Transcriptional reprogramming of neural stem cells by a PHF6/EphR signalling pathway [RNA-seq]
The plant homeodomain zinc finger protein,PHF6, is a transcriptional regulator, with its germline mutations causing the X-linked intellectual disability (XLID), Bo¨rjeson-Forssman-Lehmann syndrome (BFLS).The precise mechanisms by whichPHF6regulates transcription and how its mutant forms lead to XLID remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex, most of which overlap (CA)n-microsatellite repeats near genes involved in central nervous system development and neurogenesis. Mice harbouring BFLS patient mutations exhibit defects in neurogenesis and altered neural stem cell fate. Mechanistically, we find that PHF6 regulatesa panel of Ephrin receptors (EphRs)and that PHF6regulation ofEphRis impaired in BFLS mice and ina conditionalPhf6knock-out mouse model. We report that mice harboring BFLS mutations exhibit an increase in embryonic neural stem cell (eNSC) self-renewal, a significant attenuation of newly born neurons, and thatEphRknockdown phenocopies the PHF6 loss-of-function defects in altering eNSC fate. Our results suggest that alterations in eNSC fate specification via a PHF6/EphRtranscriptional pathway leads to impaired neurogenesis in BFLS Overall design: RNA-seq profiling of cortical progenitors treated with siCtl or siPhf6. Cortical progenitors were established from the cortex and were maintained in culture for 5 days to expand the homogeneous population prior to submitting samples for mRNA-Seq. mRNA-seq raw reads were mapped to mm10 genome using HISAT2 (Kim, Langmead, & Salzberg, 2015), followed by duplicate read removal using samtools. Gene-level read counts were obtained by HTSeq (Anders, Pyl, & Huber, 2015), using gene annotations from GENCODE (release M9). Genes with a minimum of 150 reads in at least one sample were retained. Gene set analysis was performed using ConsensusPathDB (Kamburov et al., 2011).