Replication Factor C Complexes Play Unique Pro- and Anti-Establishment Roles in Sister Chromatid Cohesion

Recent studies have lead to a rapid expansion of sister chromatid cohesion pathways. Of particular interest is the growth in classifications of anti-establishment factors—now including those that are cohesin-associated (Rad61/WAPL and Pds5) or DNA replication fork-associated (Elg1-RFC). In this study, we show that the two classes of anti-establishment complexes are indistinguishable when challenged both genetically and functionally. These findings suggest that both classes function in a singular pathway that is centered on Ctf7/Eco1 (herein termed Ctf7) regulation. The anti-establishment activity of Elg1-RFC complex is particular intriguing given that an alternate Ctf18-RFC complex exhibits robust pro-establishment activity. Here, we provide several lines of evidence, including the use of Ctf7 bypass suppressors, indicating that these activities are not simply antagonistic. Moreover, the results suggest that Ctf18-RFC is capable of promoting sister chromatid pairing reactions independent of Ctf7. The combination of these studies suggest a new model of sister chromatid pairing regulation

The Elg1-RFC Clamp-Loading Complex Performs a Role in Sister Chromatid Cohesion

It is widely accepted that of the four Replication Factor C (RFC) complexes (defined by the associations of either Rfc1p, Ctf18p, Elg1p or Rad24p with Rfc2p-Rfc5p), only Ctf18-RFC functions in sister chromatid cohesion. This model is based on findings that CTF18 deletion is lethal in combination with mutations in either CTF7ECO1 or MCD1 sister chromatid cohesion genes and that ctf18 mutant cells exhibit cohesion defects. Here, we report that Elg1-RFC not only participates in cohesion but performs a function that is distinct from that of Ctf18-RFC. The results show that deletion of ELG1 rescues both ctf7eco1 mutant cell temperature sensitivity and cohesion defects. Moreover, over-expression of ELG1 enhances ctf7eco1 mutant cell phenotypes. These findings suggest that the balance of Ctf7pEco1p activity depends on both Ctf18-RFC and Elg1-RFC. We also report that ELG1 deletion produces cohesion defects and intensifies the conditional phenotype of mcd1 mutant cells, further supporting a role for Elg1-RFC in cohesion. Attesting to the specificity of these interactions, deletion of RAD24 neither suppressed nor exacerbated cohesion defects in either ctf7eco1 or mcd1 mutant cells. While parallel analyses failed to uncover a similar role in cohesion for Rad24-RFC, it is well known that Rad24-RFC, Elg1-RFC and Ctf18-RFC play key roles in DNA damage responses. We tested and found that Ctf7pEco1p plays a significant role in Rad24-RFC-based DNA response pathways. In combination, these findings challenge current views and document new and distinct roles for RFC complexes in cohesion and for Ctf7pEco1p in DNA repair

Verification of Infinium methylation score by pyrosequencing.

<p>A) Pyrograms of GRIK1 CpG loci hypermethylated in a ccRCC but unmethylated in an oncocytoma and NRP, B) Correlation between Infinium methylation score and pyrosequencing. The R² is the Pearson coefficient. Circled red, gray, and black points correspond to ccRCC, oncocytoma, and NRP specimens respectively shown in A).</p

Genome-Wide Promoter Methylome of Small Renal Masses

<div><p>The majority of renal cell carcinoma (RCC) is now incidentally detected and presents as small renal masses (SRMs) defined as ≤4 cm in size. SRMs are heterogeneous comprising several histological types of RCC each with different biology and behavior, and benign tumors mainly oncocytoma. The varied prognosis of the different types of renal tumor has implications for management options. A key epigenetic alteration involved in the initiation and progression of cancer is aberrant methylation in the promoter region of a gene. The hypermethylation is associated with transcriptional repression and is an important mechanism of inactivation of tumor suppressor genes in neoplastic cells. We have determined the genome-wide promoter methylation profiles of 47 pT1a and 2 pT1b clear cell, papillary or chromophobe RCC, 25 benign renal oncocytoma ≤4 cm and 4 normal renal parenchyma specimens by Infinium HumanMethylation27 beadchip technology. We identify gene promoter hypermethylation signatures that distinguish clear cell and papillary from each other, from chromophobe and oncocytoma, and from normal renal cells. Pairwise comparisons revealed genes aberrantly hypermethylated in a tumor type but unmethylated in normal, and often unmethylated in the other renal tumor types. About 0.4% to 1.7% of genes comprised the promoter methylome in SRMs. The Infinium methylation score for representative genes was verified by gold standard technologies. The genes identified as differentially methylated implicate pathways involved in metabolism, tissue response to injury, epithelial to mesenchymal transition (EMT), signal transduction and G-protein coupled receptors (GPCRs), cancer, and stem cell regulation in the biology of RCC. Our findings contribute towards an improved understanding of the development of RCC, the different biology and behavior of histological types, and discovery of molecular subtypes. The differential methylation signatures may have utility in early detection and particularly differential diagnosis for prognostic stratification as well as identify novel gene and pathway targets for therapeutic intervention.</p></div

Pathways significantly overrepresented by aberrant gene methylation.

<p>IPA analysis of hypermethylated genes identified significant canonical pathways A) all RCC, B) ccRCC, C) pRCC.</p

Number of genes hypermethylated in RCC and oncocytoma.

<p>Number of genes hypermethylated in RCC and oncocytoma.</p

Barplots of genes aberrantly hypermethylated in RCC or oncocytoma.

<p>Y axis is Infinium β-value. Tumors above the horizontal blue line (delta β 0.2> mean of NRP) are hypermethylated for the gene compared to the unmethylated (β<0.15) 4 NRP. The gene name and Infinium probe ID are given above each barplot.</p

PRC target genes are overrepresented in the renal cancer methylome.

<p>Proportion of PRC target genes among genes hypermethylated in renal cancer compared to ∼4% with 3 marks or 9.5% with 1 or more marks in human ES cells from Lee et al 2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077309#pone.0077309-Lee1" target="_blank">[48]</a>.</p

Barplots of genes differentially methylated between tumor types.

<p>Y axis is Infinium β-value. For each gene, tumors above the horizontal blue line (delta β 0.2> mean of comparison tumor type) are hypermethylated in the tumor type but unmethylated (β<0.15) in the comparison tumor type.</p