ABSTRACT
Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.
ABSTRACT
Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.
ABSTRACT
The AAA+ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.
Subject(s)
Adenosine Triphosphatases , Indoles/pharmacology , Mutation, Missense , Pyrimidines/pharmacology , Valosin Containing Protein , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Substitution , HCT116 Cells , Humans , Protein Domains , Valosin Containing Protein/antagonists & inhibitors , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
The AAA ATPase p97/VCP regulates protein homeostasis using a diverse repertoire of cofactors to fulfill its biological functions. Here we use the allosteric p97 inhibitor NMS-873 to analyze its effects on enzyme composition and the ability of cells to adapt to its cytotoxicity. We found that p97 inhibition changes steady state cofactor-p97 composition, leading to the enrichment of a subset of its cofactors and polyubiquitin bound to p97. We isolated cells specifically insensitive to NMS-873 and identified a new mutation (A530T) in p97. A530T is sufficient to overcome the cytotoxicity of NMS-873 and alleviates p97 composition changes caused by the molecule but not other p97 inhibitors. This mutation does not affect NMS-873 binding but increases p97 catalytic efficiency through altered ATP and ADP binding. Collectively, these findings identify cofactor-p97 interactions sensitive to p97 inhibition and reveal a new on-target mechanism to suppress the cytotoxicity of NMS-873.
Subject(s)
Acetanilides/pharmacology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Benzothiazoles/pharmacology , Enzyme Inhibitors/pharmacology , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Small Molecule Libraries/pharmacology , Acetanilides/chemistry , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Allosteric Regulation/drug effects , Benzothiazoles/chemistry , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , HCT116 Cells , Humans , Models, Molecular , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclopentanes/pharmacology , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Leukemia/genetics , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/genetics , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cullin Proteins/metabolism , Cyclopentanes/chemistry , DNA Mutational Analysis , Enzyme Inhibitors/chemistry , Genotype , Humans , K562 Cells , Leukemia/metabolism , Models, Molecular , NEDD8 Protein , Point Mutation , Protein Binding , Protein Conformation , Pyrimidines/chemistry , Structure-Activity Relationship , U937 Cells , Ubiquitin-Activating Enzymes/chemistry , Ubiquitins/genetics , Ubiquitins/metabolismABSTRACT
The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys11- and Lys48-linked ubiquitin polymers, but not those containing Lys63 linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys11 and Lys48 ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys11- and Lys48-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys11 and Lys48 ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys11 and Lys48 polyubiquitinated proteins before their degradation by the proteasome.
Subject(s)
Adenosine Triphosphatases/metabolism , Endoplasmic Reticulum/metabolism , Nuclear Proteins/metabolism , Polyubiquitin/metabolism , Animals , Cell Line , HEK293 Cells , Humans , Insecta , Protein Binding/physiologyABSTRACT
N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.
Subject(s)
Adenosine/analogs & derivatives , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Adenosine/metabolism , Amino Acid Sequence , Animals , Cell Line , Embryonic Stem Cells/cytology , Gene Knockdown Techniques , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Sequence Homology, Amino AcidABSTRACT
The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.
Subject(s)
Bacterial Proteins/metabolism , Cullin Proteins/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/metabolism , Ubiquitins/metabolism , Bacterial Proteins/genetics , COP9 Signalosome Complex , Cullin Proteins/genetics , Glutamic Acid/genetics , Glutamic Acid/metabolism , Glutamine/genetics , Glutamine/metabolism , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , NEDD8 Protein , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/geneticsABSTRACT
The combinatorial architecture of cullin 1-RING ubiquitin ligases, in which multiple F-box containing substrate receptors compete for access to CUL1, poses special challenges to assembling cullin 1-RING ubiquitin ligase complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire F-box containing substrate receptor repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global cullin 1-RING ubiquitin ligase network by promoting the assembly of newly synthesized F-box containing substrate receptors with CUL1-RBX1 core complexes. Our studies of in vivo cullin 1-RING ubiquitin ligase dynamics and in vitro biochemical findings showing that CAND1 can displace F-box containing substrate receptors from Cul1p suggest that CAND1 functions in a cycle that serves to exchange F-box containing substrate receptors on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire F-box containing substrate receptor repertoire in order to maintain the cullin 1-RING ubiquitin ligase landscape, a function that we show to be critical for substrate degradation and normal physiology.
Subject(s)
SKP Cullin F-Box Protein Ligases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Proteolysis , Substrate SpecificityABSTRACT
Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.
Subject(s)
Cyclopentanes/pharmacology , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Amino Acid Sequence , Amino Acid Substitution , Cell Line, Tumor , Chromatography, Liquid , Cullin Proteins/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Drug Resistance, Neoplasm/genetics , HCT116 Cells , Humans , Molecular Sequence Data , Point Mutation , Sequence Alignment , Tandem Mass Spectrometry , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/physiologyABSTRACT
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Endoplasmic Reticulum/enzymology , Membrane Proteins/metabolism , Signal Transduction/physiology , Stress, Physiological/physiology , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Endoribonucleases/metabolism , HEK293 Cells , HeLa Cells , Humans , In Vitro Techniques , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/physiology , Protein Serine-Threonine Kinases/metabolism , Transfection , Ubiquitination/physiologyABSTRACT
Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.
Subject(s)
Cerebellum/abnormalities , Cerebellum/embryology , Cerebral Cortex/abnormalities , Cerebral Cortex/embryology , Lamin Type B/deficiency , Animals , Cell Movement , Cerebellum/pathology , Cerebral Cortex/pathology , Gene Silencing , Lamin Type B/metabolism , Mice , Neurons/pathologyABSTRACT
The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia-inducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-beta-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3beta. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.
Subject(s)
Cullin Proteins/metabolism , DNA-Binding Proteins/metabolism , Protein Kinases/metabolism , Transcription Factors/physiology , Carrier Proteins/metabolism , Cell Hypoxia/physiology , Cell Line , Cell Line, Tumor , Cycloheximide/pharmacology , DNA-Binding Proteins/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Immunoblotting , Phosphorylation , Protein Stability , Protein Synthesis Inhibitors/pharmacology , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/physiology , TOR Serine-Threonine Kinases , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , beta-Transducin Repeat-Containing Proteins/metabolismABSTRACT
Defects in the biogenesis of lamin A from its farnesylated precursor, prelamin A, lead to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes. A deficiency in ZMPSTE24, a protease involved in prelamin A processing, leads to prelamin A accumulation, an absence of mature lamin A, misshapen nuclei, and a lethal perinatal progeroid syndrome: restrictive dermopathy (RD). Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot be processed to lamin A. The hallmark cellular abnormality in RD and HGPS is misshapen nuclei. We hypothesized that the farnesylation of prelamin A is important for its targeting to the nuclear envelope in RD and HGPS and that blocking farnesylation would ameliorate the nuclear shape abnormalities. Indeed, when RD fibroblasts were treated with a farnesyltransferase inhibitor (FTI), prelamin A was partially mislocalized away from the nuclear envelope, and the frequency of nuclear shape abnormalities was reduced (P < 0.0001). A FTI also mislocalized prelamin A and improved nuclear shape in Zmpste24-deficient mouse embryonic fibroblasts (P < 0.0001) and improved nuclear shape in human HGPS fibroblasts (P < 0.0001). Most remarkably, a FTI significantly improved nuclear shape in two fibroblast cell lines from atypical progeria patients with lamin A missense mutations in the absence of prelamin A accumulation (P = 0.0003 and P < 0.0001). These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes and suggest a potential strategy for treating these diseases.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Cell Nucleus/enzymology , Fibroblasts/cytology , Progeria/enzymology , Progeria/pathology , Animals , Cell Nucleus/drug effects , Cells, Cultured , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Humans , Lamin Type A , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloendopeptidases/deficiency , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Mice , Nuclear Proteins/metabolism , Protein Precursors/metabolismABSTRACT
Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Cell Nucleus/pathology , Nuclear Proteins/genetics , Progeria/metabolism , Protein Precursors/genetics , Quinolines/pharmacology , Animals , Blotting, Southern , Cell Nucleus/drug effects , DNA Primers , Farnesyltranstransferase , Fibroblasts/drug effects , Fibroblasts/metabolism , Genetic Vectors , Lamin Type A , Mice , Mice, Transgenic , Microscopy, Fluorescence , Progeria/genetics , Progeria/pathology , Sequence Analysis, DNAABSTRACT
Requisite levels of intracellular cholesterol and fatty acids are maintained in part by the sterol regulatory element binding proteins (SREBPs). Three major SREBP isoforms exist; SREBP-1a and SREBP-1c are expressed from overlapping mRNAs, whereas SREBP-2 is encoded by a separate gene. The active forms of SREBP-1a and SREBP-1c differ only at their extreme N termini; SREBP-1c lacks 28 aa present in SREBP-1a and instead contains 4 unique aa of its own. While the SREBP-1a and -1c isoforms differentially activate transcription, the molecular basis of this difference is unknown. Here we define the differences between these proteins that confer the enhanced activity of SREBP-1a and demonstrate that this enhancement is a direct result of its avid binding to the coactivator CREB binding protein (CBP) and the mammalian mediator complex. While previous work determined that the C/H1 zinc finger and KIX domains of CBP bind to SREBP-1a, we provide evidence that the interaction with C/H1 is important for gene activation. We further show that the association between the activation domain of SREBP-1 and mediator is through aa 500 to 824 of DRIP150. Finally, we demonstrate the recruitment of mediator to an SREBP-responsive promoter in a sterol-dependent manner.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Transcription Factors , Amino Acid Sequence , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Corticosterone , Humans , Molecular Sequence Data , Mutagenesis , Promoter Regions, Genetic , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Sterol Regulatory Element Binding Protein 1 , Sterols/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional ActivationABSTRACT
The mRNAs for all three members of the sterol regulatory element-binding protein (SREBP) family are widely expressed, and the proteins are highly similar. They have potential to both hetero- and homodimerize through their bHLHLZ domains, so it has been difficult to definitively study the role of each one apart from the other two. In the current study, we have utilized cell lines that express only one functional SREBP and the chromatin immunoprecipitation technique to analyze individual SREBP binding to three specific target genes: hydroxymethylglutaryl-CoA reductase (Red), fatty acid synthase (FAS), and squalene synthase (SQS). Our studies show that SREBP-2 binds to promoters for all three genes, and in agreement with the original report using these cells, all three mRNAs are also induced. In the line expressing only SREBP-1a, mRNAs for Red and FAS are induced, but SQS is not. Chromatin immunoprecipitation also shows that SREBP-1a is recruited efficiently to Red and FAS promoters but not to SQS. This observation indicates SREBP-2 selectively binds the SQS promoter and is sufficient to explain the lack of SQS mRNA induction in the SREBP-1a-expressing cells. SREBP-1c protein was not stably recruited to any SREBP target promoter despite being fully active in DNA binding when purified from extracts of the corresponding cells. This is also sufficient to explain the lack of SREBP target gene induction by the singular expression of SREBP-1c. We also show that whereas SREBP-1a and -2 proteins interact efficiently with transcriptional co-activators that modify cellular chromatin, SREBP-1c does not. Taken together, our data support a model suggesting that chromatin modification is required during the initial stage of specific site recognition by SREBPs in native chromatin in vivo.
Subject(s)
DNA-Binding Proteins/metabolism , Promoter Regions, Genetic , Protein Isoforms/metabolism , Transcription Factors/metabolism , Base Sequence , Binding Sites , CCAAT-Enhancer-Binding Proteins , Cell Line , Chromatin/chemistry , Chromatin/metabolism , DNA/metabolism , DNA Primers , DNA-Binding Proteins/genetics , Polymerase Chain Reaction , Precipitin Tests , Protein Binding , Protein Isoforms/genetics , Sterol Regulatory Element Binding Protein 1 , Sterol Regulatory Element Binding Protein 2 , Transcription Factors/geneticsABSTRACT
Sterol regulatory element-binding proteins (SREBPs) activate promoters for key genes of metabolism to keep pace with the cellular demand for lipids. In each SREBP-regulated promoter, at least one ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient gene induction. Some of these putative co-regulatory proteins are members of transcription factor families that all bind to the same DNA sequence elements in vitro and are often expressed in the same cells. These two observations have made it difficult to assign specific and redundant functions to the unique members of a specific gene family. We have used the chromatin immunoprecipitation (ChIP) technique coupled with a transient complementation assay in Drosophila SL2 cells to directly compare the ability of two members of the CREB/ATF family to function as co-regulatory proteins for SREBP-dependent activation of the HMG-CoA reductase promoter. Results from both of these experimental systems demonstrate that CREB is an efficient SREBP co-regulator but ATF-2 is not.