Identification and characterization of a novel nuclear protein complex involved in nuclear hormone receptor-mediated gene regulation

Abstract

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.

FIGURE 1.

Two-step sequential purification scheme for the NIF-1 complex(es). a, 60 mg of nuclear extract protein from pLPC-Vector and pLPC-NIF-1 cells was initially dialyzed to a final salt concentration of 0.1 m KCl. The dialyzed nuclear extract was affinity-purified using an anti-FLAG affinity matrix, and the bound proteins were subsequently eluted from the beads using 3×-FLAG peptide. The eluates were further purified by HA-affinity matrix and, after extensive washing, eluted with a competitive HA peptide. b, the affinity-purified Vector- and NIF-1-complex was resolved in 4–15% SDS-polyacrylamide gels, and the associated proteins were visualized by silver staining. The molecular weight markers are as shown on the right.

FIGURE 2.

Western blotting confirmation of components of the purified NIF-1 complex. a, NIF-1-associated proteins were analyzed by Western blot with antibodies indicated on the right. Equivalent amounts of the Vector-purified fractions were used to confirm specificity. 5 μg of nuclear extract was used as Input. b, the purified NIF-1 complex does not include a number of known MLL/SET methyltransferase components. NIF-1-associated proteins were analyzed by Western blot with antibodies indicated on the right.5 μgof nuclear extract was used as Input.

FIGURE 3.

NIF-1 exists in at least two distinct molecular weight complexes and interacts with DBC-1. a, NIF-1-associated proteins purified as described were fractionated in a Superose 6 gel filtration column in buffer containing 0.2 m KCl. The collected fractions were analyzed by Western blotting with antibodies indicated on the right. Positions of protein standards are indicated on the top. The two distinct groups of NIF-1-associated proteins are detected. A high molecular weight complex with a peak of ∼1.5 MDa contains NIF-1, DBC-1, and EMSY. b, evidence for association between the two distinct sub-groups of NIF-1-associated proteins. 20 mg of nuclear extract from NIF-1-expressing HeLa S3 cells was partially purified using FLAG-affinity matrix and eluted with competing 3×-FLAG peptide. The FLAG eluates were immunoprecipitated with either rabbit IgG (control) or with antibodies against Ash2L, DBC-1, or EMSY indicated at the top. The immunoprecipitates were subjected to Western blotting with the antibodies indicated on the right. c, various deletion constructs of NIF-1 as B42 fusions (described under “Experimental Procedures”) were tested for interaction with full-length LexA-DBC-1 in two-hybrid assays as assessed by an X-gal colony response. LexA (pEGΔPL) and B42 (pJG4–5ΔPL) vectors were used as control plasmids and showed no response. The results were reproduced and verified twice. As expected, no interaction was found on dextrose plates. Those indicated by “++++” showed a strong X-gal colony response on Gal/Raf plates within 12–20 h of incubation. The interaction assay with NIF-1b indicated as negative (–) showed no interaction as judged by color reaction after several weeks of incubation even though NIF-1b is expressed as assessed by Western blotting.

FIGURE 4.

DBC-1 and EMSY are involved in stimulation of Sox9, an RAR target gene. MCF-7 cells were transfected with either an siRNA against DBC-1 (a) or an siRNA against EMSY along with a control siRNA at a concentration of 40 nm. 42 h later the cells were incubated with 1 μm RA for 24 h before whole cell lysates were prepared and immunoblotted with an anti-Sox9 antibody. The extent of knockdown of both DBC-1 and EMSY was also analyzed. β-Actin was used as a loading control. b, siRNA knockdown does not alter the levels of hSRT1 or Menin, which are less abundant and less stable than β-actin. This indicates that the effect of knockdown of DBC-1 and EMSY on stimulation of Sox9 by RA does not reflect a general effect on gene expression.

FIGURE 5.

Real-time PCR analysis of Hox1 and Sox9. MCF-7 cells were transfected with siRNAs against DBC-1, EMSY, and NIF-1 at a concentration of 40 nm as indicated. A control siRNA was included in the study. The cells were incubated with 1 μm RA for 24 h before the cells were processed. Total RNA (1 μg) was reverse-transcribed, and 1 μl of each cDNA sample was used for the quantitative PCR analysis. All samples were assayed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal normalizing control. The quantification was done using the comparative C_t method. Error bars represent ± S.D.

FIGURE 6.

Knockdown of DBC-1 and EMSY affects ligand-dependent stimulation by Gal4-RARα in MCF-7 cells. MCF-7 cells were transfected with siRNAs against DBC-1 or EMSY along with a control siRNA at a concentration of 40 nm. 48 h later, the cells were transfected with the reporter pMC110 (100 ng, all lanes) and either Gal4-RARα (200 ng) or equimolar amounts of the Gal4 vector pSG424 as indicated. The cells were incubated with or without RA (0.25 μm) and assayed for chloramphenicol acetyltransferase (CAT) activity 72 h post-siRNA transfection as described previously (28). The maximum chloramphenicol acetyltransferase activity designated as 100 was obtained with Gal4-RARα in the presence of RA with samples transfected with control siRNA. All transfections were carried out in triplicate, and error bars represent ± S.D. The figure also indicates the knockdown of DBC-1 and EMSY as confirmed by Western blotting using equal amounts of protein extracts (35 μg). In addition to the control siRNA (Con.siRNA), Western blotting was also performed with lysates from cells that did not receive siRNA (Con.lysate).

FIGURE 7.

Effect of DBC-1 on stimulation of Sox9 expression is SirT1-independent. a, MCF-7 cells were transfected with either an siRNA against DBC-1 or a control siRNA at a concentration of 40 nm. The cells were treated with 50 nm nicotinamide, a concentration that we have found fully inhibits SirT1 in MCF-7 cells. Following preincubation with nicotinamide, the cells were incubated with RA as indicated for 24 h before whole cell lysates were prepared and immunoblotted with an anti-Sox9 antibody. The extent of knockdown of DBC-1 was also analyzed using an antibody against DBC-1. b, SirT1 is not a component of the NIF-1 complex. Purified NIF-1 and Vector fractions were analyzed by Western blotting with an antibody against SirT1. 5 μg of nuclear extract was used as Input.

FIGURE 8.

One or more NIF-1 complexes contain methyltransferase activity that does not methylate H3K4. 5 and 10 μl (out of 1 ml of purified NIF-1 complex) were incubated with 2 μg of WT and recombinant H3K4A histone octamers and the methyl donor [³H]S-adenosylmethionine. The samples were resolved in 15% SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and stained with Coomassie Blue (lower panel). The polyvinylidene difluoride membrane was sprayed with EN³HANCE and analyzed by autoradiography (upper panel). The vector-purified fraction was also analyzed with 10 μl of the final preparation. Total nuclear extract (NE) served as a positive control for the in vitro methyltransferase assay.