New Functions of Ctf18-RFC in Preserving Genome Stability outside Its Role in Sister Chromatid Cohesion

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability—expansions, contractions, and fragility—with effect over a wide range of allele lengths from 20–155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair

Base J and H3.V Regulate Transcriptional Termination in <i>Trypanosoma brucei</i>

<div><p><i>Trypanosoma brucei</i> is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. <i>T</i>. <i>brucei</i> retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V) and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil), are two chromatin marks found at both transcription termination sites (TTSs) and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (<i>VSG</i>) genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.</p></div

The absence of base J and H3.V results in an increase in the number of transcripts between genes that flank convergent SSRs.

<p>(A) Schematic, the log₂(RPKM) value is computed only for the region lying between the two genes that flank the cSSR. (B-D) Scatter plots showing log₂(RPKM) values for WT and indicated mutant cells computed for regions between genes that flank the cSSR, defined as between the 3´ end of the last gene in the (+) strand PTU and the 5´ end of the first gene in the (-) strand PTU. Dotted grey lines indicate changes that are 4-fold up or down. Comparisons between WT and <i>JΔ</i> cells are shown in (B), WT and <i>H3</i>.<i>VΔ</i> cells in (C), and WT and <i>JΔ H3</i>.<i>VΔ</i> cells in (D). (E) Boxplot for log₂(RPKM) values computed for the regions plotted in B-D. Boxplots are displayed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005762#pgen.1005762.g002" target="_blank">Fig 2G</a>. * indicates a significant difference (<i>P</i> < 0.05) as measured by a Mann-Whitney U test. ** <i>P</i> < 0.01 (F) q-PCR experiment on total RNA in WT and mutant cells to determine transcripts level at selected TTSs. TTS-30, 68, 79, 100, 102, and 128 all showed higher expression of polyA+ RNA in <i>JΔ H3</i>.<i>VΔ</i> cells when compared to WT cells in the RNA-seq experiment. TTS-105 did not show upregulation in the RNA-seq dataset and was used as a negative control. MCM-BP primers were used as an additional negative control. Significance is measured using a Student’s T test.</p

Cells that are simultaneously deleted for base J and H3.V are viable.

<p>WT, <i>JΔ</i>, <i>H3</i>.<i>VΔ</i>, and <i>JΔ H3</i>.<i>VΔ</i> cells were diluted to 10,000 cells/ml and cells were counted after two days of incubation. This was repeated two more times. Cells were counted three times. Error bars are shown and there were no significant differences between WT and mutant cells.</p

H3.V is important for maintaining silencing of a subset of <i>VSG</i> genes.

<p>(A) Schematic diagrams of <i>VSG</i> locations; BES <i>VSG</i> (Bloodstream-form Expression Site <i>VSG</i>), m<i>VSG</i> (metacyclic <i>VSG</i>), and MC <i>VSG</i>, (minichromosomal <i>VSG</i>). Reads from the RNA-seq experiment were aligned to the <i>VSG</i>nome database and log₂(RPKM) values were obtained for all <i>VSG</i> genes. (B-D) log₂(RPKM) values for <i>VSG</i>s with known chromosomal locations were compared between the indicated mutant samples and WT in scatter plots. Dotted grey lines indicate changes that are 4-fold up or down. Blue dots are BES-<i>VSG</i>s, red are metacyclic promoter-associated <i>VSG</i>s (m<i>VSG</i>) and grey are minichromosomal <i>VSG</i>s. The orange circle corresponds to <i>VSG</i> 582, which is associated with a metacyclic promoter in an atypical metacyclic ES located far upstream of the telomere [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005762#pgen.1005762.ref022" target="_blank">22</a>]. MC, minichromosomal, m, metacylic promoter-associated. <i>ESAG</i>, Expression Site Associated Gene. (E-G) Notched boxplots of log₂(RPKM) values were generated for all <i>VSG</i>s located in BESs (E), metacyclic expression sites (F), or minichromosomes (G). Expression values for the <i>VSG</i> in the active BES are not shown on the BES boxplots in (E) but do not vary significantly between genotypes, ranging from 18.47–18.54. Red lines indicate median values, and are indicated with numbers on the plot. Upper and lower blue lines mark the interquartile range. Whiskers mark data falling within the 1^st and 4^th quartiles. Outliers are shown as blue dots. Notches are calculated as ±1.58 x IQR/√n where n indicates the number of samples and IQR is the inner quartile range. Significant differences are demarcated by * (<i>P</i> < 0.05) and ** (<i>P</i> < 0.01) between indicated mutant and wild-type values as measured by a Mann-Whitney U statistical test.</p

Deletion of base J and H3.V results in the production of antisense transcripts near regions of transcription termination.

<p>(A) Diagram of windows used to compute values. (B) and (C) The median difference between the indicated mutant cell log₂(RPKM) value and the WT log₂(RPKM) value was calculated for all genes within 5 kb sliding windows with step size 100bp extending from the halfway point of the region lying between the 3´ end of the last gene in the (+) strand PTU or the 5´ end of the first gene in the (-) strand PTU that flanks the cSSR. Median difference in gene expression was plotted against the distance of each 5 kb window from the halfway point. (B) Sense transcripts. (C) Antisense transcripts. The halfway point is indicated by a black line with 0 in the diagram in (A).</p

Deletion of base J and H3.V increases antisense transcription near convergent SSRs.

<p>(A) Top, schematic of computed values. log₂(RPKM) values are calculated in WT or mutant cells for each gene that falls within 1,000 bp of a cSSR. In this example, 2 genes upstream of this cSSR and one gene downstream of this cSSR (indicated by solid black frames around the gene) would be included in the analysis. Bottom left, boxplot generated from log₂(RPKM) values from sense transcripts of genes that flank cSSRs, defined as within 1,000 bp upstream of the 3´ end of the last gene in the (+) strand PTU or within 1,000 bp downstream of the 5´ end of the first gene in the (-) strand PTU in WT and mutant cells as indicated. Boxplots are displayed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005762#pgen.1005762.g002" target="_blank">Fig 2G</a>. Bottom right, same as bottom left except for antisense transcripts. ** indicates a significant difference (P < 0.01) between indicated mutant and WT values as measured by a Mann-Whitney U statistical test. (B) Top, same as diagram in A except the length has been extended to 5,000 bp so more genes are included in the analysis, in this case 4 genes upstream and 3 genes downstream of the cSSR. Bottom, boxplots were generated as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005762#pgen.1005762.g003" target="_blank">Fig 3A</a>) except that the length on either end of the cSSR has been increased to 5,000 bp. (C) Boxplots were generated as in (A) except that the length on either end of the cSSR has been increased to 10,000 bp.</p