A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway inDrosophila melanogaster

Abstract

The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.

Posttranslational mechanisms controlling centromere function and assembly

“…In this respect, the modification of the preassembled CENP-A/H4 dimer is similar to histone H3/H4, which is also acetylated on the same residues of histone H4 [26]. Depletion of RbAp46/48/Mis16 in several organisms, or the mutation of histone H4 Lys5 or Lys12 residues to non-acetylated amino acids, reduces new CENP-A deposition [36 •• ,38,39]; implying that the modification of the H4 subunit of the pre-nucleosomal heterodimer is essential for CENP-A deposition.…”

“…CENP-A is also phos-phorylated on Ser68, within the histone fold domain in a cell cycle specific manner, and is discussed below. Drosophila dCENP-A CID is phosphorylated at Ser75/77, a site that may be analogous to Ser16/18 in human CENP-A and was enriched in the nuclear fraction [39]. In contrast, dCENP-A CID is acetylated on Lys105 selectively in the prenucleosomal complex.…”

Posttranslational modifications of CENP-A: marks of distinction

“…The Drosophila counterpart of human CENP-A (a.k.a. dCENP-A CID ) has been shown to be acetylated at Lys-105, located within the N–terminus, exclusively in cytosolic prenucleosomal fraction (Boltengagen et al 2016). In addition, dCENP-A CID also undergoes phosphorylations at Ser-20, Ser-75, and Ser-77 (Boltengagen et al 2016).…”

“…dCENP-A CID ) has been shown to be acetylated at Lys-105, located within the N–terminus, exclusively in cytosolic prenucleosomal fraction (Boltengagen et al 2016). In addition, dCENP-A CID also undergoes phosphorylations at Ser-20, Ser-75, and Ser-77 (Boltengagen et al 2016). The phosphorylation of dCENP-A CID Ser-75/77 is suggestive of human CENP-A Ser-16/18 phosphorylation; however, the functional consequences of these phosphorylation events appear to be different in Drosophila and human.…”

“…The phosphorylation of dCENP-A CID Ser-75/77 is suggestive of human CENP-A Ser-16/18 phosphorylation; however, the functional consequences of these phosphorylation events appear to be different in Drosophila and human. While prenucleosomal cytosolic dCENP-A CID exhibits unphosphorylated, mono-or di-phosphorylated (Ser-20 and Ser-75) forms, the nucleoplasmic dCENP-A CID is enriched in an additional phosphorylation mark at Ser-77 (Boltengagen et al 2016). Both acetylation and phosphorylation of Drosophila dCENP-A CID occur on the N–terminus, and based on their differential patterns in prenucleosomal and nucleosomal fractions, have been suggested to determine the localization of dCENP-A CID (Boltengagen et al 2016).…”