The Impact of Whole GenomeIn SilicoScreening for Nuclear Receptor‐Binding Sites in Systems Biology

Deciphering the calcitriol-induced transcriptomic response in keratinocytes: presentation of novel target genes

“…Definite recombinant VDR-DBD binding to (bipartite) DR3 repeats could be confirmed for three out of five randomly selected candidates genes and thereby provides us with both a level of further validation and a putative mechanism of regulation. Nevertheless, definite VDRE uncovering does not signify unambiguous response element functioning in vivo, as VDRE may be covered by nucleosomes (especially elements in locations isolated from transcription factor 'modules'), while maintenance of responsiveness may, on the other hand, not demand strict binding site conservation (Toell et al 2000, Carlberg & Seuter 2009). …”

“…Because we measured changes in gene expression at only one concentration of calcitriol at a single time point for microarray analysis and reevaluated only 78 of the 86 original candidates derived from the whole-transcriptome microarray chip at two concentrations, our confirmed list of regulated genes likely reflects an underestimate of the total number of calcitriol targets in primary keratinocytes. Lastly, our RTq-PCR-evaluated data have been complemented by literature-inspired , Carlberg & Heinäniemi 2010, weighted nucleotide distribution matrix-based predictions that assign (near) consensus nuclear hormone receptor superfamily response elements (Cartharius et al 2005). As the affinity of a monomeric VDR molecule to a single-core binding motif is not sufficient to allow optimal DNA binding, transcriptional control of primary calcitriol targets is preferentially achieved by association of VDR-RXR heterodimers to the major groove of classical DR3-type (i.e.…”

“…These obvious disparities might, to a certain extent, be due to the fact that early efforts often lacked biological sample replication and statistical normalization necessary to eliminate false-positive artifacts. Because of cell-type and tissue-specific phenomena -tumor cells might exhibit reduced vitamin D responses, for instance, by displaying altered nuclear corepressor levels (Khanim et al 2004) -or transient responses , Carlberg & Seuter 2009, Carlberg 2010), these parameters not only complicate direct mutual comparative assessments but might explain the limited agreement between the resultant catalogs of experimentally detected calcitriol targets (Tavera-Mendoza et al 2006). Nonetheless, from the published studies, the number of reliable (primary) response genes can be estimated to range somewhere between 250 (Palmer et al 2003, Carlberg 2010) and 1000 (Lin et al 2002), not taking into account the plethora of secondary, delayed constituents that might contribute to the observable broad physiological actions.…”

“…Together, with complementary chromatin immunoprecipitation (ChIP and sophisticated modifications developed thereof, such as genome-wide ChIPchip hybridization of resultant chromatin fragments on microarrays or massively parallel ChIP-seq sequencings) and in silico screens for VDREs, they have largely rationalized the entirety of nuclear hormone receptor superfamily research (Tavera-Mendoza et al 2006, Carlberg & Seuter 2009, Carlberg & Heinäniemi 2010, which previously relied on the transfection of 'indirect' reporter gene chimeras (Lemon & Freedman 1996). To date, in-depth deciphering of calcitriol-triggered transcriptional changes has been achieved in both primary cells and established (tumor) cell lines derived from multiple tissues.…”