TAF7: A Check-point Regulator in Transcription Initiation and Translation

Division Of Clinical Sciences - Nci

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Abstract

The general transcription factor, TFIID, consists of the TATA binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. The largest TFIID component, TAF1 has both acetyltransferase (AT) and kinase activities. We demonstrated previously that TAF1 is necessary for the transcription of an MHC class I gene, and that its intrinsic acetyltransferase activity is essential for both in vitro transcription of naked DNA and in vivo transcription. TAF7, a 55 kD TFIID component, binds to TAF1 and inhibits its AT activity, resulting in repression of transcription. Upon completion of the assembly of the PIC, TAF7 is released from TAF1, allowing transcription to initiate. Surprisingly, TAF7 also contributes to the regulation of each of the subsequent steps of transcription. Namely it binds to TFIIH/CDK7, BRD4, and P-TEFb/CDK9, inhibiting their Pol II C-terminal domain (CTD) kinases activity that are required for early elongation, pause release and efficient elongation, respectively. Therefore, TAF7 serves as a critical checkpoint regulator that ensures each step of transcription is completed before proceeding to the next step . Interestingly, the kinetics and spectrum of genes affected by siRNA mediated depletion of TAF7 or TAF1 differs between the two. This finding supports the conclusion that TAF7 functions at multiple points in transcription, not just in PIC assembly. The importance of TAF7 in regulating normal transcription is documented by our finding that cells depleted of TAF7, by siRNA or shRNA technology, proliferate poorly. Consistent with the critical role of TAF7 in cell growth, expression profiling reveals that depletion of TAF7 by siRNA results in reduced expression of a large number of genes. We have discovered that in addition to the regulatory role it plays in transcription, TAF7 also functions as a chaperone that delivers newly synthesized RNA, in a crm-dependent manner, from the site of transcription to polysomes in the cytoplasm. Thus, TAF7 is an RNA binding protein that binds a large spectrum of RNAs and shuttles them from the nucleus to the cytoplasm. Consistent with TAF7 playing a functional role in the cytoplasm, we have mapped both NLS and NES elements within the protein. Mutation of the NES sequence results in reduced translation, consistent with a requirement for TAF7 delivery of RNA to polysomes. The cytoplasmic TA7 is found in a large 450kD complex that also contains many ribosomal proteins. Immunoprecipitation studies have validated the binding of TAF7 to ribosomal proteins and demonstrated that this binding is RNA dependent. Further validation is that TAF7 is recovered from purified polysomes. Deletion of TAF7 from the cytoplasm results in reduced translation. Taken together, these findings demonstrate that TAF7 is not only a checkpoint regulator of transcription but also functions to promote translation. Intriguingly, the TAF7 cytoplasmic/nuclear ratio differs among different cell types, increasing with increasingly malignant phenotypes. Current studies are directed at elucidating the role of TAF7 in malignancy. To further examine the role of TAF7 in global gene regulation and development we have generated conditional TAF7 knock-out mice. Complete ablation of TAF7 results in embryonic lethality; embryos do not develop beyond d.3-4. Excision of TAF7 in MEFs derived from conditional knock-outs results in aborted cell proliferation and a global reduction in transcription initiation. TAF7 is also essential for the differentiation of immature thymic precursors that undergo massive proliferation during their differentiation. Thus, TAF7 deletion mediated by cre, expressed under the control of the Lck promoter that functions in very early DN thymocytes, ablates all T cell development. In sharp contrast, TAF7 is not required for the subsequent proliferation-independent differentiation of lineage committed thymocytes. Deletion of TAF7 by cre under the control of a EIII8 enhancer, which only functions at the DP stage of thymocyte development spares the maturation of single positive cells which are able to egress into the periphery. However, although only a small number of transcripts is affected by TAF7 deletion, TAF7-deficient peripheral T cells are not able to undergo activation and expansion in response to antigenic stimuli. We conclude that TAF7 plays a critical role in transcription in cells that undergo proliferation, but that its role is more restricted in non-proliferating differentiated cells.