Functional characterization of the OFD1 protein reveals a nuclear localization and physical interaction with subunits of a chromatin remodeling complex

Abstract

Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA(+)-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment.

Figure 1.

Subcellular localization of the endogenous OFD1 protein. Localization at primary cilium was investigated by confocal microscopy in MDCK cells. (A) Staining with the anti-acetylated α-tubulin. (B) Staining with the anti-OFD1 C-ter antibody. OFD1 localizes at the basal body of the primary cilium as the merge in C shows. In Cos-7 cells studied by immunofluorescence microscopy, the anti-OFD1 detects also a diffuse signal in the nucleoplasm (E), which disappears when using the preimmune (G). Nuclei are stained with DAPI (D). (H) Nuclear and cytoplasmic fractions from whole Cos-7 cell extracts. Immunoblotting with anti-OFD1 C-ter antibody shows that the endogenous OFD1 is present in both cellular compartments. The same amount of protein from the two compartments and from whole cell was loaded. To verify the purity of the extracts, the samples were immunoblotted with anti-β-tubulin and anti-acetyl-Hystone H4.

Figure 2.

OFD1 homotypic interactions. (A) Co-IP experiments. The lysates from Cos-7 cells cotransfected with vectors expressing Flag-OFD1 (120 kDa) and MycGFP-OFD1 (175 kDa) were immunoprecipitated with an anti-Flag antibody (left panel, lane 1) and as control, the lysates from cells transfected only with MycGFP-OFD1 were used (lane 2). The total cell lysates used as input are shown (TCL, lanes 1 and 2). The same experiment was repeated in the opposite direction, using an anti-Myc antibody for IPs and, as a control, the lysates from cells transfected with Flag-OFD1 (right panel, lanes 1 and 2, respectively). Also in this case total cell lysates used as input are shown (TCL, lanes 1 and 2). (B) OFD1 fragments used for interaction mating and GST pulldown experiments are represented with the corresponding domains LisH (dark gray) and CC (light gray). (C) GST pulldown assay in which the lysates from Cos-7 cells transfected with MycGFP-OFD1 was used as input and tested with the OFD1 fragments expressed as GST fused proteins (GST-OFD1a–d) and, as a control, with GST alone (GST). In the immunoblot, the MycGFP-tagged OFD1 corresponds to a band of about 175 kDa. IP, immunoprecipitated samples; IB, immunoblot. TCL+ and TCL− indicate the total cell lysates from cotransfected and not transfected Cos-7 cells, respectively.

Figure 3.

Study of the 1757delG OFD1 mutant form. (A) The position of the 1757delG frameshift mutation is depicted with respect to the LisH (dark gray) and the CC domains (light gray). (B) GST pulldown experiments. The lysates from Cos-7 cells overexpressing the MycGFP-tagged 1757delG mutant was used as input and tested with the OFD1b fragment expressed as GST fused protein and with GST alone. As control, lysates from cells overexpressing MycGFP-tagged OFD1 wild type were used. The band of about 175 kDa corresponds to the MycGFP-tagged OFD1 wild type, whereas the MycGFP-tagged 1757delG form (asterisk) is detectable as a band of about 100 kDa.

Figure 4.

OFD1 interaction with RuvBl1. (A) Left panels, the lysates from Cos-7 cells transfected with a vector expressing HA-RuvBl1 (top) or, as control, with the empty vector (bottom) were immunoprecipitated with a monoclonal anti-HA antibody, and the samples were immunoblotted with the polyclonal anti-OFD1. The experiment was repeated in the opposite direction (right panels) by using the anti-OFD1 C-ter antibody to immunoprecipitate extracts from cells transfected with a vector expressing MycGFP-RuvBl1 (top) or with the empty vector (bottom). In B the lysates from MDCK cells were immunoprecipitated with the anti-OFD1 C-ter, the OFD1-associated proteins were eluted after competition with the antigenic peptide, and the samples were immunoblotted with the same anti-OFD1 antibody (left panel). The samples were then analyzed with anti-RuvBl1 (right panel). As a control, the OFD1 antibody was incubated with the antigenic peptide prior IP (Ip control, both panels). In C, GST pulldown experiments. Overexpressed MycGFP-OFD1 was used as input and tested with GST-RuvBl1 and GST alone. In (D) the different OFD1 fragments were expressed as GST fused proteins, and the lysates from Cos-7 cells overexpressing MycGFP-RuvBl1 were used as input. IB, immunoblot; IP, immunoprecipitated samples; TCL, total cell lysate.

Figure 5.

OFD1 coimmunoprecipitates with subunits of the human TIP60 complex. (A) Lysates from MDCK cells were immunoprecipitated with the anti-OFD1 Cter antibody, eluted with OFD1 peptide, and analyzed by Western blot with antibodies recognizing four subunits of the TIP60 complex. OFD1 coimmunoprecipitates with TRRAP, TIP60, DMAP, and RuvBl2. (B) Two examples of the control experiments in which the same lysates were immunoprecipitated with the anti-OFD1 antibody preincubated with the peptide (Ip control). (C) Immunoprecipitation with an anti-TIP60 antibody recovered the endogenous OFD1 protein. IP, immunoprecipitated samples; TCL, total cell lysate.