HA Epitope Research Articles

Topology of Transmembrane Channel-like Gene 1 Protein

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?

Mitochondrial-DNA diseases have no effective treatments. Allotopic expression—synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment and its importation into mitochondria—has been proposed as a gene-therapy approach. Allotopic expression has been successfully demonstrated in yeast, but in mammalian mitochondria results are contradictory. The evidence available is based on partial phenotype rescue, not on the incorporation of a functional protein into mitochondria. Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression. We recoded mitochondrial mt-Nd6 to the universal genetic code, and added the N-terminal mitochondrial-targeting sequence of cytochrome c oxidase VIII (C8) and the HA epitope (C8Nd6HA). The protein apparently co-localized with mitochondria, but a significant part of it seemed to be located outside mitochondria. Complex I activity and assembly was restored, suggesting successful allotopic expression. However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation. These findings demonstrate the need for extreme caution in the interpretation of functional rescue experiments and for clear-cut controls to demonstrate true rescue of mitochondrial function by allotopic expression.

The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis

Cytokinins affect plant immunity to various pathogens; however, the mechanisms coupling plant-derived cytokinins to pathogen responses have been elusive. Here, we found that plant-derived cytokinins promote resistance of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst). Modulated cytokinin levels or signaling activity in CKX- or IPT-overexpressing plants or in ahk2 ahk3 mutants correlated with altered resistance. In fact, the cytokinin-activated transcription factor ARR2 contributes specifically to Pst resistance. The salicylic acid (SA) response factor TGA3 binds ARR2, and mutation of TGA-binding cis-elements in the Pr1 promoter abolished cytokinin- and ARR2-dependent Pr1 activation. Cytokinin treatment did not increase pathogen resistance in tga3 plants, as the cytokinin-dependent induction of Pr1 was eliminated. Moreover, SA signaling enhanced binding of ARR2/TGA3 to the Pr1 promoter. Taken together, these results show that cytokinins modulate the SA signaling to augment resistance against Pst, a process in which the interaction between TGA3 and ARR2 is important.

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.

Heparan Sulfate Chain Valency Controls Syndecan-4 Function in Cell Adhesion

Fibroblasts null for the transmembrane proteoglycan, syndecan-4, have an altered actin cytoskeleton, compared with matching wild-type cells. They do not organize α-smooth muscle actin into bundles, but will do so when full-length syndecan-4 is re-expressed. This requires the central V region of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild-type phenotype, whereas those expressing two or three were competent. However, clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and α-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains.

Molecular and Cellular Mechanisms Underlying Neural Tube Defects in the Loop-tail Mutant Mouse

Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, S464N). Mammalian Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as establishment of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis and cardiogenesis. Vangl proteins are thought to assemble with other PCP proteins (Dvl, Pk) to form membrane-bound PCP signaling complexes that provide polarity information to the cell. In the present study, we show that Vangl1 is expressed exclusively at the plasma membrane of transfected MDCK cells, where it is targeted to the basolateral membrane. Experiments with an inserted exofacial HA epitope indicate that the segment delimited by the predicted transmembrane domains 1 and 2 is exposed to the extracellular milieu. Comparative studies of the Lp-associated pathogenic mutation D255E indicate that the targeting of the mutant variant at the plasma membrane is greatly reduced; the mutant variant is predominantly retained intracellularly in endoplasmic reticulum (ER) vesicles colocalizing with the ER marker calreticulin. In addition, the D255E variant shows drastically reduced stability with a half-life of approximately 2 h, compared to >9 h for its wild type counterpart and is rapidly degraded in a proteasome-dependent and MG132 sensitive pathway. These findings highlight a critical role for D255 for normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at that site. Our study provide an experimental framework for the analysis of human VANGL mutations recently identified in familial and sporadic cases of spina bifida.

IFN-γ exerts antiviral activity against polyomavirus infection (42.14)

Abstract CD8 T cells use both cytolytic and noncytolytic effector mechanisms to eliminate infectious pathogens. Using the mouse polyoma virus (MPyV) system, we have previously shown that TNFa, Fas/FasL, and the perforin/granzyme pathways are dispensable for viral control and susceptibility to MPyV-induced tumors. Here, we investigated whether IFN-γ may be an important effector mechanism for limiting viral burden and tumorigenesis. Using Western analyses for early region nonstructural viral T antigens, as well as FACS analyses of cells infected by a novel recombinant MPyV carrying an intra-T antigen HA epitope tag, we found that IFN-γ reduces expression of viral T antigens in a dose-dependent fashion. Compared to MPyV-infected wild type C57BL/6 mice, IFN-γ receptor KO mice had a 1-log higher viral load during acute infection in most organs, as well as during the persistent phase of MPyV infection; notably, 3-log higher viral genome copies were present in the kidneys, a major site of persistence by both murine and human polyomaviruses. This increase in viral burden was not due to a lack of responding cells, as fully functional virus-specific CD8 T cells were found during the entire course of infection in IFN-γR KO mice at levels similar to those seen in wild type mice. Studies are underway to determine if IFN-γ receptor KO mice are susceptible to PyV tumorigenesis and to investigate the importance of IFN-γ as a critical CD8 antiviral effector function in MPyV infection.

Characterization of a Coenzyme Q biosynthetic complex in Saccharomyces cerevisiae

Coenzyme Q (Q) is a critical molecule for proper functioning of the electron transport chain in mitochondrial respiration. Q is synthesized by a series of enzymes in the mitochondria and the human Q biosynthetic proteins are well conserved in the yeast Saccharomyces cerevisiae, with several capable of functionally substituting for their yeast homologues. Yeast is thus a good model for understanding Q biosynthesis in higher eukaryotes. There is evidence that several of the Q biosynthetic proteins, the Coq proteins, are associated in a large complex in the mitochondria. All of the Coq proteins are peripheral membrane proteins on the matrix face of the inner mitochondrial membrane, except for Coq2p which is an integral membrane protein. It is thus possible that Coq2p serves as the membrane anchor for the other Coq polypeptides in the complex. Coq2p has also been demonstrated to exist in a high molecular mass complex via gel filtration chromatography and 2D Blue Native‐PAGE, although its partner proteins have not been identified. One of our aims is to characterize the Coq2p‐containing complex to determine its role in the organization and stabilization of the complex. BN‐PAGE will be used to determine whether other Coq proteins co‐migrate with Coq2p in a high molecular mass complex. Immunoprecipitation of Coq2p tagged with the HA epitope will identify interacting proteins.

A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription

Set2-mediated H3 Lys(36) methylation is a histone modification that has been demonstrated to function in transcriptional elongation by recruiting the Rpd3S histone deacetylase complex to repress intragenic cryptic transcription. Recently, we identified a trans-histone pathway in which the interaction between the N terminus of Set2 and histone H4 Lys(44) is needed to mediate trans-histone H3 Lys(36) di- and trimethylation. In the current study, we demonstrate that mutation of the lysine 44 residue in histone H4 or the Set2 mutant lacking the histone H4 interaction motif leads to intragenic cryptic transcripts, indicating that the Set2 and histone H4 interaction is important to repress intragenic cryptic transcription. We also determine that histone H2A residues (Leu(116) and Leu(117)), which are in close proximity to histone H4 Lys(44), are needed for proper trans-histone H3 Lys(36) methylation. Similar to H4 Lys(44) mutants, histone H2A Leu(116) and Leu(117) mutations exhibited decreased H3 Lys(36) di- and trimethylation, increased histone H4 acetylation, increased resistance to 6-azauracil, and cryptic transcription. Interestingly, the combined histone H4 Lys(44) and H2A mutations have more severe methylation defects and increased H4 acetylation levels. Furthermore, we identify that additional histone H2A and H3 core residues are also needed for H3 Lys(36) di- and trimethylation. Overall, our results show and suggest that multiple H4, H2A, and H3 residues contribute to and form a Set2 docking/recognition site on the nucleosomal surface so that proper Set2-mediated H3 Lys(36) di- and trimethylation, histone acetylation, and transcriptional elongation can occur.

Insoluble DISC1–Boymaw fusion proteins generated by DISC1 translocation

Insoluble DISC1–Boymaw fusion proteins generated by DISC1 translocation

Quantitative GPCR Assay Using Time-Resolved Fret

Quantitative study of membrane proteins presents considerable technical challenges. Here, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to quantify functional Chemokine (C-C motif) Receptor 5 (CCR5). CCR5 is a seven-transmembrane helical G protein-coupled receptor (GPCR) expressed primarily on immune cells. It is of significant interest due to its role as the co-receptor of R5-tropic HIV-1. The TR-FRET assay exploits energy transfer between a long-lived europium cryptate donor fluorophore and an appropriate acceptor. We have developed a homogeneous sandwich-type immunoassay with labeled antibodies against a conformationally-sensitive epitope on the extracellular domain of CCR5 and an engineered C-terminal 1D4-mAb epitope. The assay yields a quantitative FRET signal corresponding to the total amount receptor. To quantitate “functional” receptor, a labeled anti-HA (hemagglutinin) antibody against an engineered N-terminal HA epitope is used in conjunction with labeled MIP-1a, a chemokine ligand for CCR5. We are also interested in quantifying the degree of chemical labeling of unnatural amino acids incorporated into expressed GPCRs by amber codon suppression technology. The Staudinger-Bertozzi ligation links a commercially-available FLAG-phosphine reagent to p-L-azidophenylalanine. A europium-labeled anti-FLAG antibody can then be used in another version of the sandwich assay. The resulting FRET signals are directly proportional to amount of total, functional, and labeled receptor, but must be calibrated precisely to extract an absolute concentration. Calibration is accomplished by measuring binding of a fluorescent derivative of a small-molecule CCR5 antagonist. The assay is highly specific due to the long lifetime of the europium donor, and nanomolar concentrations of receptor are detectable. This GPCR assay technology can be used to optimize CCR5 reconstitution conditions and can be readily extended to other members of the chemokine receptor family.

Amino Acid Signaling in Yeast: Post-genome Duplication Divergence of the Stp1 and Stp2 Transcription Factors

When yeast cells detect external amino acids via their permease-like Ssy1 sensor, the cytosolic precursor forms of Stp1 and Stp2 transcription factors are activated by endoproteolytic removal of their N-terminal domains, a reaction catalyzed by the Ssy5 endoprotease. The processed Stp factors then migrate into the nucleus, where they activate transcription of several amino acid permease genes including AGP1. We report here that the STP1 and STP2 genes most likely derive from the whole genome duplication that occurred in a yeast ancestor. Although Stp1 and Stp2 have been considered redundant, we provide evidence that they functionally diverged during evolution. Stp2 is the only factor processed when amino acids are present at low concentration, and the transcriptional activation of AGP1 promoted by Stp2 is moderate. Furthermore, only Stp2 can sustain Agp1-dependent utilization of amino acids at low concentration. In contrast, Stp1 is only processed when amino acids are present at high concentration, and it promotes higher level transcriptional activation of AGP1. Domain swapping experiments show that the N-terminal domains of Stp1 and Stp2 are responsible for these proteins being cleaved at different amino acid concentrations. Last, induction of the DIP5 permease gene by amino acids depends on Stp2 but not Stp1. We propose that post-whole genome duplication co-conservation of the STP1 and STP2 genes was favored by functional divergence of their products, likely conferring to cells an increased ability to adapt to various amino acid supply conditions.

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

Identification of Nucleases and Phosphatases by Direct Biochemical Screen of the Saccharomyces cerevisiae Proteome

The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His6, an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins.

The RNA-binding Motif Protein 15B (RBM15B/OTT3) Acts as Cofactor of the Nuclear Export Receptor NXF1

The human SPEN family proteins SHARP, RBM15/OTT1, and RBM15B/OTT3 share the structural domain architecture but show distinct functional properties. Here, we examined the function of OTT3 and compared it with its paralogues RBM15 and SHARP. We found that OTT3, like RBM15, has post-transcriptional regulatory activity, whereas SHARP does not, supporting a divergent role of RBM15 and OTT3. OTT3 shares with RBM15 the association with the splicing factor compartment and the nuclear envelope as well as the binding to mRNA export factors NXF1 and Aly/REF. Mutational analysis revealed direct interaction of OTT3 and RBM15 with NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knockdown of RBM15 in mouse is embryonically lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles.

Presynaptic Targeting of α4β2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1β

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.

Common human alpha2a&c adrenergic receptor polymorphisms do not affect cellular processing nor surface trafficking

Naturally occurring variant genetic forms of α2a and/or α2c adrenergic receptors (α2a&c ARs) have been identified and linked to congestive heart failure. Two well described α2a&c AR variants occur within the 3rd intracellular loop, α2aLys251 and α2cDel322‐325. We investigated if these variant forms exhibited alterations in localization or cellular processing to account for their phenotypic differences.Human cDNA encoding wild‐type and polymorphic α2a&c ARs were either purchased commercially or created using standard Quick Change mutagenesis techniques, incorporating an amino terminal HA epitope. Each construct was examined with respect to drug binding, biochemical processing (e.g. glycosylation), and surface and total expression (e.g. immunocytochemistry and FACS analysis).All four cDNA constructs were successfully transfected and expressed in HEK293 cells. RX 821002 binding did not differ greatly between wild‐type and polymorphic forms. Transfected cells were analyzed by immunocytochemistry and FACS; neither polymorphic form demonstrated differences in cell surface or intracellular expression patterns. Lastly, endoglycosidase digestions of transfected cell membranes revealed that the polymorphic forms were processed in a similar manner as their wild‐type counterparts.It has been shown previously that certain GPCR polymorphisms can affect receptor stability and trafficking. We examined the two most common α2a&c AR polymorphisms and found no discernable difference in receptor expression, drug binding, surface trafficking, or biochemical processing. Therefore, phenotypic differences between wild‐type and polymorphic forms are most likely due to signal transduction, or possibly neuronal specific influences.This work was supported in part by NINDS K08 NS050654‐01A1 (TA)

Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

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.