The C-terminal domain of CENP-C displays multiple and critical functions for mammalian centromere formation

Abstract

CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III). The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP) we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III) displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin.

Figure 1. Sequence similarity among Mif2p homology domains of CENP-C orthologs.

Amino acid sequences of human CENP-C Mif2p homology domains II and III, contained in the HA::638/819 and HA::760/943 constructs respectively, are compared to those of orthologous CENP-C proteins. Mif2p homology domains II and III are indicated in light grey and dark grey, respectively. Percent conservation is represented as follows: 100% conservation (red), 90% conservation (blue), 80% conservation (cyan); 70% conservation or less (white). Multiple sequence alignments of the CENP-C protein families were built with ClustalW, Multialin version 5.4.1 and T-Coffee and edited by hand –. Accession Numbers: Human CENP-C (GenBank M95724); Chimpanzee CENP-C (GenBank XM517266-7); Dog CENP-C (GenBank XM532388); Cow CENP-C (GenBank XM598358); Mouse CENP-C (GenBank U03113); Rat CENP-C (GenBank AAU04621.1); Chicken CENP-C (GenBank BAA24110.1); Sheep CENP-C (GenBank AAA79099.1); A. thaliana CENP-C (GenBank AAU04629.1); S. cerevisiae Mif2p (GenBank NP012834.1).

Figure 2. Both Mif2p homology domains II and III target centromeres.

(A) The indicated chimeric HA::CENP-C proteins were expressed in human HEK-293T cells and revealed by an anti-HA monoclonal antibody (red signal), while endogenous CENP-B was detected with an anti-CENP-B polyclonal antibody (green signal). Co-localization of the HA::CENP-C proteins and CENP-B is shown in yellow in the merged image. In the diagram, bars describe the different CENP-C truncated proteins as compared to the wt protein; Mif2p homology domain II (light grey), Mif2p homology domain III (dark grey), central DNA binding domain (dotted box), HA-tag (black box). Localization of proteins within the nuclei has been determined by epifluorescent microscopy. (B) Centromere localization of HA::CENP-C fusion proteins in interphase nuclei. For each mutant at lest 100 transfected cells were scored for colocalization of HA and CENP-B signals.

Figure 3. The Mif2p homology domain II of CENP-C binds the alpha satellite DNA in vivo.

HA::CENP-C constructs were independently transfected into HEK-293T cells and their binding to alpha satellite DNA was analyzed by ChIP assay. Chromatin was immunoprecipitated by using anti-HA (IP anti-HA) or anti-CENP-C antibodies (IP anti-CENP-C). Immunoprecipitated DNAs along with total DNA were hybridized with alpha-satellite or Alu DNA probes. The relative enrichment of alpha-satellite DNA (left panel) was normalized to that of Alu sequences (right panel) and compared to that obtained from cells transfected with the empty vector, pcDNA3.1HA (IP background). Values are the mean±s.e.m. of at least three experiments.***P<0,01; **P<0,1.

Figure 4. The Mif2p homology domain III of CENP-C possesses a self-associating activity in vivo.

Cells HEK-293T were transiently transfected with the entire C-terminal region of CENP-C (HA::638/943) and FLAG::CENP-C constructs as indicated in the diagrams (A, FLAG::23/410; B, FLAG::638/943; C, FLAG::638/819 and D, FLAG::760/943). After 24 hours of expression, nuclear extracts were performed and proteins were immunoprecipitated through an anti-HA affinity matrix. Interactions between CENP-C domains are revealed by the presence of FLAG-tagged proteins in the IP anti-HA sample. The diagram in each panel illustrates the HA and FLAG CENP-C derivatives as compared to the full length protein: Mif2p homology domain II (light grey), Mif2p homology domain III (dark grey), central DNA binding domain (dotted box), HA-tag (black box), FLAG-tag (white box).

Figure 5. C-terminal domain of CENP-C can form dimers/oligomers in vitro.

HA CENP-C truncated proteins were expressed in HEK-293T cells and immunopurified with anti-HA affinity matrix. Purified proteins were recovered by elution with an HA peptide and crosslinked (C) or not (NC) with glutaraldehyde to stabilize possible protein aggregates. Formation of possible oligomers was determined by western blot probed with an anti-HA antibody. Dimers are indicated by arrows whereas trimers and tetramers are indicated by asterisks.

Figure 6. CENP-C can interact with CENP-A and histone H3 through its C-terminal domain.

(A) HA::CENP-A coimmunoprecipitates the endogenous CENP-C. HEK-293 cells were transfected with HA::CENP-A or with the HA empty vector (pcDNA3.1HA) and HA proteins were purified from nuclear extracts with anti-HA affinity matrix. The presence of the endogenous CENP-C in the immunoprecipitated samples was determined by western blot with an anti-CENP-C antibody (B) Overexpressed HA::CENP-A coimmunoprecipitates FLAG C-terminal CENP-C domain. (C) Coimmunoprecipitation of overexpressed HA::CENP-C domains along with FLAG-tagged histone proteins and c-Myc protein. Only the construct that contains the C-terminal CENP-C region is able to interact with both CENP-A and histone H3.

Figure 7. The Mif2p homology domain III mediates the interaction of CENP-C with CENP-A and histone H3.

Coimmunoprecipitation of overexpressed HA::CENP-C C-terminal domains along with FLAG-tagged CENP-A and histone H3. Constructs containing the Mif2 homology domain III shows the ability to interact with both CENP-A and histone H3.

Figure 8. Interaction between CENP-C and CENP-A takes place at centromere positions.

HEK293 cells transfected with plasmids expressing the chimeric protein combinations indicated at the top of each column A–E (A: N-GFP::HA::CENPC and HA::CENPA::C-GFP; B: N-GFP::HA::CENPC; C: HA::CENPA::C-GFP; D: N-GFP::HA::CENPCΔ890-943 and HA::CENPA::C-GFP; E: N-GFP::HA::CENPCΔ890-943). After fixation cells were immunolabelled with a monoclonal anti-HA antibody and polyclonal antibodies used as centromere markers (A and D: anti-CENP-B; B and E anti-CENP-A; C: anti-CENP-C). Row I shows complementing GFP (green); row II shows merge between complementing GFP and the centromeric marker (red); row III shows HA-tagged chimeric proteins (cyan); row IV shows merge between HA-tagged chimeric proteins and the centromeric marker; row V shows merge between centromeric marker and DNA (blue). F: diagrams of the chimeric proteins expressed in cells. G: quantification of colocalization between the green signal due to complementing GFP and the centromeric marker in cells that coexpress HA::CENPA::C-GFP and N-GFP::HA::CENPC full length or the truncated mutant N-GFP::HA::CENPCΔ890-943. 10 to 15 nuclei for each experiment were analyzed and the data expressed as ratio between GFP signal corresponding to centromeres and number of visible centromeres. Statistical significance have been checked with T-student test and indicated in the graph. The bright green punctate signals in panel AI indicate efficient GFP complementation in cells coexpressing N-GFP::HA::CENPC and HA::CENPA::C-GFP. The green signals in AI well correspond to centromere positions as indicated by merge in panel AII and to HA-tagged proteins shown in panels AIII-IV. The green signal shown by cells expressing respectively only N-GFP::HA::CENPC or HA::CENPA::C-GFP is very low (BI) or absent (CI) although the chimeric HA-tagged proteins are expressed. In cells expressing N-GFP::A::CENPCΔ890-943 and HA::CENPA::C-GFP few green signals are visible corresponding to centromere position (green and red in DI–II) suggesting lower interaction when CENP-C Mif2 homology domain III is deleted although N-GFP::HA::CENPCΔ890-943 localizes at centromeres (EIII–IV).