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Results: 9

1.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  3.—

F igure 3.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

The rtf1Δ1 mutation suppresses spt5-242. Serial dilutions of SPT5 rtf1Δ and spt5-242 rtf1Δ strains transformed with the indicated RTF1 plasmids were spotted to SC −Trp media and grown at 22° or 30° for 3 days. Only rtf1Δ1, which disrupts Rtf1–Chd1 interactions, suppressed the cold-sensitive phenotype of spt5-242.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
2.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  9.—

F igure 9.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

Loss of Chd1 leads to increased H3 acetylation. Wild-type, chd1Δ, and set2Δ strains were subjected to ChIP with anti-H3Ac and anti-H3 antisera followed by QPCR analysis using primers directed against the promoters and transcribed regions of FLO8, STE11, PMA1, and TEF2. Bar graphs present H3Ac values relative to total histone H3 and are normalized to wild type.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
3.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  2.—

F igure 2.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

chd1 mutations suppress growth defects of spt dst1 mutants. Genetic crosses were performed to create strains carrying combinations of complete deletions of DST1 and CHD1 with the cold-sensitive spt5-242 mutation, a complete deletion of SPT4, and the temperature-sensitive spt6-14 mutation. Serial dilutions of these strains were spotted to YPD media and grown at the indicated temperature. (A) chd1Δ suppresses the growth defect of spt5-242 dst1Δ cells at 22° and 30°. (B) chd1Δ suppresses the growth defect of spt4Δ dst1Δ cells at 39°. (C) chd1Δ suppresses the growth defect of spt6-14 dst1Δ cells at 37°.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
4.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  4.—

F igure 4.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

spt5-242 is suppressed by loss of H3K4 or H3K36 methylation. (A) Spt5-242 is suppressed by loss of the H3K4 or H3K36 methyltransferases Set1 and Set2 but not by loss of the H3K79 methyltrasferase Dot1 nor the ubiquitin conjugating enzyme Rad6. Strains with the indicated genotypes were spotted to YPD and incubated at 30° for 2 days or 22° for 4 days. (B) Mutations altering H3K4 or H3K36 suppress spt5-242. SPT5 and spt5-242 strains carrying deletions of both histone H3–H4 loci and a CEN URA3 HHT1-HHF1 plasmid were transformed with plasmids carrying the indicated histone H3 allele. Trp+ transformants were spotted directly to 5FOA and incubated at 30° for 2 days or 22° for 3 days.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
5.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  6.—

F igure 6.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

Mutations that disrupt Rpd3S function suppress spt5-242. (A) To determine whether loss or reduced recruitment of the Rpd3S complex leads to suppression of spt5-242, strains carrying set2Δ, rpd3Δ, eaf3Δ, or rco1Δ mutations in combination with wild-type SPT5 or spt5-242 were spotted on YPD and incubated at 22° or 30° for 2 days. (B) Loss of Set2 suppresses the growth defect of spt5-242 dst1Δ double mutants. Strains carrying the indicated combinations of set2Δ, dst1Δ, and spt5-242 mutations were serially diluted on YPD media and incubated at 22°, 30°, or 37° for 3 days. (C) Loss of Rco1, a subunit of Rpd3S, suppresses the growth defect of spt5-242 dst1Δ double mutants. Strains carrying the indicated combinations of rco1Δ, dst1Δ, and spt5-242 mutations were serially diluted on YPD media and incubated at 22°, 30°, or 37° for 3 days.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
6.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  1.—

F igure 1.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

Reduced rate of RNA polymerase II elongation in spt5-242 mutant. (A) Location of ChIP probes on hybrid GAL1-YLR454W gene. (B) Measurement of RNAPII processivity. ChIP of RNAPII across the hybrid GAL1-YLR454W gene in wild-type cells and the indicated mutants was performed under inducing conditions. For each mutant, IP/Input values for each ChIP probe were determined and normalized to the corresponding value measured in the wild-type strain. (C) Measurement of elongation rate. Galactose-induced cells were treated with glucose to repress transcription from the GAL1-YLR454W gene and samples were processed for ChIP 5 min later. IP/Input values for each probe are expressed relative to the corresponding value for that probe measure just prior to addition of glucose.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
7.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  7.—

F igure 7.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

Mutations that interfere with histone acetylation do not suppress spt5-242. (A) Loss of the SAGA subunit Spt8 decreases the viability of spt5-242 mutants. A strain containing spt5-242 was crossed to a strain lacking SPT8. The resulting diploid was transformed with a URA3 SPT5 plasmid and then followed through sporulation. The parental strains, a wild-type control, and three representative double mutants were selected and spotted to YPD and 5FOA and incubated at 30° for 2 days. (B) Mutation of H3K9 or H3K14 fails to suppress spt5-242, but mutation of H3K9/14/18/23 leads to enhanced spt5-242 phenotypes. SPT5 and spt5-242 strains carrying deletions of both histone H3–H4 loci and a URA3 HHT1-HHF1 plasmid were transformed with plasmids carrying the indicated histone H3 allele. Trp+ transformants were spotted directly to 5FOA and incubated at 30° for 2 days or 22° for 3 days.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
8.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  5.—

F igure 5.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

All three conserved domains of Chd1 are required for its function and localization to chromatin. (A) The location of the conserved sequence motifs of Chd1 as well as sites targeted for mutations. (B) Deletion of either or both chromodomains of Chd1 is sufficient for suppression of spt5-242. SPT5 chd1Δ and spt5-242 chd1Δ strains were transformed with URA3 CEN plasmids carrying the indicated chd1 mutations expressed from the normal CHD1 promoter. Serial dilutions of cells with the indicated genotypes were spotted to SC −Ura media and grown at the indicated temperature for 4 days. (C and D) All three conserved domains of Chd1 are required for its association with chromatin. Strains expressing HA3-tagged forms of Chd1 were subjected to anti-HA1 ChIP followed by QPCR analysis using primers directed against the promoters and transcribed regions of PMA1 (C) and TEF2 (D).

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.
9.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  8.—

F igure 8.—. From: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription.

Cryptic, internal initiation of transcription in a chd1 mutant. Two approaches were used to determine whether loss of Chd1 leads to the appearance of cryptic, internally initiated transcripts over transcribed sequences. (A) Diagram of the pGAL1-FLO8-HIS3 reporter gene. Transcription initiation from the normal FLO8 start site produces a transcript in which HIS3 is out of frame and not translated. Internal initiation of this transcript produces in frame transcripts and a His+ phenotype. (B) Diagram showing the pattern of cells carrying the pGAL1-FLO8-HIS3 reporter and indicated genotypes that were patched onto YPD media. The strains labeled hht1Δ or hht2Δ lack one HHT-HHF locus. [HHT1] and [HHT2] refer to a strain with deletions of both HHTHHF loci complemented by a plasmid-borne copy of one of these two loci; in the patch labeled Δ4-30, this strain carries an hht2HHF2 plasmid, carrying a deletion of codons 4–30 of histone H3. Patches K4A and K4R indicate similar strains with plasmids carrying H3K4 mutations. (C) The YPD plate described in B was replica plated onto SC −His media utilizing either glucose or galactose as the carbon source and incubated at 30° for the indicated number of days. (D) RNA was isolated from the indicated strains and subjected to Northern blot analysis to detect short transcripts from STE11 (top) and FLO8 (middle). Hybridization to a RAD18 probe (bottom) was used as a loading control.

Tiffani Kiyoko Quan, et al. Genetics. 2010 February;184(2):321-334.

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