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TAF8 regions important for TFIID lobe B assembly are required for embryonic stem cell survival

ABSTRACT: The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 is forming a histone fold pair in lobe B and connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF core-interacting TAF8 domain, and the proline rich domain of TAF8 interacting with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that TAF8 regions involving in the connection of lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jie Luo

LAB HEAD: Jeff Ranish

PROVIDER: PXD026575 | Pride | 2021-10-02

REPOSITORIES: Pride

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			Action	DRS
	201703-TBP.inter1.xls	Xls
	201703-TBP.intra1.xls	Xls
	JL030217_030317_Fusion_MS2_TBP_100D.mzXML	Mzxml
	JL030217_030317_Fusion_MS2_TBP_2uL.mzXML	Mzxml
	JL030217_030317_Fusion_MS2_TBP_50B.mzXML	Mzxml

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Crosslinking Mapping of yeast Komagataella phaffii TFIID complex

Project description:Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. We used CXMS to study the organization and structure of the purified TFIID complex from Komagataella phaffii. Together with Cryo-EM, We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a HF domain. Interaction with promoter DNA highlights two non-selective binding sites consistent with a DNA scanning mode.

2018-10-08 | PXD011092 | Pride

Importance of TAF8 regions for TFIID lobe B assembly, or for TAF2 interactions, are required for embryonic stem cell survival

Project description:The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 is forming a histone fold pair in lobe B and connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF core-interacting TAF8 domain, and the proline rich domain of TAF8 interacting with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that TAF8 regions involving in the connection of lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

2022-02-15 | PXD028352 | Pride

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Project description:General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic nuclei. Human TFIID is a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). The cellular mechanism of TFIID assembly is poorly understood. In the cytoplasm of human cells, we discovered a heterotrimeric TFIID sub-complex consisting of the TAF2, TAF8 and TAF10 proteins. Native mass-spectrometry uncovered the interactions between the TAFs, defining a central role of TAF8 in nucleating the complex. X-ray crystallography revealed a non-canonical arrangement of the TAF8-TAF10 histone fold domains (HFDs). TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a step-wise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.

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TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing [CUT&RUN]

Project description:TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

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TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing [RNA-Seq]

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TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing

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