Transcriptional regulator Taf14 binds DNA and is required for the function of transcription factor TFIID in the absence of histone H2A.Z.

The transcriptional regulator Taf14 is a component of multiple protein complexes involved in transcription initiation and chromatin remodeling in yeast cells. Although Taf14 is not required for cell viability, it becomes essential in conditions where the formation of the transcription preinitiation complex is hampered. The specific role of Taf14 in mediating transcription initiation and preinitiation complex formation is unclear. Here, we explored its role in the general transcription factor IID by mapping Taf14 genetic and proteomic interactions and found that it was needed for the function of the complex if Htz1, the yeast homolog of histone H2A.Z, was absent from chromatin. Dissecting the functional domains of Taf14 revealed that the linker region between the YEATS and ET domains was required for cell viability in the absence of Htz1 protein. We further show that the linker region of Taf14 interacts with DNA. We propose that providing additional DNA binding capacity might be a general role of Taf14 in the recruitment of protein complexes to DNA and chromatin.

Taf14 is required for the stabilization of transcription pre-initiation complex in Saccharomyces cerevisiae.

BACKGROUND: The YEATS domain is a highly conserved protein structure that interacts with acetylated and crotonylated lysine residues in N-terminal tails of histones. The budding yeast genome encodes three YEATS domain proteins (Taf14, Yaf9, and Sas5) that are all the subunits of different complexes involved in histone acetylation, gene transcription, and chromatin remodeling. As the strains deficient in all these three genes are inviable, it has been proposed that the YEATS domain is essential in yeast. In this study we investigate in more detail the requirement of YEATS domain proteins for yeast survival and the possible roles of Taf14 YEATS domain in the regulation of gene transcription. RESULTS: We found that YEATS domains are not essential for the survival of Saccharomyces cerevisiae cells. Although the full deletion of all YEATS proteins is lethal in yeast, we show that the viability of cells can be restored by the expression of the YEATS-less version of Taf14 protein. We also explore the in vivo functions of Taf14 protein and show that the primary role of its YEATS domain is to stabilize the transcription pre-initiation complex (PIC). Our results indicate that Taf14-mediated interactions become crucial for PIC formation in rpb9Δ cells, where the recruitment of TFIIF to the PIC is hampered. Although H3 K9 residue has been identified as the interaction site of the Taf14 YEATS domain in vitro, we found that it is not the only interaction target in vivo. CONCLUSIONS: Lethality of YEATS-deficient cells can be rescued by the expression of truncated Taf14 protein lacking the entire YEATS domain, indicating that the YEATS domains are not required for cell survival. The YEATS domain of Taf14 participates in PIC stabilization and acetylated/crotonylated H3K9 is not the critical target of the Taf14 YEATS domain in vivo.

Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival.

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective.

Uniform distribution of elongating RNA polymerase II complexes in transcribed gene locus.

The intensity of gene transcription is generally reflected by the level of RNA polymerase II (RNAPII) recruitment to the gene. However, genome-wide studies of polymerase occupancy indicate that RNAPII distribution varies among genes. In some loci more polymerases are found in the 5' region, whereas in other loci, in the 3' region of the gene. We studied the distribution of elongating RNAPII complexes at highly transcribed GAL-VPS13 locus in Saccharomyces cerevisiae and found that in the cell population the amount of polymerases gradually decreased toward the 3' end of the gene. However, the conventional chromatin immunoprecipitation assay averages the signal from the cell population, and no data on single cell level can be gathered. To study the spacing of elongating polymerases on single chromosomes, we used a sequential chromatin immunoprecipitation assay for the detection of multiple RNAPII complexes on the same DNA fragment. Our results demonstrate uniform distribution of elongating polymerases throughout all regions of the GAL-VPS13 gene.

Relicensing of transcriptionally inactivated replication origins in budding yeast.

DNA replication origins are licensed in early G(1) phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G(1) to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G(1) might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.

Acetylation of H3 K56 is required for RNA polymerase II transcript elongation through heterochromatin in yeast.

In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription.