Centromeric histone CENP-A, a variant of canonical histone H3, plays a central role in proper chromosome segregation. Loading of CENP-A at centromeres is cell cycle-regulated: parental CENP-A is deposited at centromeres during S phase, whereas newly synthesized CENP-A is deposited during later stages of the cell cycle. The mechanisms involved in deposition of CENP-A at centromeres during S phase remain poorly understood. In fission yeast, loading of CENP-A during S phase is regulated by the GATA-type factor, Ams2. Here we show that the Dos1/2-Cdc20 complex, previously characterized as a silencing complex essential for inheritance of H3K9 methylation during S phase, is also required for localization of CENP-A cnp1 at centromeres at this stage. Disruption of Dos1 (also known as Raf1/Clr8/Cmc1), Dos2 (also known as Raf2/Clr7/Cmc2), or Cdc20, a DNA polymerase epsilon subunit, results in dissociation of CENP-A from centromeres and mislocalization of the protein to noncentromeric sites. All three mutants display spindle disorganization and mitotic defects. Inactivation of Dos1 or Cdc20 also results in accumulation of noncoding RNA transcripts from centromeric cores, a feature common to mutants affecting kinetochore integrity. We further find that Dos1 physically associates with Ams2 and is required for the association of Ams2 with centromeric cores during S phase. Finally, we show that Dos2 associates with centromeric cores during S phase and that its recruitment to centromeric cores depends on Cdc20. This study identifies a physical link between DNA replication and CENP-A assembly machinery and provides mechanistic insight into how CENP-A is faithfully inherited during S phase.
Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.
Centromere is a specialized chromatin domain that plays a vital role in chromosome segregation. In most eukaryotes, centromere is surrounded by the epigenetically distinct heterochromatin domain. Heterochromatin has been shown to contribute to centromere function, but the precise role of heterochromatin in centromere specification remains elusive. Centromeres in most eukaryotes, including fission yeast (Schizosaccharomyces pombe), are defined epigenetically by the histone H3 (H3) variant CENP-A. In contrast, the budding yeast Saccharomyces cerevisiae has genetically-defined point centromeres. The transition between regional centromeres and point centromeres is considered as one of the most dramatic evolutionary events in centromere evolution. Here we demonstrated that Cse4, the budding yeast CENP-A homolog, can localize to centromeres in fission yeast and partially substitute fission yeast CENP-ACnp1. But overexpression of Cse4 results in its localization to heterochromatic regions. Cse4 is subject to efficient ubiquitin-dependent degradation in S. pombe, and its N-terminal domain dictates its centromere distribution via ubiquitination. Notably, without heterochromatin and RNA interference (RNAi), Cse4 fails to associate with centromeres. We showed that RNAi-dependent heterochromatin mediates centromeric localization of Cse4 by protecting Cse4 from ubiquitin-dependent degradation. Heterochromatin also contributes to the association of native CENP-ACnp1 with centromeres via the same mechanism. These findings suggest that protection of CENP-A from degradation by heterochromatin is a general mechanism used for centromere assembly, and also provide novel insights into centromere evolution.
Abstract The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-ACnp1 in fission yeast results in the assembly of CENP-ACnp1 at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-ACnp1 exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-ACnp1 overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-Acnp1 is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-Acnp1 results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitin-dependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A.
Many of the genes and enzymes critical for assembly and biogenesis of yeast cell walls remain unidentified or poorly characterized. Therefore, we designed a high throughput genomic screen for defects in anchoring of GPI-cell wall proteins (GPI-CWPs), based on quantification of a secreted GFP-Sag1p fusion protein. Saccharomyces cerevisiae diploid deletion strains were transformed with a plasmid expressing the fusion protein under a GPD promoter, then GFP fluorescence was determined in culture supernatants after mid-exponential growth. Variability in the amount of fluorescent marker secreted into the medium was reduced by growth at 18 degrees C in buffered defined medium in the presence of sorbitol. Secondary screens included immunoblotting for GFP, fluorescence emission spectra, cell surface fluorescence, and cell integrity. Of 167 mutants deleted for genes affecting cell wall biogenesis or structure, eight showed consistent hyper-secretion of GFP relative to parental strain BY4743: tdh3 (glyceraldehyde-3-phosphate dehydrogenase), gda1 (guanosine diphosphatase), gpi13 and mcd4 (both ethanolamine phosphate-GPI-transferases), kre5 and kre1 (involved in synthesis of beta1,6 glucan), dcw1(implicated in GPI-CWP cross-linking to cell wall glucan), and cwp1 (a major cell wall protein). In addition, deletion of a number of genes caused decreased secretion of GFP. These results elucidate specific roles for specific genes in cell wall biogenesis, including differentiating among paralogous genes.
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)