| 1992 |
Yeast SPT3 physically associates with TFIID (TBP) in yeast cell extracts, as demonstrated by coimmunoprecipitation. Allele-specific suppression of spt15-21 (a TBP mutation) by spt3 mutations indicates a direct functional interaction between SPT3 and TBP required for transcription at particular promoters in vivo. |
Coimmunoprecipitation from yeast extracts; allele-specific extragenic suppressor genetics |
Genes & development |
High |
1628834
|
| 1984 |
SPT3 is required for normal transcription initiation from delta (Ty LTR) sequences in S. cerevisiae; in spt3 null mutants, Ty delta-delta transcripts are absent and initiation shifts ~800 bp into the epsilon region, establishing SPT3 as a positive regulator of Ty element transcription. |
Genetic null mutant analysis; Northern blot / transcript mapping |
Cell |
High |
6096019
|
| 1998 |
Human SUPT3H (hSPT3) is not associated in vivo with human TBP/TFIID or with a TBP-free TAFII complex; instead, hSPT3 co-purifies in vivo with TAFII31 and the long form of human GCN5 (hGCN5-L) in a novel complex (STAGA) that possesses histone acetyltransferase activity. This established STAGA as the human homologue of yeast SAGA. |
Immunoprecipitation from HeLa cell nuclear extracts; histone acetyltransferase activity assay; molecular cloning and sequence analysis |
The Journal of biological chemistry |
High |
9726987
|
| 2000 |
Within SAGA, Spt3 (and Spt8) inhibit TBP binding to the HIS3 promoter in vitro; SAGA lacking Spt3 or Spt8 loses this inhibitory activity. Two distinct forms of SAGA exist in cell extracts, one lacking Spt8, and conditions that induce transcription shift the balance toward the Spt8-lacking form, indicating that SAGA composition is dynamic and that Spt3/Spt8 function as inhibitory subunits for TBP recruitment under non-induced conditions. |
In vitro TBP-DNA binding assay with purified SAGA complexes; biochemical fractionation of SAGA isoforms; genetic analysis of spt3 and spt8 deletion effects on transcription |
Molecular and cellular biology |
High |
10611242
|
| 1997 |
Genetic epistasis experiments show that SPT3 functionally interacts with MOT1 (an ATP-dependent TBP inhibitor) and TFIIA to regulate TBP-DNA interactions and TATA-box selection in vivo. Double mutant lethality (spt3Δ mot1) and suppression of spt3Δ by TFIIA overexpression define a cooperative pathway controlling TBP distribution at promoters. |
Synthetic lethality screen; genetic suppressor analysis; in vivo transcription assays |
Molecular and cellular biology |
Medium |
8972209
|
| 1994 |
Genetic analysis shows that spt8 null mutations are suppressed by particular spt3 alleles, suggesting that SPT8 promotes a functional SPT3–TBP interaction. Both SPT8 and SPT3 are required for TBP function at specific promoters. |
Genetic suppressor analysis; null mutant combination phenotyping |
Genetics |
Medium |
8088510
|
| 2008 |
Site-specific in vivo and in vitro cross-linking using the non-natural photoreactive amino acid BPA substituted onto TBP surface residues demonstrates a direct physical interaction between TBP and SAGA subunits Spt3 and Spt8. Mutations on the Spt3-interacting surface of TBP reduce TBP–SAGA interaction, decrease transcriptional activation, and impair TBP recruitment to a SAGA-dependent promoter, proving that a direct Spt3–TBP contact is required for activated transcription. |
Site-specific photocrosslinking with non-natural amino acid BPA in vivo and in vitro; chromatin immunoprecipitation; in vivo transcription assays; mutagenesis |
Genes & development |
High |
18981477
|
| 2004 |
Spt3 (within SAGA) is required for nucleosome remodeling at the GAL1 promoter upon transcriptional induction, and this function is independent of TBP recruitment. Spt3 and Mot1 are both required for nucleosome remodeling and are recruited to GAL1 promoter (and a non-promoter nucleosome near an activator-binding site) in an interdependent manner, revealing a chromatin remodeling role for Spt3 distinct from its TBP-delivery role. |
Chromatin immunoprecipitation; nucleosome remodeling assays; genetic analysis of spt3 and mot1 mutants; synthetic promoter experiments |
The EMBO journal |
Medium |
15057269
|
| 2007 |
New dominant-negative spt3 mutations cluster in a conserved region of Spt3 and allele-specifically interact with spt15 (TBP) mutations, confirming a direct Spt3–TBP interface in vivo. One spt3 mutation (spt3-401) greatly increases SAGA–TBP physical association, while most spt3, spt8, and spt15 mutations do not alter bulk SAGA–TBP interaction, suggesting that direct Spt3–TBP contact is required for normal TBP levels at Spt3-dependent promoters. |
Dominant-negative overexpression genetics; extragenic suppressor isolation; TBP ChIP; SAGA–TBP co-immunoprecipitation |
Genetics |
Medium |
18073420
|
| 2007 |
A SAGA-independent function of SPT3 is identified: deletion of SPT3, but not deletion of other SAGA subunits (SPT7), suppresses transcriptional defects of a not1-2 (Ccr4-Not scaffold) mutant, and spt3Δ shows synthetic phenotypes with spt7Δ. This indicates SPT3 has functions outside the SAGA complex that are functionally linked to the Ccr4-Not complex. |
Genetic suppressor analysis; transcriptional assays; genetic epistasis with SAGA and Ccr4-Not mutants |
Genetics |
Medium |
17660549
|
| 1998 |
The human SUPT3H cDNA encodes a protein sharing 30% identity with yeast Spt3 across three conserved domains. Full-length SUPT3H cannot complement yeast spt3Δ, but a human-yeast chimeric gene containing 42% human sequences can partially complement, indicating partial functional conservation of transcriptional control from yeast to human. |
Molecular cloning; yeast complementation assay with full-length and chimeric constructs |
Genomics |
Medium |
9787080
|
| 2022 |
In mammalian cells (human U2OS and mouse ESCs), SAGA can assemble without SUPT3H. Loss of SUPT3H does not cause major changes in TBP accumulation at gene promoters and does not globally impair RNA Pol II transcription; instead, SUPT3H affects transcription of only a specific gene subset and is required for mESC growth and self-renewal. This contrasts with yeast where Spt3 broadly controls TBP recruitment. |
Biochemical purification of SAGA from SUPT3H-knockout cells; ChIP for TBP; RNA-seq; mESC growth/self-renewal assays |
Nucleic acids research |
High |
35871303
|
| 2020 |
The spliceosomal ATPase Prp5p directly interacts with SAGA subunit Spt8p (but not Spt3p) in vitro. However, both spt8Δ and spt3Δ rescue Prp5 splicing defects and restore Pol II recruitment to an intron-containing gene. This interaction mediates reciprocal coupling between transcription initiation/elongation (via the SAGA TBP-binding module containing Spt3/Spt8) and pre-spliceosome assembly. |
In vitro binding assay (Prp5–Spt8 direct interaction); genetic suppressor analysis; chromatin immunoprecipitation (ChIP and ChIP-seq) |
Nucleic acids research |
Medium |
32399566
|
| 2023 |
In S. cerevisiae, Spt3 and Spt8 (SAGA subunits) block the spread of telomeric silencing regions at the right arm of chromosome III in a TBP-interaction-dependent manner; mutants altering the Spt3–TBP interaction impair boundary formation. Spt3 has a greater genome-wide transcriptional effect than Spt8, and boundary formation is DNA sequence-independent. |
Microarray transcriptome analysis; RT-qPCR of subtelomeric genes; genetic analysis of spt3–TBP interaction mutants |
Biomolecules |
Medium |
37189367
|
| 2025 |
ChIP-seq in amino acid-starved S. cerevisiae shows that deleting SPT3 or SPT8 (but not GCN5) reduces TBP binding at many Gcn4 target genes, while deleting GCN5 (but not SPT3 or SPT8) impairs promoter histone eviction. Nuclear depletion of TFIID subunit Taf1 further reduces TBP recruitment at SAGA-dependent genes only when Spt3 or Spt8 are absent, demonstrating that SAGA's TBP-recruitment function via Spt3/Spt8 is non-redundant with TFIID in the Gcn4 transcriptome. |
ChIP-seq for TBP and Pol II; auxin-inducible degron nuclear depletion of Taf1; genetic deletion of SPT3, SPT8, GCN5 |
Nucleic acids research |
High |
40637224
|
| 2014 |
In murine pre-osteoblastic MC3T3-E1 cells, the Supt3h promoter physically contacts the bone-specific Runx2-P1 promoter (located in the first intron of which Supt3h resides) with increased contact frequency during osteoblast differentiation. RUNX2 and CTCF bind the Supt3h promoter, and interplasmid-3C plus luciferase reporter assays show that the Supt3h promoter can modulate Runx2-P1 transcriptional activity via direct association. |
Chromosome conformation capture (3C); DNaseI hypersensitivity assay; ChIP for RUNX2 and CTCF; luciferase reporter assay |
Nucleic acids research |
Medium |
25120271
|