Identification and characterization of CENP-Aᶜⁿᵖ¹-associated proteins in Schizosaccharomyces pombe
Item statusRestricted Access
Embargo end date06/07/2020
Singh, Puneet Prabhakar
Centromeres are chromosomal regions on each chromosome crucial for the assembly of the kinetochore which facilitates the binding of the microtubules and subsequently leads to accurate chromosome segregation during mitosis and meiosis. Centromeres are epigenetically defined by exclusive localization of the centromere-specific histone H3-variant CENP-A. Although centromere sequence is neither necessary nor sufficient for centromere function, centromeres are generally associated with particular sequences. The mechanism by which CENP-A is deposited and inherited exclusively at centromeric chromatin is not fully understood. Schizosaccharomyces pombe provides an excellent model system to dissect the structure and function of a complex eukaryotic centromere due to its epigenetically regulated centromeres that are structurally related to those of metazoa. The main aim of this thesis was to study the proteome of CENP-ACnp1 chromatin and gain insights into potential role of CENP-ACnp1-associated proteins in CENP-ACnp1 chromatin establishment and maintenance. Affinity purification and mass spectrometry analysis of GFP-CENP-ACnp1 was used to identify centromeric chromatin-enriched proteins based on their relative abundance in GFP-CENP-ACnp1 compared to mock affinity-purifications. The analysis yielded 199 proteins enriched in centromere chromatin preparations and 23 non-essential proteins were selected for further investigation. Here, I investigate the involvement of Hap2 and Dbl5 in CENP-ACnp1 chromatin establishment and maintenance. Hap2, an Ino80 complex subunit, is enriched over the CENP-A chromatin and is required to maintain normal nucleosome occupancy at centromeres. Cells lacking the hap2 gene fail to establish CENP-ACnp1 chromatin on a naïve centromeric sequence and show attenuated transcriptional silencing at centromeres. Surprisingly, loss of different Ino80 complex subunits showed an opposing effect on centromere establishment frequency suggesting that some Ino80 subunits could have different effects on de novo centromere establishment. CENP-ACnp1 is deposited at centromeres in late S/early G2 phase of the cell cycle in a replication-independent manner (Lando et al., 2012; Shukla et al., 2017). Eviction of histone H3 is postulated to provide an opportunity for CENP-ACnp1 nucleosome deposition (Allshire and Karpen, 2008; Shukla et al., 2017). Hap2 is required to promote replication-independent histone H3 turnover at ectopically inserted centromere 2 central domain DNA, endogenous centromeres and other genomic regions; this could explain the loss of centromere establishment and maintenance observed in hap2Δ. In budding yeast, the Psh1 E3 ubiquitin ligase regulates CENP-ACse4 levels. Dbl5 is a Psh1 homolog and thus may also regulate CENP-ACse4 levels via ubiquitin-mediated proteolysis. Deletion of the dbl5 gene partially suppressed a cnp1-1 mutant and completely suppressed a mis6-306 mutant temperature sensitivity suggesting a genetic interaction with this kinetochore component. Overexpression of CENP-ACnp1 causes segregation defects and toxicity in dbl5Δ with spreading of CENP-ACnp1 into pericentric outer repeat regions. Similar to budding yeast Psh1, deletion of the dbl5 gene stabilized CENP-ACnp1 protein levels in vivo suggesting a role in CENP-ACnp1 degradation. In budding yeast, CENP-ACse4 was shown to be ubiquitylated, in a Psh1- dependent manner, and targeted for degradation (Hewawasam et al., 2010; Ranjitkar et al., 2010). Human CENP-A is ubiquitylated on lysine 124 by the CUL4A-RBX1- COPS8 complex which has been proposed to be required for the deposition of CENPA at centromeres (Niikura et al., 2015). In fission yeast CENP-ACnp1 is ubiquitylated in Dbl5-dependent manner. Mass Spectrometric analysis of affinity selected GFP-CENPACnp1 reproducibly detected ubiquitylation of CENP-ACnp1 lysine 4 (K4), however its biological function remains to be determined. Studying non-kinetochore CENP-A chromatin associated proteins is important for understanding the regulatory mechanisms involved in controlling the dynamics of nucleosome assembly and disassembly at centromeres. It remains to be determined whether, as shown here in S. pombe, different Ino80 subunits have opposing roles at metazoan centromeres. Further analysis are required to determine the role of Dbl5 and CENP-ACnp1 K4 ubiquitylation in ensuring normal patterns of CENP-ACnp1 deposition.