Functional genetic characterization by CRISPR-Cas9 of two enhancers ofFOXP2in a child with speech and language impairment

Abstract

Mutations in the coding region of FOXP2 are known to cause speech and language impairment.Microdeletions involving the region downstream the gene have been also associated to speech and cognitive deficits. We recently described a girl harbouring a complex chromosomal rearrangement with one breakpoint downstream the gene that might affect their speech and cognitive abilities via physical separation of distant regulatory DNA elements. In this study, we have used highly efficient targeted chromosomal deletions induced by the CRISPR/Cas9 genome editing tool to demonstrate the functionality of two enhancers (FOXP2-E proximal and FOXP2-E distal ) located in the intergenic region between FOXP2 and its adjacent MDFIC gene. Deletion of any of these two functional enhancers in the neuroblastomic cell line SK-N-MC downregulates FOXP2 and decreases FOXP2 protein levels, conversely it upregulates MDFIC and increases MDFIC protein levels. This suggests that both regulatory elements may be shared between FOXP2 and MDFIC. We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting speech and language. KEYWORDSFOXP2, MDFIC, speech and language impairment, Spanish, CRISP-Cas9, functional enhancer

The untold stories of the speech gene, the FOXP2 cancer gene

“…• Two promoters telomeric to the most downstream transcript were identified and functionally validated [ 37 ].…”

“…A recent genomic analysis reports the presence of two additional regulatory elements with enhancer function in the telomeric territory separating FOXP2 from its neighbor, MDF1C [ 37 ]. These enhancers have been observed to be disrupted in a child with language and speech disorder.…”

“…These enhancers have been observed to be disrupted in a child with language and speech disorder. The requirement for these two elements in driving proper FOXP2 expression levels was functionally validated in human cell lines [ 37 ]. These data lend support to the hypothesis that FOXP2 expression falls under a large array of regulatory elements, which may increase the probability of dysregulation during oncogenic processes.…”

No Evidence for Recent Selection at FOXP2 among Diverse Human Populations

“…Any modified function of the ROI does not appear to be related to language, however, as modern southern African populations tolerate high minor allele frequencies with no apparent consequences to language faculty. We do not dispute the extensive functional evidence supporting FOXP2 ’s important role in the neurological processes related to language production (Lai et al, 2001; MacDermot et al, 2005; Torres-Ruiz et al, 2016). However, we show here that recent natural selection in the ancestral Homo sapiens population cannot be attributed to the FOXP2 locus and thus Homo sapiens’ development of spoken language.…”

What Lenneberg Got Right: A Homological Program for the Study of Language Evolution

“…Progress has also been made in the identification of genes targeted by FOXP2 during the corresponding developmental loop, which reasonably enough contribute either to keep it active or to realize the form/function specificities of the resulting human characteristic circuitry (Johnson et al 2009, Konopka et al 2009, Vernes & Fisher 2009, Roll et al 2010, Konopka et al 2012, Ayub et al 2013, Chiu et al 2014, Rodenas-Cuadrado et al 2014, Webb 2015. Finally, important insights have been gained with regard to enhancers and protein regulators-for example, SUMO proteins-of FOXP2's own activity (Bonkowsky et al 2008, Becker et al 2015, Estruch et al 2006, Becker 2016, Torres-Ruiz et al 2016, Usui et al 2016.…”