Neocentromeres Form Efficiently at Multiple Possible Loci in Candida albicans

Abstract

Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-ACse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.

Euchromatic Subdomains in Rice Centromeres Are Associated with Genes and Transcription

“…Human neocentromeres and evolutionarily new centromeres are often associated with gene deserts (Lomiento et al, 2008;Alonso et al, 2010). Similarly, induced neocentromeres in Candida albicans were preferentially associated with gene-poor regions (Ketel et al, 2009). …”

“…Neocentromere formation in S. pombe can negatively impact the transcription of the underlying endogenous genes (Ishii et al, 2008). Similarly, neocentromere formation can cause silencing of the underlying transgene in C. albicans (Ketel et al, 2009). Transcripts derived from centromeric repetitive DNA were reported in a number of plant and animal species (Topp et al, 2004;May et al, 2005;Wong et al, 2007;Carone et al, 2009).…”

Maize centromeres expand and adopt a uniform size in the genetic background of oat

“…Insertion of a marker gene in the centromeres of Schizosaccharomyces pombe chromosomes results in complete silencing of the gene (Allshire et al 1995). Similarly, neocentromeres in multiple species generally form in gene-poor regions (Lomiento et al 2008;Alonso et al 2010;Shang et al 2013); when they do form over genic areas, the affected genes are suppressed or silenced (Ishii et al 2008;Ketel et al 2009;Shang et al 2013). Why does centromeric chromatin avoid actively transcribed genes?…”

“…One way to study centromere repositioning is to focus on newly established centromeres known as neocentromeres. There are many known neocentromere examples in human clinical samples (Voullaire et al 1993;Marshall et al 2008) as well as in different animal and plant species (Williams et al 1998;Maggert and Karpen 2001;Nasuda et al 2005;Ishii et al 2008;Ketel et al 2009;Topp et al 2009;Fu et al 2013). Most newly formed neocentromeres lie in moderately repetitive genomic regions interspersed with single-copy sequences (Marshall et al 2008), whereas nearly all mature centromeres contain long arrays of satellite repeats (Henikoff et al 2001;Jiang et al 2003).…”

“…Some human neocentromeres and several plant centromeres contain genes embedded as islands within centromeres (Saffery et al 2003;Nagaki et al 2004;Gong et al 2012). While genes may closely border CENH3-containing nucleosomes, gene transcription is generally incompatible with CENH3 (Ketel et al 2009). Centromeres in higher eukaryotes usually span hundreds of kilobases of sequence and often do not appear to have sharp edges, at least as determined by chromatin immunoprecipitation (ChIP) of CENH3 from complex plant tissues (Yan et al 2008;Gong et al 2012).…”