Abstract: Background: Intervening sequences (introns) have significant effects on genomic regulations and molecular evolution. So, it deserves a deeper analysis for better understanding the possible regulato-ry roles of these regions. Objective and Method: Accordingly, the intron 2 (In-2) of the human B-cell lymphoma 2 (hBcl2) gene, with regard to the size of the In-2 as well as critical roles of the gene in the homeostatic of the cellular balance, was analyzed by using in-silico approaches to identify In-2 transcription factor binding (In2-TFBs) motifs. Results: Our analysis revealed 966 motifs of 118 different TFBs types which were scattered throughout both the strands of the complete sequence of the gene, in particular on the In-2, with significant pattern of distribution and repetition. Distribution pattern of these motifs revealed that most of them were ac-cumulated in narrow regions of the In-2, far from the area of the splicing sites. Moreover, it was ob-served that except for WT1-TFBs, Gfi-1-TFBs, GAGA-TFBs, all other motifs were sporadic, with ir-regular and random distribution. Among these motifs, WT1-TFBs showed the highest frequencies which were situated in four neighboring regions of the In-2, by a close linear relationship to Sp1-TFBs. Furthermore, the sequence logos of the WT1-TFBs showed that they ranged in size from 22 up to 45 bps and were enriched with G and T nucleotides. Meanwhile, the binding affinity of WT1-TF to WT1-TFBs revealed significant differences compared to the other sequences of the gene as negative control. Conclusion: In general, this data provides supporting evidences for the existence of regulatory regions in the intronic sequences of the hBcl2 gene especially in the In-2, and also represents new targets for WT1-TF which might contribute to hBcl2 regulation and apoptosis process.

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