transfections, the total amount of expression vector was maintained at a constant level by the inclusion, where necessary, of a compensatory amount of empty vector (pAc5.1). The reporter gene activities with the expression vectors are given relative to those obtained with the empty vector alone. The error bars represent the standard deviation.

Figure 3.

Figure 3.

Mot1 and NC2 have opposite effects as TBP upon transcription of DPE- versus TATA-containing endogenous genes in Drosophila cells. These studies examined secondary/late ecdysone-responsive genes that are activated upon ecdysone induction. Drosophila Kc167 cells were depleted of the indicated factors by RNAi, and then induced with 20-hydroxyecdysone (20HE; 1 μM) for 24 h. The transcription levels of two TATA-containing genes (that lack DPE motifs) and three DPE-containing genes (that lack TATA elements) were determined by real-time RT–PCR. The error bars represent the SEM.

Figure 4.

Figure 4.

TBP ChIP increases in TATA- but not DPE-containing promoters upon ecdysone induction. ChIP analyses of TBP and RNA polymerase II (Rpb3 subunit) were carried out with Drosophila Kc167 cells in the absence or presence of 20HE. Three ecdysone-responsive genes as well as hsp70, as a reference/control, were analyzed. The amounts of immunoprecipitated DNA with each set of the indicated primers were quantitated by real-time PCR. The enrichments observed with anti-TBP or anti-Rpb3 relative to a control nonimmune serum are shown. The error bars represent the SEM from three independent sets of experiments. The diagrams of the genes are not drawn to scale.

Figure 5.

Figure 5.

Mot1, NC2, and TBP create a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription. In this model, TBP activates TATA transcription and represses DPE transcription, and Mot1 and NC2 act to inhibit the function of TBP. Thus, a decrease in TBP and/or an increase in Mot1/NC2 favors DPE transcription, whereas a decrease in Mot1/NC2 and/or an increase in TBP favors TBP transcription.

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