p97/VCP drives turnover of SUMOylated centromeric CCAN proteins and CENP-A.

The centromere is a unique chromatin domain that links sister chromatids and forms the attachment site for spindle microtubules in mitosis. Centromere inheritance is largely DNA sequence-independent but strongly reliant on a self-propagating chromatin domain featuring nucleosomes containing the H3 variant CENP-A. Unlike other histones, CENP-A is maintained with unusually high stability in chromatin.

Stable inheritance of CENP-A chromatin: Inner strength versus dynamic control.

Chromosome segregation during cell division is driven by mitotic spindle attachment to the centromere region on each chromosome. Centromeres form a protein scaffold defined by chromatin featuring CENP-A, a conserved histone H3 variant, in a manner largely independent of local DNA cis elements. CENP-A nucleosomes fulfill two essential criteria to epigenetically identify the centromere.

Spt6 is a maintenance factor for centromeric CENP-A.

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.

Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics.

Novel small molecule binders of human N-glycanase 1, a key player in the endoplasmic reticulum associated degradation pathway.

Peptide:N-glycanase (NGLY1) is an enzyme responsible for cleaving oligosaccharide moieties from misfolded glycoproteins to enable their proper degradation. Deletion and truncation mutations in this gene are responsible for an inherited disorder of the endoplasmic reticulum-associated degradation pathway. However, the literature is unclear whether the disorder is a result of mutations leading to loss-of-function, loss of substrate specificity, loss of protein stability or a combination of these factors.

Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity.

Chromatin Immunoprecipitation (ChIP) Assay in Candida albicans.

Chromatin immunoprecipitation (ChIP) is a widely used technique which can determine the in vivo association of a specific protein on a particular DNA locus in the genome. In this method cross-linked chromatin is sheared and immunoprecipitated with antibodies raised against a target protein of interest. The end result of this process is the enrichment of DNA fragments associated with the desired protein.

Rad51-Rad52 mediated maintenance of centromeric chromatin in Candida albicans.

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity.