Transcription factor binding site analysis

The activity of the Hedgehog (Hh) signaling pathway is primarily mediated through the Gli1,2 and 3 transcription factors. To look for Gli target genes that could be under the control of the Hh pathway in retina, we selected a number of murine genes with consensus Gli binding sites conserved in human and that were expressed in the central nervous system according to the EST database. Out of 390 candidates, 46 were examined and 30 were were modulated by Hh pathway activation in E14.5 and P0.5 retinal explants. Gli2 was verified to bind to the Sox8 promoter depending on Hh-pathway activation [1].

The NF-kappa-B family of transcription factors (TFs) is composed of the members RelA (p65), RelB, c-Rel and the precursor proteins p105 and p100, which are processed to p50 and p52, respectively. These proteins can form various homo- and heterodimers, which regulate gene expression by binding to kappa-B sites in the promoters and enhancers of genes in a variety of mammalian cell types. We examined the DNA-binding sites of RelA, RelB, p50 and p52 in HL cells using ChIP-seq and complemented these results with analysis of differential gene expression by microarrays of knock downs of the genes encoding the respectively tested TFs [2]. HL cells were used because these have the Nf-kappa-B pathway constitutively active and thus constitute a good model for the study of the gene regulatory effects of the pathway. DNA binding patterns were largely inter-related: RelA shared more than 94% and RelB more than 85% of their target regions with at least one other NF-kappa-B subunit. Binding regions for p50 and p52 overlapped by about 70%. p50 or p52 were uniquely binding 41% of all regions. De novo searches for motifs in binding sites indicated that p50 has a higher conservation of a G in the first position compared to p52-selective motif. Analysis of binding sites for other TFs indicated co-occurrence with motifs for AP-1, IRF, ETS, FOX and CTCF. Gene expression effects of DNA depletion were tested for pairs p50-RelA and p52-RelB indicating that they inhibit the intrinsic (mitochondrial driven) and extrinsic (receptor driven) apoptosis pathways, respectively.



[1] McNeill, B., C. Perez-Iratxeta, C. Mazerolle, M. Furimsky, Y. Mishina, M.A. Andrade-Navarro, V.A. Wallace. 2012. Comparative genomics identification of a novel set of temporally regulated Hedgehog target genes in the retina. Molecular and Cellular Neuroscience. 49, 333-340.

[2] de Oliveira, K.A.P., E. Kaergel, M. Heinig, J.F. Fontaine, G. Patone, E.M. Muro, S. Mathas, M. Hummel, M.A. Andrade-Navarro, N. Hübner, and C. Scheidereit. 2016. A roadmap of constitutive NF-κB activity in Hodgkin lymphoma: dominant roles of p50 and p52 revealed by genome-wide analyses. Genome Medicine. In press.