Ortholog replacement reveals stable TFIIIC complexes are required for proper mitotic chromosome segregation
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ABSTRACT:
PROVIDER: PRJNA1211566 | ENA |
REPOSITORIES: ENA
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Project description:Transcription factors are speculated to play crucial roles in adaptive evolution. Here we investigated how essential transcription factors (eTFs) change using ortholog replacement assays. Several orthologous eTFs from other yeast species could not fully complement the mutants in Saccharomyces cerevisiae, suggesting that these eTFs have changed their functions or interactions to become incompatible. We further characterized TFIIIC, a protein complex assisting RNA polymerase III transcription, that exhibits complete or partial incompatibility in several subunits. In the orthologous Tfc7-replacement line, the binding of TFIIIC to tRNA genes is reduced, but the abundance of tRNAs is not severely affected. However, Tfc7-replacement cells often mis-segregate chromosomes during mitosis and their fitness is further reduced in the spindle checkpoint mutant. Chromatin immunoprecipitation experiments showed that unstable TFIIIC binding results in defective cohesion loading, leading to chromosome mis-segregation. Swapping the highly divergent C-terminal domain of Tfc7 orthologs rescues its interaction with Tfc1 and cell fitness, suggesting that incompatibility is caused by altered interactions between complex subunits. Our results reveal separatable essential functions of a well-studied protein complex.
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Project description:Condensin complexes are highly conserved for chromosome compaction to ensure their faithful segregation in mitosis. However, little is known about the role of condensin complexes in interphase. Condensins exists in two complexes, condensins I and II, in higher eukayotic cells. During interphase, condensin II is predominantly localized in the nucleus throughout the cell cycle, whereas condensin I is localized at the cytoplasm in interphase. The distinct localization patterns suggest that condensin II, but not condensin I, may contribute to genome organization in interphase. Our results suggest that condensin II is associated with TFIIIC complex in vivo. The aim of these experiments is to unravel the dependency of each other on binding to chromatin.
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Project description:Condensin complexes are highly conserved for chromosome compaction to ensure their faithful segregation in mitosis. However, little is known about the role of condensin complexes in interphase. Condensins exists in two complexes, condensins I and II, in higher eukayotic cells. During interphase, condensin II is predominantly localized in the nucleus throughout the cell cycle, whereas condensin I is localized at the cytoplasm in interphase. The distinct localization patterns suggest that condensin II, but not condensin I, may contribute to genome organization in interphase. Our results suggest that condensin II is associated with TFIIIC complex in humans.
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Project description:How repetitive elements, epigenetic modifications and architectural proteins interact ensuring proper genome expression remains poorly understood. Here we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the Activity-Dependent Neuroprotector Homeobox protein (ADNP) and located near cell cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.
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Project description:Folding of the mammalian genome is governed by architectural proteins, such asCTCF. TFIIIC, a RNA polymerase III transcription factor, has been identified as aninsulator but its role in genome topology is totally unknown. Here, we show that TFIIICestablishes long-range genomic interactions that affect gene expression. Upon serumstarvation (SS), TFIIIC occupancy increases at Alu elements (AEs) near promoters ofcell cycle-related genes. Bound AEs become H3K18 hyper-acetylated and fold tocontact distal pre-loaded CTCF sites near other cell cycle genes. The promoters ofthese genes also become hyper-acetylated ensuring their basal transcription during SSand their increased expression during serum re-exposure. Ablation of TFIIIC ordeletion of the TFIIIC-bound AE that loops to the G2/M cycling F (CCNF) locus affectsits expression and nuclear positioning. These results illustrate a novel function ofhuman TFIIIC in changing 3D genome topology through the epigenetic state of AEs.
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Project description:RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a single putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131 and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major.
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Project description:Eukaryotic chromosomes reach their stable rod-shaped appearance in mitosis in a reaction dependent on the evolutionarily conserved condensin complex. Little is known about how and where condensin associates with chromosomes. Here, we analyse condensin binding to budding yeast chromosomes using high resolution oligonucleotide tiling arrays. Condensin binding sites coincide with those of the loading factor Scc2/4 of the related cohesin complex. The sites map to tRNA genes, ribosomal protein genes, and other places characterised by the RNA polymerase III transcription factor TFIIIC. An ectopic B-Box element, recognised by TFIIIC, constitutes a minimal condensin binding site, and TFIIIC and the Scc2/4 complex promote productive condensin association with chromosomes. A similar pattern of condensin binding is conserved along fission yeast chromosomes. This reveals that TFIIIC binding sites, including tRNA genes, constitute a hitherto unknown chromosomal feature with important implications for chromosome architecture during both interphase and mitosis. Keywords: Chip-chip, cell type comparison
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