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1.
Cell Adh Migr ; 17(1): 1-14, 2023 12.
Article in English | MEDLINE | ID: mdl-37749865

ABSTRACT

The LMNA gene encoding lamin A/C is amplified in some clear cell renal cell carcinoma (ccRCC) samples. Our data showed that depletion of the tumor suppressor PBRM1 can upregulate lamin A/C levels, and lamin A/C could interact with PBRM1. However, the role of lamin A/C in ccRCC is not yet fully understood. Our functional assays showed that although the proliferation ability was slightly impaired after LMNA depletion, the migration and invasion of ccRCC cells were significantly inhibited. This suppression was accompanied by a reduction in MMP2, MMP9, AKT/p-AKT, and Wnt/ß-catenin protein levels. Our data therefore suggest that lamin A/C, as an interaction partner of the tumor suppressor PBRM1, plays a crucial role in tumor invasion and metastasis in ccRCC.


Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , beta Catenin/genetics , Proto-Oncogene Proteins c-akt/metabolism , Lamin Type A/genetics , Lamin Type A/metabolism , Cell Proliferation/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic
2.
Nat Cell Biol ; 25(5): 765-777, 2023 05.
Article in English | MEDLINE | ID: mdl-37095322

ABSTRACT

PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.


Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Genomics , Kidney Neoplasms/metabolism , NF-kappa B/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics
3.
J Cell Mol Med ; 26(14): 3837-3849, 2022 07.
Article in English | MEDLINE | ID: mdl-35672925

ABSTRACT

The PBRM1 (PB1) gene which encodes the specific subunit BAF180 of the PBAF SWI/SNF complex, is highly mutated (~ 40%) in clear cell renal cell carcinoma (ccRCC). However, its functions and impact on cell signalling are still not fully understood. Aerobic glycolysis, also known as the 'Warburg Effect', is a hallmark of cancer, whether PB1 is involved in this metabolic shift in clear cell renal cell carcinoma remains unclear. Here, with established stable knockdown PB1 cell lines, we performed functional assays to access the effects on 786-O and SN12C cells. Based on the RNA-seq data, we selected some genes encoding key glycolytic enzymes, including PFKP, ENO1, PKM and LDHA, and examined the expression levels. The AKT-mTOR signalling pathway activity and expression of HIF1α were also analysed. Our data demonstrate that PB1 deficiency promotes the proliferation, migration, Xenograft growth of 786-O and SN12C cells. Notably, knockdown of PB1 activates AKT-mTOR signalling and increases the expression of key glycolytic enzymes at both mRNA and protein levels. Furthermore, we provide evidence that deficient PB1 and hypoxic conditions exert a synergistic effect on HIF 1α expression and lactate production. Thus, our study provides novel insights into the roles of tumour suppressor PB1 and suggests that the AKT-mTOR signalling pathway, as well as glycolysis, is a potential drug target for ccRCC patients with deficient PB1.


Subject(s)
Carcinoma, Renal Cell , DNA-Binding Proteins , Kidney Neoplasms , Transcription Factors , Animals , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Glycolysis/genetics , Humans , Kidney Neoplasms/pathology , Oncogene Addiction , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
4.
EMBO J ; 30(13): 2569-81, 2011 May 13.
Article in English | MEDLINE | ID: mdl-21572391

ABSTRACT

Oestrogen receptor α (ERα) is key player in the progression of breast cancer. Recently, the cistrome and interactome of ERα were mapped in breast cancer cells, revealing the importance of spatial organization in oestrogen-mediated transcription. However, the underlying mechanism of this process is unclear. Here, we show that ERα binding sites (ERBS) identified from the Chromatin Interaction Analysis-Paired End DiTag of ERα are enriched for AP-2 motifs. We demonstrate the transcription factor, AP-2γ, which has been implicated in breast cancer oncogenesis, binds to ERBS in a ligand-independent manner. Furthermore, perturbation of AP-2γ expression impaired ERα DNA binding, long-range chromatin interactions, and gene transcription. In genome-wide analyses, we show that a large number of AP-2γ and ERα binding events converge together across the genome. The majority of these shared regions are also occupied by the pioneer factor, FoxA1. Molecular studies indicate there is functional interplay between AP-2γ and FoxA1. Finally, we show that most ERBS associated with long-range chromatin interactions are colocalized with AP-2γ and FoxA1. Together, our results suggest AP-2γ is a novel collaborative factor in ERα-mediated transcription.


Subject(s)
Chromatin/metabolism , Estrogen Receptor alpha/physiology , Transcription Factor AP-2/physiology , Transcription, Genetic , Binding Sites/genetics , Chromatin/genetics , Chromatin Immunoprecipitation , Cluster Analysis , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Microarray Analysis , Models, Biological , Protein Binding/drug effects , Protein Binding/genetics , Protein Binding/physiology , RNA, Small Interfering/pharmacology , Response Elements/genetics , Transcription Factor AP-2/antagonists & inhibitors , Transcription Factor AP-2/genetics , Transcription, Genetic/genetics , Transcription, Genetic/physiology , Tumor Cells, Cultured
5.
Mol Syst Biol ; 6: 456, 2010 Dec 21.
Article in English | MEDLINE | ID: mdl-21179027

ABSTRACT

A major question in transcription factor (TF) biology is why a TF binds to only a small fraction of motif eligible binding sites in the genome. Using the estrogen receptor-α as a model system, we sought to explicitly define parameters that determine TF-binding site selection. By examining 12 genetic and epigenetic parameters, we find that an energetically favorable estrogen response element (ERE) motif sequence, co-occupancy by the TF FOXA1, the presence of the H3K4me1 mark and an open chromatin configuration in the pre-ligand state provide specificity for ER binding. These factors can model estrogen-induced ER binding with high accuracy (ROC-AUC=0.95 and 0.88 using different genomic backgrounds). Moreover, when assessed in another estrogen-responsive cell line, this model was highly predictive for ERα binding (ROC-AUC=0.86). Variance in binding site selection between MCF-7 and T47D resides in sites with suboptimal ERE motifs, but modulated by the chromatin configuration. These results suggest a definable interplay between sequence motifs and local chromatin in selecting TF binding.


Subject(s)
Chromatin/metabolism , Estrogen Receptor alpha/metabolism , Transcription Factors/metabolism , Base Sequence , Binding Sites , Cell Line , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/genetics , Hepatocyte Nuclear Factor 3-alpha , Humans , Ligands , Models, Biological , Protein Binding , RNA Polymerase II/metabolism , Response Elements , Transcription Initiation Site
6.
Int J Biol Sci ; 6(2): 163-71, 2010 Mar 29.
Article in English | MEDLINE | ID: mdl-20376207

ABSTRACT

Ulcerative colitis (UC) is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to alpha-appendage of the adaptor protein complex 2 (AP2), which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV). The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.


Subject(s)
Colitis, Ulcerative/pathology , Endocytosis , Tacrolimus Binding Proteins/metabolism , Transcription Factor AP-2/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Cell Line , Cell Line, Tumor , Chromatography, Liquid/methods , Colorectal Neoplasms/pathology , Humans , Macrophages/metabolism , Mass Spectrometry/methods , Models, Biological , Proteins/metabolism , Proteomics/methods , Wiskott-Aldrich Syndrome Protein/metabolism
7.
Nature ; 462(7269): 58-64, 2009 Nov 05.
Article in English | MEDLINE | ID: mdl-19890323

ABSTRACT

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.


Subject(s)
Chromatin/genetics , Chromatin/metabolism , Estrogen Receptor alpha/metabolism , Genome, Human/genetics , Binding Sites , Cell Line , Chromatin Immunoprecipitation , Cross-Linking Reagents , Formaldehyde , Humans , Promoter Regions, Genetic/genetics , Protein Binding , Reproducibility of Results , Sequence Analysis, DNA , Transcription, Genetic , Transcriptional Activation
8.
J Biol Chem ; 283(47): 32977-88, 2008 Nov 21.
Article in English | MEDLINE | ID: mdl-18728018

ABSTRACT

Nuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans.


Subject(s)
Receptors, Estrogen/metabolism , Response Elements , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Cell Line, Tumor , Chromatin/chemistry , Enhancer Elements, Genetic , Estrogens/metabolism , Evolution, Molecular , Genome , Histones/chemistry , Humans , Molecular Sequence Data , Protein Binding , Sequence Homology, Nucleic Acid
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