Identification of parafibromin-associated proteins.
To explore the protein complexes associated with parafibromin, we generated and validated an antipeptide rabbit polyclonal antibody, Ab648, that specifically recognizes both transfected (Fig. , lanes 2 and 4) and endogenous (Fig. , lanes 1 and 7) parafibromin protein. To identify endogenous parafibromin-associated proteins, we immunoprecipitated protein from 293T cells with the Ab648 antiparafibromin antibody; as a control, we preincubated the antibody with a blocking peptide control (Fig. ). Immunoprecipitated protein bands were excised, and peptides were sequenced by tandem mass spectrometry.
FIG. 1. Identification of parafibromin (Parafib) interacting proteins. (A) Characterization of antiparafibromin antibody Ab648 in transfected and untransfected cells. An immunoblot with Ab648 is shown. Whole-cell lysate (150 μg) of untransfected 293T (more ...)
The 64-kDa band (Fig. , band 1) was identified as parafibromin. Mass spectrometric analysis identified 42 peptides comprising 62.9% of the predicted parafibromin protein sequence. The proteins coimmunoprecipitated with antiparafibromin antibody include human homologs of the yeast Paf1 complex: an approximately 80-kDa band (Fig. , band 2) includes peptides from hPaf1, a 105-kDa band (Fig. , band 3) includes peptides from hLeo1, and an approximately 150-kDa band (Fig. , band 4) includes peptides from hCtr9, with predicted amino acid sequence similarities of 40, 52, and 44%, respectively. No peptides corresponding to the human homolog of Rtf1 were identified in the immunoprecipitates. To our knowledge, the human hPaf1 and hLeo1 proteins have not been described in the literature. hCtr9 has been described as an SH2 domain binding nuclear phosphoprotein that has TPR repeats and is thought to be involved in protein-protein interaction (16
). Identification of the remaining protein bands of the antiparafibromin immunoprecipitates is still in progress.
To verify interactions between parafibromin and the human homologs of the yeast Paf1 complex, we generated polyclonal antibodies against the parafibromin interacting proteins in the Paf1 complex (anti-hPaf1, anti-hLeo1, anti-hCtr9, and anti-hRtf1). These antibodies were validated for the ability to immunoprecipitate antigens and associated proteins from metabolically labeled cells (not shown). To reconfirm interactions among putative parafibromin complex members, we immunoprecipitated protein from 293T cells with anti-hLeo1 antibody Ab677, with peptide-blocked antibody as a negative control (Fig. ). Immunoprecipitated proteins were excised from the gel and sequenced by tandem mass spectrometry. As expected, the 105-kDa band (Fig. , band 3) corresponded to hLeo1, with five peptides covering 6.8% of the predicted sequence. Multiple peptides were identified from each of the other five proteins precipitated with the anti-hLeo1 antibody. Three of these proteins are human homologues of the yeast Paf1 complex that we had found in the parafibromin-associated complex. The 64-kDa protein (Fig. , band 1) is parafibromin, the ~80-kDa protein (Fig. , band 2) is hPaf1, and the 150-kDa protein (Fig. , band 4) is hCtr9 (11 peptides, 7.9% coverage). Analysis of two higher-molecular-weight bands (Fig. , bands 5 and 6) revealed peptides corresponding to Ranbp2, a nuclear pore complex protein (36
Parafibromin is associated with a human Paf1 complex.
To verify interaction among the hPaf1 complex proteins more thoroughly, reciprocal coimmunoprecipitation experiments were performed with antibodies to the endogenous proteins (Fig. ). Antiparafibromin antibody Ab648 could specifically precipitate parafibromin, hLeo1, hPaf1, and hCtr9 (Fig. , lane 4). These proteins were not detectable with peptide-blocked antibody, the negative control (Fig. , lane 3). Similarly, immunoprecipitation with anti-hLeo1 antibody but not the peptide-blocked control could isolate all four hPaf1 complex members (Fig. , lanes 5 and 6). The anti-hPaf1 antibodies, while effective for immunoblotting, were not as efficient for immunoprecipitation but could still precipitate hPaf1 and parafibromin (Fig. , lanes 7 and 8). Taken together, the above results indicate that parafibromin, hPaf1, hLeo1, and hCtr9 form a stable complex. The antiparafibromin, anti-hLeo1, and anti-hPaf1 antibodies did not immunoprecipitate a band that could be detected by anti-hRtf1 antibodies in immunoblot analysis. These interactions remained intact following treatment with DNase (not shown), indicating that the complex is likely to be assembled via protein-protein interactions rather than protein-DNA interactions.
FIG. 2. Parafibromin (Parafib) is part of a human Paf1 complex. Antiparafibromin antibody Ab648 (lane 4), anti-hLeo1 antibody Ab677 (lane 6), and anti-hPaf1 antibody Ab674 (lane 8) immunoprecipitates from 293T cell lysates were immunoblotted with antiparafibromin (more ...) Parafibromin interacts with the RNA polymerase II large subunit.
Yeast Paf1 complex members have been reported to associate with RNA polymerase II (14
). The repetitive CTD of the RNA polymerase II large subunit, Rpb1, has two important serine residues that become highly phosphorylated during transcription. Phosphorylation on Ser5 is correlated with initiation and early elongation, while phosphorylation on Ser2 is linked to established elongation (11
To test whether the parafibromin complex interacts with any of the phosphorylated forms of Rpb1, we immunoprecipitated parafibromin and blotted the immunoprecipitates with specific antibodies to detect the Rpb1 CTD that is unphosphorylated, phosphorylated on Ser5, or phosphorylated on Ser2. Parafibromin specifically coprecipitated all three forms of Rpb1 (Fig. ), suggesting that it may be involved in both initiation and elongation.
FIG. 3. Parafibromin (Parafib) interacts with the RNA polymerase large subunit. (A) 293T cell lysates were immunoprecipitated (IP) with antiparafibromin antibody Ab648, a peptide-blocked control (C), or a normal rabbit serum control and immunoblotted with antibodies (more ...)
To further explore the relationship between the hPaf1 complex and RNA polymerase II, 293T cell extract was fractionated with a glycerol gradient and fractions from the gradient were separated on a polyacrylamide gel and immunoblotted for the indicated proteins (Fig. ). The hPaf1 complex members mainly comigrated in fractions 11 through 13. The Rpb1 Ser5 CTD phosphorylated form overlapped with the parafibromin complex, but most of the RNA polymerase II appears to be in a higher-molecular-weight complex. In addition, we also tested the fractions for hRtf1. The hRtf1 reactivity is mainly found in fractions 5 to 9, not overlapping the parafibromin/Paf1 complex.
Parafibromin is a nuclear protein.
To determine the subcellular localization of parafibromin and the hPaf1 complex proteins, we performed immunofluorescence studies. As neither of the antiparafibromin antibodies could detect endogenous protein by immunofluorescence, we overexpressed WT parafibromin in HeLa cells with a Flag epitope tag on its N terminus. Confocal microscopic detection of immunofluorescence showed significant overlap between overexpressed Flag-tagged parafibromin (green fluorescence) and DAPI staining (Fig. , right side), indicating that parafibromin is a nuclear protein. Control cells that were not transfected did not exhibit any staining (Fig. , left side) In addition, immunofluorescence was performed with the anti-hLeo1 Ab677 antibody. Confocal microscopy shows that hLeo1 is a nuclear protein with a punctate distribution (Fig. ). Taken together, these results suggest that parafibromin and hPaf1 complex members localize to the nucleus.
FIG. 4. Parafibromin and hLeo1 are nuclear proteins. (A) Immunofluorescence assay with FITC-conjugated anti-Flag antibody on untransfected HeLa cells (Untransfected) or HeLa cells transfected with Flag-tagged WT parafibromin (Parafibromin). The left side of each (more ...) Some mutant forms of parafibromin lack association with Paf1 complex members.
Several germ line and somatic mutations have been identified in HRPT2
). Most of these are truncation and frameshift mutations that result in premature stop codons and are predicted to cause deficient or impaired parafibromin protein function.
We generated expression constructs for several truncation mutant forms of parafibromin—136X, 227X, and 413X—by PCR (Fig. ). 136X is a truncation found in the germ line of HPT-JT patients. We chose to make the 227X and 413X truncations because some patients have germ line nucleotide insertions or deletions at codons 226 and 413 that result in a frame shift and early termination. In addition, we made an L64P point mutation that was identified in germ line HPT-JT. This leucine residue is conserved in Drosophila, Caenorhabditis elegans, mice, and humans, suggesting that it has an important function. A Flag epitope tag was introduced into the 5′ end of each parafibromin-encoding truncation mutant construct. The parafibromin-encoding truncation constructs were transfected into 293T cells. Cell lysates from these transfections were fractioned by SDS-PAGE, and expression was assessed by immunoblotting with anti-Flag antibody (Fig. ). All of the constructs gave rise to detectable protein corresponding to the expected size.
FIG. 5. Characterization of parafibromin truncation mutant constructs. (A) Schematic diagram of parafibromin truncation mutant constructs and the L64P mutant construct representing germ line alterations identified from HPT-JT patients. (B) 293T cell lysates from (more ...)
To test the abilities of the mutant forms of parafibromin to associate with hPaf1 complex members, we cotransfected 293T cells with Flag-tagged WT parafibromin or parafibromin mutant constructs together with an expression plasmid encoding either hLeo1, T7-hPaf1, or hCtr9 protein and immunoprecipitated the parafibromin complex with anti-Flag antibody (Fig. ). A portion of this immunoprecipitation was blotted with anti-Flag antibody to verify expression and precipitation of the various parafibromin proteins. Although hPaf1, hLeo1, and hCtr9 were expressed in all of the cells (not shown), only WT parafibromin, L64P, and 413X could immunoprecipitate hPaf1 complex proteins while 227X and 136X (not shown) could not, indicating that a portion of parafibromin between amino acids 226 and 413 is required for binding to the hPaf1 complex.
Parafibromin can associate with an HMTase complex.
Because yeast Cdc73 has been reported to associate with a Set1 HMTase complex, we decided to test the association of both WT and mutant parafibromin with HMTase activity. To test the association of parafibromin and the mutant forms of parafibromin with HMTase activity, we transfected 293T cells with Flag-tagged WT parafibromin or parafibromin mutant constructs and performed an HMTase assay (Fig. ). WT parafibromin and the 413X mutant construct are associated with HMTase activity, while the 227X mutant construct is not. The L64P mutant form of parafibromin shows reduced activity compared to that of the WT and 413X forms (Fig. ); this result was obtained consistently in three experiments.
FIG. 6. Interaction between parafibromin and HMTase activity and complex members. (A) HMTase assays were performed on immunoprecipitates (IP) with anti-Flag antibodies from 293T cell lysates transfected with the indicated Flag-tagged WT parafibromin or Flag-tagged (more ...)
Given the HMTase activity associated with parafibromin, we tested whether WT parafibromin and the mutant forms of parafibromin can interact with components of Set1-like complexes, including Rbbp5 and Ash2L. We cotransfected 293T cells with Flag-tagged WT parafibromin or parafibromin mutant constructs together with an expression plasmid encoding either Rbbp5 or Ash2L protein and immunoprecipitated the parafibromin-containing complex with anti-Flag antibody (Fig. ). Similar to the association with HMTase activity, WT parafibromin and 413X could immunoprecipitate the Set1 complex proteins Rbbp5 and Ash2L while 227X could not. L64P exhibits reduced binding to these proteins, consistent with the HMTase activity of this mutant construct.
In addition, we determined the subcellular localization of the various mutant forms of parafibromin (data not shown). The L64P, 413X, and 227X mutant constructs exhibited predominantly nuclear staining, while the K136X mutant construct typically showed some even nuclear staining but also significant cytoplasmic and cell membrane localization. These results indicate that the putative classic nuclear localization signal, at amino acids 136 to 139 of parafibromin, may be functional. These results suggest that binding to hPaf1 complex components and to HMTase complex components, as well as nuclear localization, may be essential to parafibromin function and that elimination of complex assembly may contribute to tumor formation in the setting of HRPT2 mutation.
Recently we have generated a second antiparafibromin polyclonal antibody, Ab649. After checking the specificity of this antibody, we found that in addition to parafibromin it precipitates two other major bands, possibly corresponding to the angiomotin protein (Amot). Angiomotin has been reported to bind to angiostatin and to regulate endothelial cell migration and tube formation and has not been shown to be involved in chromatin remodeling (33
). Since the overexpressed parafibromin protein is associated with HMTase complex components and activity, we assayed parafibromin-containing complexes precipitated by Ab649 for HMTase activity. To that end, we immunoprecipitated protein from 293T cell lysates with antiparafibromin Ab649 or peptide-blocked or IgG controls and performed an HMTase assay. The antiparafibromin immunoprecipitate specifically methylated histone H3 (Fig. , lane 3), while the controls did not (Fig. , lanes 1 and 2).
FIG. 7. Parafibromin (Parafib) interacts with an HMTase complex that methylates histone H3 on lysine 4. (A) Antiparafibromin antibody Ab649 (lane 3), normal rabbit IgG (lane 1), and peptide-blocked antibody Ab649 control (ctl; lane 2) immunoprecipitates were (more ...)
To determine which lysine residue in histone H3 is methylated by the parafibromin-associated HMTase complex, we performed Edman degradation on labeled histone H3 with antiparafibromin Ab649, peptide-blocked, or IgG immunoprecipitates prepared from lysates of 293T cells. The radioactivity released by the degradation products was counted (Fig. ). Histone H3 incubated with the antiparafibromin immunoprecipitate was specifically labeled on lysine 4 (Fig. ), while the control was not specifically methylated (not shown). Some methylation was also detected on H3 lysine 9, but this appeared to be nonspecific. Taken together, theses results suggest that endogenous parafibromin is associated with a Set1-like HMTase complex that methylates histone H3 on Lys4.