Formation of functional centromeric chromatin is specified epigenetically in Candida albicans

Abstract

In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragments derived in vivo by homologous recombination of a native chromosome with linear DNA fragments containing a telomere and a selectable marker. An in vivo derived 85-kb truncated chromosome containing the 3-kb CEN7 locus on 69 kb of chromosome 7 DNA was stably and autonomously maintained in mitosis, indicating that preexisting active CEN chromatin remains functional through many generations. This same 85-kb chromosome fragment, isolated as naked DNA (devoid of chromatin proteins) from C. albicans and reintroduced back into C. albicans cells by standard DNA transformation techniques, was unable to reform functional CEN chromatin and was mitotically unstable. Comparison of active and inactive CEN chromatin digested with micrococcal nuclease revealed that periodic nucleosome arrays are disrupted at active centromeres. Chromatin immunoprecipitation with antibodies against CaCse4p confirmed that CEN7 introduced into C. albicans cells as naked DNA did not recruit CaCse4p or induce its spread to a duplicate region only 7 kb away from active CEN7 chromatin. These results indicate that CaCse4p recruitment and centromere activation are epigenetically specified and maintained in C. albicans.

Fig. 1.

Targeted in vivo truncation of chromosomes 6 and 7. (A) Strategy to evaluate the contribution of CEN DNA, CEN-proximal DNA, and epigenetic DNA modifications to de novo kinetochore assembly in C. albicans. The resulting phenotypes (boxed), CF CEN DNA (marked with black dot) and possible methylated bases (Me) are shown. (B) Derivation of CF6-95 and CF7-85. The regions are drawn to scale and numbered in kilobases. CEN DNA (filled oval), targeting vector sequences (thick lines), and ORFs (boxes) are indicated for chromosomes (Chr) 6 and 7. Pertinent ORFs are labeled with their assembly 19 designations or their putative homologous genes. Open and shaded boxes are transcribed from top to bottom and bottom to top, respectively. Chromosome maps are based on the following resources: Chibana et al. (22), http://candida.bri.nrc.ca, and www.candidagenome.org. (C and D) Electrophoretic karyotypes of C. albicans strains containing CF6-95 or CF7-85. Undigested chromosomal DNA prepared in agarose plugs was separated by clamped homogeneous electrical field electrophoresis to resolve CFs from native chromosomes. A reverse image of each ethidium-stained gel is compared with a Southern blot hybridized with a ³²P-labeled probe as indicated. Lane M, S. cerevisiae chromosome size markers; WT, strain BWP17; in vivo, CF strain derived by in vivo truncation; de novo, strain CF7-85 de novo, containing the CF introduced as naked DNA prepared from the in vivo truncation strain.

Fig. 2.

CaCse4p is present only at native CEN loci. Formaldehyde cross-linked chromatin from C. albicans strain CAKS5, carrying an extra 3-kb CEN7 sequence (CEN7*) integrated 6.7 kb away from its native location, was fragmented and immunoprecipitated with antibodies against CaCse4p. Enrichment of CaCse4p bound to endogenous CEN7 and exogenous CEN7* was assayed by PCR using primers corresponding to the numbered black arrowheads (Table 3). Cross-hatched box, native CEN7 region; hatched box, CEN7* region introduced by recombination as naked DNA; open box, target sequence for integration; targeting site Pa, PacI; SM, starting material; +Ab, ChIP with anti-CaCse4p; −Ab, mock ChIP. Primers 1′ and 6″ anneal to vector sequences adjacent to CEN7*. Primers 1–6 anneal to both CEN7 and CEN7*. Primers 13 and 14 anneal within the non-ORF region between CEN7 and Orf19.6522.

Fig. 3.

Micrococcal nuclease treatment reveals an unusual chromatin structure at the C. albicans CEN. Chromatin from strain CAI4 was digested with MNase for 2, 5, 10, 20, and 40 min. Purified DNA was separated by gel electrophoresis, Southern transferred to membranes, and hybridized to labeled probes (see below). Bulk nucleosomes are shown in a reverse image of one ethidium bromide-stained gel for comparison to the nucleosome profiles detected by hybridization with ³²P-labeled probes 7A–7G. Probes are aligned with a map of the CEN7 region drawn to scale. The boundary map positions of CEN7 are shown in base pairs. Arrowheads, PCR primers used to amplify probes (7A, 1,434 bp; 7B, 1,141 bp; 7C, 1,346 bp; 7D, 1,130 bp;7E, 882 bp; 7F, 1,216 bp; 7G, 872 bp).

Fig. 4.

Chromatin structure of CEN7 on CEN-active and CEN-inactive CFs. (A) CF7-85 schematic. Black box, CEN7 probe 7C (see Fig. 3). (B–D) Chromatin from the indicated strains (WT, strain BWP17) were digested with MNase for the length of time (min) shown above each lane. Purified DNA was separated by electrophoresis and a reverse image of each ethidium bromide (eth)-stained gel is shown next to its nucleosome profile detected with a ³²P-labeled 1.4-kb CEN7 probe.