Identification of TFAP2A and MITF binding sites in the melanoma cell line SK-MEL-28
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ABSTRACT:
PROVIDER: PRJNA641275 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12073342_1.fastq.gz | Fastqsanger.gz | |||
| SRR12073342_2.fastq.gz | Fastqsanger.gz | |||
| SRR12073350_1.fastq.gz | Fastqsanger.gz | |||
| SRR12073350_2.fastq.gz | Fastqsanger.gz | |||
| SRR12073351_1.fastq.gz | Fastqsanger.gz |
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Project description:TFAP2A and MITF binding sites were identified in SK-MEL-28 cell lines using Cleavage Under Targets and Release Using Nuclease (CUT&RUN).
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Project description:In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that Tfap2 facilitates MITF's ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express Tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the latter necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells.
2022-04-26 | GSE190610 | GEO
Project description:Though mitogen activated protein kinase kinases (MKK or MEK) 1 and 2 are widely assumed to be functionally redundant some reports indicate they possess distinct biologic activities. To test the hypothesis that MEK1 and MEK2 signaling pathways are interchangeable we used two complementary approaches to determine the necessity and sufficiency of individual MEK1 and MEK2 signaling pathways for human melanoma SK-MEL-28 cell proliferation. To test the necessity we targeted MEK1 and/or MEK2 using specific siRNAs. An effect on proliferation was observed only when both MEK1 and MEK2 were knocked down indicating that neither of the individual MEK isoforms is necessary for SK-MEL-28 cell proliferation. To test the sufficiency we inhibited multiple MEK and MKK signaling pathways in SK-MEL-28 cells with anthrax lethal toxin (LeTx) a MEK/MKK-specific protease and rescued individual MEK signaling pathways by expressing a cleavage-resistant form of MEK (MEKcr). In this fashion ERK activation was retained only in MEK2cr-expressing cells but not in MEK1cr-expressing cells following LeTx treatment. Microarray analysis revealed groups of non-overlapping downstream transcriptional targets of MEK1 and MEK2 and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results not only indicate that in this cellular context MEK2 signaling pathway alone is sufficient for ERK activation melanoma cell proliferation and anchorage-independent growth but MEK1 is not but also demonstrate that MEK1 and MEK2 signaling pathways are not redundant and interchangeable for melanoma cell proliferation. We conclude that while MEK2 alone is sufficient for SK-MEL-28 cell proliferation MEK1 can conditionally compensate for loss of MEK2. SK-MEL-28 melanoma cells +/- cleavage resistant MKK1/MKK2
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Project description:Melanoma tumor antigen p97 or melanotransferrin (MTf) is an iron (Fe)-binding protein with high homology to serum transferrin. MTf is expressed at very low levels in normal tissues and in high amounts in melanoma cells. The over-expression of MTf in tumor cells was hypothesized to assist rapidly proliferating neoplastic cells with their increased Fe requirements. However, our recent characterization of the MTf knockout (MTf -/-) mouse demonstrated that MTf did not have an essential role in Fe metabolism. To understand the function of MTf, we utilized whole-genome microarray analysis to examine the gene expression profile of five models after modulating MTf expression. These models included two new stably transfected MTf hyper-expression models (SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type (SK-Mel-28 melanoma) where MTf was down-regulated by post-transcriptional gene silencing. These findings were compared to alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the gene array data were also assessed in a new model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased cellular proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and up-regulation, we identified three genes modulated by MTf expression. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and transcription factor 4 (Tcf4) were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and cell proliferation/survival, respectively. This study identifies novel molecular targets directly or indirectly regulated by MTf and potential pathways involved in its function. These molecular targets could be involved, at least in part, to the role of MTf in modulating proliferation. Experiment Overall Design: To prepare a construct capable of generating hairpin siRNA specific for human MTf mRNA, the expression vector, pSilencer⢠3.1-H1 neo (Ambion, Texas, USA) was used. The vector was used to clone transgene of 66-bp that transcribe 19-mer double-stranded hairpin RNAs of the target gene. The transgene were specifically targeted to positions 2031-2049-bp in the MTf gene (Genbank Accession: NM_005929). Transfections of pS-MTf transgenes or pS-scrambled vectors into human SK-Mel-28 melanoma cells were performed with LipofectamineTM 2000 reagent (Invitrogen, Melbourne, Australia). Total RNA was isolated from the cells using TRIzol Reagent® (Sigma-Aldrich) according to the manufacturerâs protocol. Total RNA from the stably-transfected SK-Mel-28 melanoma cell lines were prepared and hybridized onto Human Genome U133 Plus 2.0 Array. The human GeneChip® U133 Plus 2.0 consists of greater than 47,000 transcripts and variants from over 38,500 well characterized human genes (Affymetrix, Santa Clara, CA).
2008-06-15 | E-GEOD-6817 | biostudies-arrayexpress
Project description:Though mitogen activated protein kinase kinases (MKK or MEK) 1 and 2 are widely assumed to be functionally redundant some reports indicate they possess distinct biologic activities. To test the hypothesis that MEK1 and MEK2 signaling pathways are interchangeable we used two complementary approaches to determine the necessity and sufficiency of individual MEK1 and MEK2 signaling pathways for human melanoma SK-MEL-28 cell proliferation. To test the necessity we targeted MEK1 and/or MEK2 using specific siRNAs. An effect on proliferation was observed only when both MEK1 and MEK2 were knocked down indicating that neither of the individual MEK isoforms is necessary for SK-MEL-28 cell proliferation. To test the sufficiency we inhibited multiple MEK and MKK signaling pathways in SK-MEL-28 cells with anthrax lethal toxin (LeTx) a MEK/MKK-specific protease and rescued individual MEK signaling pathways by expressing a cleavage-resistant form of MEK (MEKcr). In this fashion ERK activation was retained only in MEK2cr-expressing cells but not in MEK1cr-expressing cells following LeTx treatment. Microarray analysis revealed groups of non-overlapping downstream transcriptional targets of MEK1 and MEK2 and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results not only indicate that in this cellular context MEK2 signaling pathway alone is sufficient for ERK activation melanoma cell proliferation and anchorage-independent growth but MEK1 is not but also demonstrate that MEK1 and MEK2 signaling pathways are not redundant and interchangeable for melanoma cell proliferation. We conclude that while MEK2 alone is sufficient for SK-MEL-28 cell proliferation MEK1 can conditionally compensate for loss of MEK2.
2011-06-24 | GSE23930 | GEO
Project description:We have deleted TFAP2A and its redundantly-acting paralog TFAP2C in human melanoma SkMel28 cell lines and are assessing MITF binding using Cleavage Under Targets and Release Using Nuclease (CUT&RUN). Conversely, we deleted all copies of MITF in SkMEL28 cells and are similarly profiling TFAP2A bound loci. We predict TFAP2A functions as a pioneer factor for MITF during melanocyte development and that in the absence of TFAP2A, MITF binding at loci normally co-bound by MITF/TFAP2A will be disrupted.
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2022-03-22 | PXD030206 | Pride