Elucidating the events of transcription factor binding in human brain enhancers

Abstract

Background: The information responsible for animal diversity, complexity and embryonic patterning lies in non-coding regions of a genome. The non-coding portion of a human genome has become into a light now-a-days because of its regulatory aspects. Sequence acceleration in the human lineage has been found to harbour cis regulatory elements among them enhancer constitute the most portion play a major role in gene regulation These sequences of the human genome compared with other vertebrates identified genomic regions that were highly conserved among vertebrates but fast-evolving in the human lineage called Human accelerated regions (HARs). HARs are short conserved genomic portions that have attained considerably more nucleotide variations than would be predicted in the human lineage following the divergence from chimpanzee. The rapid evolution of HARs is the reflection of their crucial role in the emergence of human-specific attributes. Human accelerated genomic regions were mostly non-coding in nature, and upon subjection to in vivo testing, confirmed the presence of cis-regulatory enhancers which regulate the expression of numerous developmental genes. Recent study has reported the positively selected single nucleotide variants (SNVs) within brain exclusive human accelerated enhancers (BE-HAEs) hs563 (hindbrain), hs304 (midbrain/forebrain) and hs1210 (forebrain). Furthermore, these SNVs were revealed to be modern human-specific by incorporating data from archaic hominins, since they were residing within the transcriptional factors binding sites (TFBSs) for RUNX1/3 (hs563), FOS/JUND (hs304) and SOX2 (hs1210). Although these results imply that these predicted alterations in TFBSs are crucial for determining current brain structure, much more research is necessary to confirm whether these changes actually correspond to functional modification. Here, in order to empirically test the binding events and binding affinity of SOX2 protein with the modern human (derived A allele) and archaic hominin (ancestral T allele) carrying DNA, we employed electrophoretic mobility shift assay (EMSA). In further analysis, molecular docking was explored to analyze how binding domain (DBD) of SOX2 TF interacts with the respective TFBSs. Moreover, exploiting the dynamic binding features for modern human and archaic hominin (Neanderthal) alleles based complexes, all atoms biomolecular simulation was performed for the solvated systems using FF19SB force-field in AMBER20. Results: We investigated the SOX2 SNV, with strongest results of positive selection in human population that was relevant for forebrain expression. We demonstrated that the viii binding domain of SOX2 (HMG) binds in vitro with modern human-specific derived A-allele and ancestral T-allele carrying DNA sites. In further analysis, DNA-protein interaction studies indicated that HMG domain of SOX2 directly interacts with the minor groove of the derived A-allele as well with the ancestral T-allele type. Energetic profile evaluation through HADDOCK score calculations revealed higher affinity of SOX2 (HMG) for the derived A-allele carrying target DNA site (-281.6 ± 4.2 kcal/mol), whereas relatively lower affinity was noticed for the ancestral T-allele carrying target DNA site (-270.3 ± 4.0 kcal/mol). The interface analysis also demonstrated a better interface in the derived A-allele-SOX2 complex as compared to ancestral T-allele, as evident by energetics and the number of hydrogen bonds. Furthermore Simulation analysis indicated the significant dynamic behavior and binding differences of THE DNA binding domain of HMG box with derived A-allele containing DNA target site when compared to site carrying ancestral T-allele. We speculate that such changes in TF affinity within BE-HAEs hs1210 and other enhancers could accumulate during recent history of human evolution to produce modifications in gene expression with functional consequences during forebrain formation. Conclusion: Taken together with the combinations of in vitro and bioinformatics analysis to comparatively characterize the evolutionary significance of positively selected modern human specific substitution (T>A) within BE-HAEs hs1210. Our comparative molecular structural analysis showed that modern human-specific single nucleotide substitution has increased the affinity of SOX2 transcriptional factor for its target binding site within BE-HAEs hs1210. These findings suggest that this predicted enhanced affinity of SOX2 towards its target site could drive the target gene expression more robustly within forebrain of modern humans compared with the archaic humans or alternatively within novel territories in the forebrain of modern humans. These findings imply that adoptive changes in TF affinity within BE-HAE (hs1210) and other HAR enhancers may have altered gene expression patterns, which may have functional effects on the development and evolution of the forebrain.