This SNP is located in a putative transcription factor 1 (TCF-1) binding site upstream of the transcription start site and may potentially be a functional SNP.
The canonical Wnt-signaling pathway involves the transcription factors T-cell factor 1 (TCF-1) and lymphoid enhancer-binding factor 1 (LEF-1), which reside in the nucleus where they are bound to Wnt-response elements.
Recently, T-cell factor 1 (TCF-1; also known as transcription factor 7, T-cell specific, TCF7) was identified as a messenger that transmit the commands from Notch signal to downstream transcriptional factor like Gata3 and Bcl11b during T cell specification and differentiation [ 76].
Analysis of ISP progression parameters showed normal TCR and IL-7R α/CD127 levels but a moderate decrease in the expression of LEF-1, TCF and ROR γ transcription factors, essential for ISP conversion to DP.
The key effector of the Wnt signaling pathway is the bipartite transcription factor β-cat/TCF, formed by free β-cat and a member of the TCF family [TCF-1/TCF7, LEF-1, TCF-3/TCF7L1 and TCF-4/TCF7L2].
TOP/FOPflash (T-cell factor-lymphoid enhancer factor-1 (TCF-LEF) reporter plasmid) was from Upstate (Lake Plasid, NY).
In "canonical" pathways, Wnt signaling is mediated by POP-1/TCF and β-catenin, which acts as a transcriptional coactivator for TCF.
Twenty-four of the investigated genes in the Notch pathway were found to contain one or several putative LEF-1/TCF sites (Fig. 1 and Supplemental Information S1).
In the present study, in silico analysis of Notch and Wnt pathway gene promoters resulted in the identification of several putative β-catenin regulated LEF-1/TCF-sites in Notch pathway gene promoters and RBP-Jκ-sites in Wnt pathway gene promoters (Fig. 1 and Supplemental Information S1).
In order to develop the in silico and DNA binding analyses and functionally investigate the effects of activated Wnt signaling on Notch pathway genes, nineteen genes in the Notch pathway, known to be important for canonical Notch signaling (Fig. 1 and Table S1), containing putative LEF-1/TCF-sites, were selected for further studies with semi-quantitative RT-PCR.
To investigate binding of LEF-1/TCF-family members Lef-1 and Tcf-4 to Notch-2 LEF-1/TCF-site −110, chromatin immunoprecipitation (ChIP) assays were carried out using a ChIP Assay Kit (Millipore).
