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Nature Structural & Molecular Biology Feb 2011Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers....
Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers. Transcriptional repression complexes, assembled by the corepressor NCoR and its homolog SMRT, are crucial in many processes, including development and metabolic physiology. The core repression complex involves the recruitment of three proteins, HDAC3, GPS2 and TBL1, to a highly conserved repression domain within SMRT and NCoR. We have used structural and functional approaches to gain insight into the architecture and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the corepressor complex.
Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Line; Crystallography, X-Ray; Humans; Intracellular Signaling Peptides and Proteins; Mice; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Nuclear Receptor Co-Repressor 2; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Transducin
PubMed: 21240272
DOI: 10.1038/nsmb.1983 -
Modern Pathology : An Official Journal... Nov 2022Xanthogranulomatous epithelial tumor (XGET) and keratin-positive giant cell-rich soft tissue tumor with HMGA2-NCOR2 fusion (KPGCT) are two recently described neoplasms...
Xanthogranulomatous epithelial tumor (XGET) and keratin-positive giant cell-rich soft tissue tumor with HMGA2-NCOR2 fusion (KPGCT) are two recently described neoplasms with both distinct and overlapping clinical and histopathologic features. We hypothesized that XGET and KPGCT may be related and represent a histologic spectrum of a single entity. To test this, we sought to characterize the clinical, radiographic, immunohistochemical, ultrastructural and molecular features of additional tumors with features of XGET and/or KPGCT, which we refer to descriptively as keratin-positive xanthogranulomatous/giant cell-rich tumors (KPXG/GCT). The archives were searched for potential cases of KPXG/GCT. Clinical and imaging features were noted. Slides were assessed for histologic and immunohistochemical findings. Ultrastructural and next generation RNA sequencing-based analysis were also performed. Nine cases were identified arising in seven women and two men [median age of 33 years (range: 12-87)]. Median tumor size was 4 cm (range: 2.4-14.0 cm) and tumors presented in the thigh (2), buttock (1), forearm (2), groin (1), cranial fossa (1), ilium (1), and tibia (1). Morphologically, tumors were most frequently characterized by a fibrous capsule, with associated lymphoid reaction, enclosing a polymorphous proliferation of histiocytes, giant cells (Touton and osteoclast-types), mixed inflammatory infiltrate, hemorrhage and hemosiderin deposition, which imparted a variably xanthogranulomatous to giant cell tumor-like appearance. One case clearly showed mononuclear cells with eosinophilic cytoplasm characteristic of XGET. All cases expressed keratin and 7 of 9 were found to harbor HMGA2-NCOR2 fusions including cases with xanthogranulomatous appearance. One patient developed local recurrence and multifocal pulmonary lesions, which were radiographically suspicious for metastases. Shared clinical, histologic and immunohistochemical features, and the shared presence of HMGA2-NCOR2 fusions supports interpretation of KPXG/GCT as a single entity which includes XGET and KPGCT. Given limited clinical follow-up to date and rare cases with apparently aggressive findings, we provisionally regard these tumors as having uncertain biologic potential.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Female; Humans; Male; Middle Aged; Young Adult; Giant Cell Tumors; Giant Cells; Hemosiderin; Keratins; Neoplasms, Glandular and Epithelial; Nuclear Receptor Co-Repressor 2; Soft Tissue Neoplasms; Oncogene Proteins, Fusion; HMGA2 Protein
PubMed: 35690644
DOI: 10.1038/s41379-022-01115-6 -
Nucleic Acids Research Jan 2016Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of...
Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.
Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Chromosomes, Bacterial; Databases, Genetic; Gene Expression Profiling; Genome, Bacterial; Systems Biology
PubMed: 26476443
DOI: 10.1093/nar/gkv1050 -
Biochemical and Biophysical Research... Jul 2020A majority of acute promyelocytic leukaemia (APL) cases are characterized by the PML-RARα fusion gene. Previous studies have shown that neutrophil elastase (NE) can...
A majority of acute promyelocytic leukaemia (APL) cases are characterized by the PML-RARα fusion gene. Previous studies have shown that neutrophil elastase (NE) can cleave PML-RARα and is important for the development of APL. Here, we demonstrate that one of the cleavage products of PML-RARα, NLS-RARα, can block cell differentiation by repressing the expression of the target genes within the retinoic acid signalling pathway. The results of reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis showed that NLS-RARα depressed the expression of the cell differentiation marker protein, CD11b and CEBPβ, as well as the retinoic acid signalling pathway target genes, RARβ and CEBPε. Studies have shown that NLS-RARα forms heterodimers with retinoid X receptor α(RXRα) and interacts with SMRT. When treated with all-trans retinoic acid (ATRA), NLS-RARα exhibits diminished transcriptional activity compared to RARα. Moreover, in the presence of high doses of ATRA, NLS-RARα could be degraded along with the consequent transactivation of retinoic acid signalling pathway target genes and cell differentiation induction in a dose- and time-dependent manner. Together, these results indicate that NLS-RARα blocks cell differentiation by inhibiting the retinoic acid signalling pathway.
Topics: Biomarkers; Cell Differentiation; Cell Line; Gene Expression Regulation; Humans; Models, Biological; Nuclear Localization Signals; Nuclear Receptor Co-Repressor 2; Protein Binding; Proteolysis; Retinoic Acid Receptor alpha; Signal Transduction; Structure-Activity Relationship; Tretinoin
PubMed: 32475642
DOI: 10.1016/j.bbrc.2020.05.076 -
American Journal of Physiology.... Apr 2016In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins....
In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTβ, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostβ promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in the promoters of FXR-target genes and possibly through direct interaction with FXR.
Topics: ATP-Binding Cassette Transporters; Animals; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Histone Deacetylases; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Nuclear Receptor Co-Repressor 2; Protein Binding; Response Elements; Transcription Factor RelA; Transcriptional Activation
PubMed: 26867564
DOI: 10.1152/ajpgi.00363.2015 -
American Journal of Physiology. Renal... Aug 2015ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides...
ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.
Topics: Acetylation; Angiotensin II Type 1 Receptor Blockers; Apoptosis Regulatory Proteins; Co-Repressor Proteins; DNA Damage; Dose-Response Relationship, Drug; Histones; Humans; Losartan; Nuclear Receptor Co-Repressor 2; Podocytes; Proteasome Endopeptidase Complex; Protective Agents; Receptors, Calcitriol; Vitamin D3 24-Hydroxylase
PubMed: 26084932
DOI: 10.1152/ajprenal.00476.2014 -
The Journal of Clinical Investigation Jan 2013Low-grade chronic inflammation is a major characteristic of obesity and results from deregulated white adipose tissue function. Consequently, there is interest in...
Low-grade chronic inflammation is a major characteristic of obesity and results from deregulated white adipose tissue function. Consequently, there is interest in identifying the underlying regulatory mechanisms and components that drive adipocyte inflammation. Here, we report that expression of the transcriptional corepressor complex subunits GPS2 and SMRT was significantly reduced in obese adipose tissue, inversely correlated to inflammatory status, and was restored upon gastric bypass surgery-induced weight loss in morbid obesity. These alterations correlated with reduced occupancy of the corepressor complex at inflammatory promoters, providing a mechanistic explanation for elevated inflammatory transcription. In support of these correlations, RNAi-mediated depletion of GPS2 and SMRT from cultured human adipocytes promoted derepression of inflammatory transcription and elevation of obesity-associated inflammatory markers, such as IL-6 and MCP-1. Furthermore, we identified a regulatory cascade containing PPARγ and TWIST1 that controlled the expression of GPS2 and SMRT in human adipocytes. These findings were clinically relevant, because treatment of diabetic obese patients with pioglitazone, an antidiabetic and antiinflammatory PPARγ agonist, restored expression of TWIST1, GPS2, and SMRT in adipose tissue. Collectively, our findings identify alterations in a regulatory transcriptional network in adipocytes involving the dysregulation of a specific corepressor complex as among the initiating events promoting adipose tissue inflammation in human obesity.
Topics: Adipocytes; Cells, Cultured; Chemokine CCL2; Female; Gene Expression Regulation; Humans; Inflammation; Interleukin-6; Intracellular Signaling Peptides and Proteins; Male; Nuclear Proteins; Nuclear Receptor Co-Repressor 2; Obesity, Morbid; PPAR gamma; Transcription, Genetic; Twist-Related Protein 1
PubMed: 23221346
DOI: 10.1172/JCI64052 -
Frontiers in Oncology 2022The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with a Ku70/Ku80 heterodimer constitutes the intact DNA-PK kinase, which is an upstream component of the...
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with a Ku70/Ku80 heterodimer constitutes the intact DNA-PK kinase, which is an upstream component of the DNA repair machinery that signals the DNA damage, orchestrates the DNA repair, and serves to maintain genome integrity. Beyond its role in DNA damage repair, the DNA-PK kinase is also implicated in transcriptional regulation and RNA metabolism, with an illuminated impact on tumor progression and therapeutic responses. However, the efforts to identify DNA-PK regulated transcriptomes are limited by short-read sequencing to resolve the full complexity of the transcriptome. Therefore, we leveraged the PacBio Single Molecule, Real-Time (SMRT) Sequencing platform to study the transcriptome after DNA-PK inactivation to further underscore the importance of its role in diseases. Our analysis revealed additional novel transcriptome and complex gene structures in the DNA-PK inactivated cells, identifying 8,355 high-confidence new isoforms from 3,197 annotated genes and 523 novel genes. Among them, 380 lncRNAs were identified. We validated these findings using computational approaches and confirmatory transcript quantification with short-read sequencing. Several novel isoforms representing distinct splicing events have been validated through PCR experiments. Our analyses provide novel insights into DNA-PK function in transcriptome regulation and RNA metabolism.
PubMed: 35992789
DOI: 10.3389/fonc.2022.941638 -
Blood Cancer Journal Dec 2021MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression...
MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.
Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Knockout Techniques; Histone Deacetylase Inhibitors; Humans; Multiple Myeloma; Nuclear Receptor Co-Repressor 2; Proto-Oncogene Proteins c-myc; Thalidomide; Up-Regulation
PubMed: 34864816
DOI: 10.1038/s41408-021-00589-y -
Proceedings of the National Academy of... Sep 1997The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding...
The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.
Topics: Animals; Cells, Cultured; DNA-Binding Proteins; Mice; Nuclear Receptor Co-Repressor 2; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-6; Repressor Proteins; Transcription Factors; Transcription, Genetic
PubMed: 9380707
DOI: 10.1073/pnas.94.20.10762