Project description:TAF7, a component of the TFIID complex that nucleates the assembly of transcription preinitiation complexes, also independently interacts with and regulates the enzymatic activities of other transcription factors, including P-TEFb, TFIIH and CIITA, ensuring an orderly progression in transcription initiation. Since not all TAFs are required in terminally differentiated cells, we examined the essentiality of TAF7 in cells at different developmental stages in vivo. Germ-line disruption of the TAF7 gene is embryonic lethal between 3.5 and 5.5 days post coitus. TAF7-deleted mouse embryonic fibroblasts (MEFs) globally cease transcription and stop proliferating. In contrast, whereas TAF7 is essential for the differentiation and proliferation of immature thymocytes, it is not required for subsequent, proliferation-independent differentiation of lineage committed thymocytes or their egress into the periphery. TAF7 deletion in peripheral CD4+ T cells affects only a small number of transcripts. However, TAF7-deleted T cells are not able to undergo activation and expansion in response to antigenic stimuli. These findings suggest that TAF7 is essential for proliferation but not for proliferation-independent differentiation. We previously provided evidence that TAF7 is a regulator of the early steps of transcription initiation, that it affects the expression of about 65% of transcripts in 293 kidney cells, and of those, some are TAF1-dependent while others are TAF1-independent. Therefore, we next asked whether TAF7 is dispensable for a subset of embryonic fibroblast genes or whether it is required globally. Both TAF7f/f and TAF7+/+ MEF lines were infected by MSCV Cre-GFP retroviruses, and the infected cells (GFP+) were isolated by FACS at 24, 48 and 72 hours post infection. Uninfected cells from each line were harvested at the same time, collected by FACS and processed simultaneously to be used as controls. The experiments were performed in triplicate. All RNAs were extracted using shredders and the Qiagen RNeasy mini kit and their quality assayed before using for hybridization. Total RNA from each of the triplicates was hybridized on the Affymetrix exon array. All exon array data were analyzed with Affymetrix Expression Console SoftwareTM (version 1.1). The Robust Multi-array Analysis (RMA) algorithm was used for gene intensity analysis. Only genes in the M-bM-^@M-^\coreM-bM-^@M-^] set, which represents RefSeq and full-length GenBank mRNAs, were included in the analysis.

Project description:TAF7, a component of the TFIID complex that nucleates the assembly of transcription preinitiation complexes, also independently interacts with and regulates the enzymatic activities of other transcription factors, including P-TEFb, TFIIH and CIITA, ensuring an orderly progression in transcription initiation. Since not all TAFs are required in terminally differentiated cells, we examined the essentiality of TAF7 in cells at different developmental stages in vivo. Germ-line disruption of the TAF7 gene is embryonic lethal between 3.5 and 5.5 days post coitus. TAF7-deleted mouse embryonic fibroblasts (MEFs) globally cease transcription and stop proliferating. In contrast, whereas TAF7 is essential for the differentiation and proliferation of immature thymocytes, it is not required for subsequent, proliferation-independent differentiation of lineage committed thymocytes or their egress into the periphery. TAF7 deletion in peripheral CD4+ T cells affects only a small number of transcripts. However, TAF7-deleted T cells are not able to undergo activation and expansion in response to antigenic stimuli. These findings suggest that TAF7 is essential for proliferation but not for proliferation-independent differentiation. The T cells are purified spleen CD4+ T cells coming either from TAF7f/- 8EIII-cre+ (TAF7 -/-) or TAF7 f/- 8EIII-cre - (TAF7+/-). The cells were purified by FACS based on cell surface marker expression: high TCRbeta and CD4 expression. The cells are enriched in naive CD4 T cells based on their low level of CD44 surface expression. All RNAs were extracted using shredders and the Qiagen RNeasy mini kit and their quality assayed before using for hybridization. Total RNA was hybridized on the Affymetrix exon array. All exon array data were analyzed with Affymetrix Expression Console SoftwareTM (version 1.1). The Robust Multi-array Analysis (RMA) algorithm was used for gene intensity analysis. Only genes in the "core" set, which represents RefSeq and full-length GenBank mRNAs, were included in the analysis.

Project description:RNA polymerase II (Pol II) transcription initiation starts with the assembly of the preinitiation complex (PIC) on core promoters. The PIC is composed of six general transcription factors (GTFs). The recognition of the core promoter sequences by the TFIID GTF complex is the first step of the PIC assembly. In metazoans, holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Genetic depletion of different murine TAFs have shown that TAFs such as TAF7 or TAF10, can be either required, or dispensable for Pol II transcription, depending on different cellular contexts. In this report, we depleted TAF7 and/or TAF10 in the same cellular system; either in mesodermal progenitors during mouse development or in mESCs. In these two models, TAF7 depletion leads to a milder phenotype compared to TAF10 depletion. As TAF10 is also a subunit of the transcriptional co-activator Spt-Ada-Gcn5 acetyl transferase (SAGA), we first showed that the difference in phenotype between the Taf7 and Taf10 mutant is not due to the SAGA effect, at least for mESCs. Immunoprecipitations coupled with mass spectrometry analyses from mESCs lysates assembly of holo-TFIID complex is rapidly affected after induction of the depletion. In line with the model of holo-TFIID sequential assembly, TAF10 depletion leads to an early defect with formation of the core-TFIID, while TAF7 depletion results in the formation of a TAF7-less TFIID. Thus, the difference in phenotype severity correlates with the degree of TFIID disassembly. Surprisingly, no major global changes in Pol II transcription could be observed after either TAF7 or TAF10 depletion. Our data suggest that the inducible loss of fully assembled canonical TFIID does not correlate with the lack of global Pol II transcription activity changes suggesting that partially assembled TFIID complexes can participate in Pol II transcription initiation, with only limited effect on Pol II nascent transcription.

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Mus musculus

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