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Bioinformatic analysis of upstream regulatory regions identified conserved modules of putative transcription factor binding sites shared by genes expressed in the same trophoblast subtype.
We used the bioinformatics software program Toucan2 [ 37] to identify groups, or modules, of putative transcription factor binding sites present in multiple promoter sequences from genes co-expressed in a particular trophoblast subtype, rather than a single promoter in isolation.
Our comprehensive expression data set allowed us for the first time to try such an in silico approach and we compared the promoters of genes co-expressed in the poorly characterized glycogen trophoblast and sinusoidal TGC populations as examples, identifying several modules of putative transcription factor binding sites common to the promoters of each group.
Bioinformatic analysis of upstream regulatory regions identified conserved combinations (modules) of putative transcription factor binding sites shared by genes expressed in the same trophoblast subtype, supporting the notion that local regulatory elements, rather than locus control regions, specify subtype-specific expression.
We then used the Toucan2 bioinformatics program [ 37] to examine the upstream regulatory regions (~3000 nt) of genes co-expressed within trophoblast subtypes in an attempt to identify conserved groups, or modules, of putative transcription factor binding sites involved in subtype-specific expression.
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The in silico analysis carried out in this study showed that promoters of almost all the sHsp genes harbor minimum one module of putative HSE.
Striking examples of putative modules include the 6 cis-element module AP2F EGRF HESF MAZF SP1F ZBPF in genes highly expressed in thymus; the 6 cis-element module E2FF EBOX ETSF MAZF SP1F ZFSF in genes highly expressed in activated T-cells; and the 6 element module AP2F CDEF EGRF SP1F ZBPF ZF5F in genes highly expressed in stimulated lymph nodes (Table 3).
Many of the approaches listed above only generate lists of putative modules but lack the ability to render sophisticated figures.
In order to estimate significance of putative modules predicted function for Gene Ontology [ 27] annotations, Fishers test was applied.
Joung et al. [ 13, 14] discovered miRNA-mRNA modules using a combination of putative miRNA-mRNA pairs and expression data; however, correlations between the expression of miRNAs and mRNAs were not considered.
Based on these properties, previously developed computational methods have shown some success in identifying motifs and motif modules in a group of putative co-regulated genes as well as on a genome-wide scale.
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