Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata

Abstract

Adherence, an important virulence factor, is mediated by the (Epithelial Adhesin) genes in the opportunistic pathogen Expression of adhesin-encoding genes requires tight regulation to respond to harsh environmental conditions within the host. The majority of genes are localized in subtelomeric regions regulated by subtelomeric silencing, which depends mainly on Rap1 and the Sir proteins. adhesion to epithelial cells is primarily mediated by Epa1. forms a cluster with and in the right telomere of chromosome E (E). This telomere contains a -acting regulatory element, the protosilencer Sil2126 between and the telomere. Interestingly, Sil2126 is only active in the context of its native telomere. Replacement of the intergenic regions between genes in E revealed that -acting elements between and are required for Sil2126 activity when placed 32 kb away from the telomere (Sil@-32kb). Sil2126 contains several putative binding sites for Rap1 and Abf1, and its activity depends on these proteins. Indeed, Sil2126 binds Rap1 and Abf1 at its native position and also when inserted at -32 kb, a silencing-free environment in the parental strain. In addition, we found that Sil@-32kb and Sil2126 at its native position can physically interact with the intergenic regions between and respectively, by chromosome conformation capture assays. We speculate that Rap1 and Abf1 bound to Sil2126 can recruit the Silent Information Regulator complex, and together mediate silencing in this region, probably through the formation of a chromatin loop.

Candida glabrata Antifungal Resistance and Virulence Factors, a Perfect Pathogenic Combination

“…Epa gene expression is complex and is regulated by subtelomeric silencing based on chromatin and transcriptional factors. Subtelomeric silencing is composed of the SIR complex ( Sir2, Sir3, and Sir4 ), which requires NAD+ as a cofactor, Rap1 that recruits the SIR complex, as well as Rif1 , yKu70, and yKu80, which may respond to various environmental factors [ 85 , 154 , 156 , 158 , 166 ].…”

Telomeric and Sub-Telomeric Structure and Implications in Fungal Opportunistic Pathogens

“…Through these interactions and the recruitment of the various proteins responsible for sub-telomeric regions silencing, it is believed that expression of EPA1 , EPA2 , and EPA3 is kept under strict control by the cell. It is possible that cis -elements are present, particularly at other telomeres, since the mechanisms seem to be different on different chromosomes [ 74 ]. The phenotype for the rif1 Δ, which showed a decrease of liver and spleen colonization, seems to be more complex.…”

Abf1 Is an Essential Protein That Participates in Cell Cycle Progression and Subtelomeric Silencing in Candida glabrata

“…The fragment obtained was cloned into the integrative vector pYC44, generating plasmid pCI30. A 714 bp fragment containing the 3 UTR region of ABF1 was cloned into pCI30 flanking the nourseothricin cassette (pCI32) [23]. Plasmid pCI32 was digested with BsgI within the region of C. glabrata homology and transformed into the parental BG14 selecting for Nat R .…”

“…We amplified the full-length ABF1 gene with primers #2353 and #2354 (Supplementary Table S3), both of which contain ClaI restriction sites, to clone it into the plasmid pGH3 [23], which contains the P MT1 promoter fusion with the c-Myc tag and the GA linker to generate translational fusions at the N-terminal end of the protein of interest. The final plasmid (pGH8: pP MT-1 ::c-Myc-ABF1-T HIS3 .Nat R ) [23] was transformed into E. coli, and after verifying that the gene fragment was cloned in the correct orientation, it was transformed into C. glabrata (see yeast transformation section). All final plasmids were sequenced to confirm that constructs were correct and had no mutations.…”

“…The EPA1 gene encodes the major adhesin (Epa1) and is the only member of the family expressed in vitro in the standard laboratory strain BG14 [16,17]. Expression of EPA1 is negatively controlled by several layers of negative regulation, including subtelomeric silencing that spreads from the telomere through up to 20 Kb [18][19][20], promoter-specific repression by a cis-acting negative element (NE) downstream from EPA1 [21], and repression by the formation of a chromatin loop between a subtelomeric, cis-acting protosilencer (Sil2126) and sequences flanking the EPA1 ORF [22,23]. We have shown that the C. glabrata ortholog of ScABF1 (CAGL0J01177g) is required for the formation of the chromatin loop that represses EPA1 along with other silencing proteins that participate in subtelomeric silencing such as CgSir2, CgSir3, and CgSir4 (forming the SIR complex), and CgRap1 [23].…”