Non-coding genetic regions of accelerated human evolutionary change; relationships of human specific traits to alterations in development

FRANCHINI L.F.

Conferencia; NIH-NIA Comparative Primate Workshop; 2021

NIH

Non-coding genetic regions of accelerated human evolutionary change; relationships of human specific traits to alterations in developmentLucía F. FranchiniInstituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
email: franchini.lucia@gmail.com; franchini@dna.ubar.arIt has been proposed that the phenotypic differences in cognitive abilities among modern and archaic humans and also with our closest living relatives, the chimpanzees, are largely due to changes in the regulation of genes involved in the development and function of the brain. To investigate this hypothesis, in our lab we study noncoding conserved regions that underwent accelerated evolution in the human lineage and have been named Human Accelerated Regions (HARs). These sequences have been identified using several bioinformatics approaches and constitute a rich dataset to investigate the impact of human specific evolution on gene regulatory function. We have mapped HARs in the human genome and found that they are not distributed randomly but they accumulate in particular genes and genomic territories. We have identified the gene Neuronal PAS Domain Protein 3 (NPAS3) as the one that accumulates the largest number of HAR in its transcriptional unit. We consider the transcriptional unit as the genomic region contained between the transcription start to the transcription end and including introns. NPAS3 encodes a transcription factor of the bHLH-PAS family that is widely expressed in the developing nervous system of vertebrates, and its dysfunction has been associated with the etiology of schizophrenia and bipolar disorder in humans. Thus NPAS3 is an ideal candidate to study human-specific gene regulation, evolution and function. We have functionally characterized the 14 NPAS3-HARs as regulatory regions through a transgenic zebrafish enhancer assay comparing the function of the human and the chimpanzee version of each sequence. Through this approach we have identified at least three HARs that lost or gained function in the human lineage. We have particularly focused on HAR202, since its human version displays a loss of function compared to the chimpanzee version of the sequence. Moreover, we found a lack of reporter EGFP expression in the brains of fishes carrying the Homo sapiens version of HAR202, while the rest of vertebrate orthologs tested (chimpanzee, macaque, mouse, chicken and zebrafish) showed strong expression in this organ. Remarkably, we also found that the HAR202 Homo neanderthalensis ortholog sequence, displaying just one substitution compared to the H. sapiens, showed also strong expression in the brain. We also observed this modern human-specific loss of activity in mouse transgenic reporter assays, comparing human and neanderthal HAR202 enhancer activity. Our results suggest that the HAR202 element lost its enhancer function constituting one of the few examples of a HAR that displays functional evolution in the brain as a result of the fast molecular evolution process undergone in the H. sapiens lineage. Furthermore, investigating other gene regions that accumulate exceptionally high numbers of HARs, we found that the topologically associating domain (TAD) determined using developing human cerebral cortex containing the FOXP2 locus includes two clusters of 12 HARs, placing the locus occupied by FOXP2 (forkhead box P2) among the top regions showing fast acceleration rates in non-coding regions in the human genome. FOXP2/foxp2 encodes a transcription factor that is highly conserved among vertebrates and that is widely expressed in the nervous system and other organs and tissues throughout development and in adults. FOXP2 has been linked to the ability of spoken language in humans since it was discovered that mutations affecting this gene in a large family impacted directly on the acquisition of speech. Moreover, this gene has been associated to the evolution of language in humans because evolutionary studies found that human FOXP2 displays two amino acid changes compared to our closest living relatives chimpanzees, gorillas and rhesus macaques and it has been suggested that this gene underwent positive selection in the human lineage. Using in vivo enhancer assays in zebrafish, we found that at least five FOXP2-HARs behave as transcriptional enhancers throughout different developmental stages. Moreover, we uncovered two FOXP2-HARs showing reporter expression gain of function in the nervous system when compared with the chimpanzee ortholog sequences. In addition, we found that these FOXP2-HARs direct the expression of the reporter gene EGFP to foxP2 expressing regions and cells. Our results indicate that regulatory sequences in the FOXP2 locus underwent a human-specific evolutionary process suggesting that the transcriptional machinery controlling this gene could have also evolved differentially in the human lineage. In summary, we found several HARs linked to NPAS3 and FOXP2 displaying functional changes in comparative studies. These HARs constitute a basement for more complex analysis in mammalian models to identify the phenotypic impact they could have had in the human brain. What is the importance of our work? First, understanding our species origins and also shedding light on the genetic mechanisms underlying morphological evolution through the evolution of gene regulation. Additionally, since the genes involved in human brain evolution that we study are also involved in human specific mental diseases such as bipolar disorder, schizophrenia, autism, etc, we expect that our studies will help to better know these genes function and thus to illuminate our understanding of complex mental diseases.