HUMAN-SPECIFIC EVOLUTION OF THE TRANSCRIPTIONAL REGULATION OF FOXP2

ALFREDO LEANDRO CAPORALE; FRANCHINI, LUCIA FLORENCIA

Conferencia; EVOLANG; 2018

IntroductionUnique human features such as the use of a complex language are considered to be the result of a series of molecular and phenotypic changes in the developmental program of our brain that occurred in the last 6 million years of human lineage evolution (Pääbo 2014). Due to the increasing availability of different primate genomes, genomic comparative analysis have estimated that the identity of sequence between humans and our closest living primate relative, the chimpanzee (Pan troglodytes) could reach 95-99%, and most of the divergence occurs over non-coding regions (Franchini and Pollard 2015). Functional non-coding regions such as promoters, enhancers and introns can differentially regulate the expression of genes and thus play a role in human evolution. Using public databases of human accelerated elements (HAEs) (Pollard et al. 2006; Prabhakar et al. 2006; Bird et al. 2007; Bush and Lahn 2008; Lindblad-Toh et al. 2011), we found that the locus of FOXP2 gene contains 6 HAEs, being one of the genes that shows one of the fastest acceleration rates in the human genome. This transcription factor has been shown to be essential for normal development of human language, and it has been extensively studied in human evolution (Scharff and Petri 2011; Deriziotis and Fisher 2017), but its transcriptional regulation is poorly understood (Maricic et al. 2013). Methods & ResultsIn this work, we characterized potential enhancers of FOXP2 by using in vivo transcriptional enhancer assay in zebrafish (Danio rerio), a useful vertebrate model for such analysis and successfully used in our lab (Kamm et al. 2013). Through this approach, we functionally and comparatively tested the activity of each FOXP2-HAE orthologs of the human and chimpanzee loci.We have found differences between some orthologs in the expression of the reporter protein during developmental stages of transgenic fishes (24, 48 and 72hpf), such as extended expression patterns in the nervous system. The human-specific changes fixed in these elements can mean gain or loss of transcription factor binding sites (TFBS), which in turn could drive FOXP2 expression to novel territories and thus acquire new regulatory functions. Therefore, we analyzed bioinformatically putative TFBS contained in HAEs and differentially represented. We found out some candidates like POU6F1 and MYT1 which are transcription factors that play a role in brain development and they are expressed in zebrafish developing brain (Johansen et al. 1993; Lopez et al. 2016), hence they would be responsible for the extended pattern in the human sequences.Furthermore, we have validated the expression of the endogenous foxP2 protein in zebrafish. This has allowed us to perform double immunohistochemistry assay and colocalize the reporter with foxP2 in the transgenic fishes. We have found colocalization only in the human transgenic fishes in cerebral areas like diencephalon and cerebellum which are relevant for the control of the orofacial movements required for speech.Discussion & ConclusionWe characterized enhancers located in the locus of FOXP2 using an in vivo approach that colocalize the reporter with the endogenous foxP2 protein in zebrafish, a suitable model to rapidly test activity of non-coding elements during early stages of development (Fisher et al. 2006). We will select those sequences that show differences of expression for a similar analysis using transgenic mice, a mammalian model with homologous brain structures to those present in primates, but more laboriously for working at lab.We expect that this analysis contributes to a better understanding of the evolutionary changes in the regulatory landscape of FOXP2, which seemingly contributed to refine the biological machinery supporting human speech and language.ReferencesBird CP, Stranger BE, Liu M, Thomas DJ, Ingle CE, Beazley C, Miller W, Hurles M, Dermitzakis ET. (2007). Fast-evolving noncoding sequences in the human genome. Genome Biology 8 (6): 118. Bush EC, Lahn BT. (2008). A genome-wide screen for noncoding elements important in primate evolution. BMC Evolutionary Biology: 8-17.Deriziotis P, Fisher SE. (2017). Speech and Language: Translating the Genome. Trends in Genetics 33, (9): 642-656.Fisher S, Grice EA, Vinton RM, Bessling SL, Urasaki A, Kawakami K, McCallion AS. (2006). 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