Enhancer ID: | E_01_003 | |
Enhancer symbol: | NEC1 Enhancer | |
Species: | Human | |
Position : | chr2:223164458-223164688 |
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Biosample name: | Melanoma | |
Experiment class : | Low throughput |
Enhancer type: | Enhancer | |
Disease: | Melanoma | |
DO: | DOID:1909 | |
Mesh: | D008545 | |
Distance from TSS: | >2KB | |
Pubmed ID: | 26252164 |
Enhancer experiment: | Phylogenetic Footprinting,Luciferase Reporter Assay | |
Enhancer experiment description: | Through phylogenic footprinting and in silico promoter analys islands of homology within the proximal 1.6 Kb PAX3 promo referred to as either NCE1 and NCE2 ([Milewski et al., 2004], shown schematically and by sequence in Fig. 4A,B) or sites I and II [Natoli et al., 1997]. Two FOX long sites were identified in the PAX3 promoter, one in NCE1 (F1) and another in NCE2 (F2). To determine if these FOXD3 sites are active in melanoma cells, PAX3pm constructs containing intact or mutated F1 and F2 sites were transfected into A375 and SKMEL-28 melanoma cell lines (Fig. 6C,D). While mutation of the F1 site reduced reporter expression to 78.5% ±18.6% (A375) and 82.1% ±19.4% (SKMEL-28) of the PAX3pm levels, this reduction was not significant. However, if the F2 or both F1 and F2 sites were mutated, the reporter levels dropped to 51.1% ±13.8% and 45.6% ±11.6% for A375 cells and 32.4% ±9.8% and 37.5% ±10.2% of the PAX3pm vector levels, respectively. This supports that FOXD3 can drive PAX3 expression from both the F1 and F2 enhancers, but the F2 enhancer is the site utilized in melanoma cells. |
Target gene : | PAX3(CDHS,HUP2,WS1,WS3) | |
Strong evidence: | -- | |
Less strong evidence: | Luciferase Reporter Assay | |
Target gene experiment description: | PAX3pm constructs with or without mutated F1 and/or F2 sites were also transfected into 293T cells in the presence or absence of a FOXD3 expression vector (Fig. 5D). FOXD3 was able to drive reporter expression at significant levels when either or both sites were present in comparison to promoter vector alone. However, when both sites were mutated, the ability of FOXD3 to drive this construct was attenuated. |
TF name : | FOXD3(AIS1,Genesis,HFH2,VAMAS2) | |
TF experiment: | EMSA | |
TF experiment description: | EMSA analysis for FOXD3 binding of PAX3 promoter sequences in vitro. Labeled probe containing the PAX3 promoter F1 site (lanes 1-3) or F2 site (lanes 4-6) were mixed with 293T lysate without (lanes 1, 4) or with (lanes 2, 3, 5, 6) exogenous FOXD3 protein. Binding of the labeled probe was inhibited with cold probe competition (lanes 3, 6). |
Enhancer function : | The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. |
Enhancer function experiment: | ChIP,PCR |
Enhancer function experiment description: |
The PAX3 gene contains two FOX binding motifs within highly conserved Enhancer regulatory elements that are essential for neural crest development. |
SNP ID: | -- | |
SNP position: | -- |
SNP experiment: | -- |
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