RLS-associated MEIS transcription factors control distinct processes in human neural stem cells [RNA-Seq]

MEIS1 and MEIS2 encode highly conserved homeodomain transcription factors crucial for developmental processes in a wide range of tissues, including the brain. They can execute redundant functions when co-expressed in the same cell types, but their roles during early stages of neural differentiation have not been systematically compared. By separate knockout and overexpression of MEIS1 and MEIS2 in human neural stem cells, we find they control specific sets of target genes, associated with distinct biological processes. Integration of DNA binding sites with differential transcriptomics implicates MEIS1 to co-regulate gene expression by interaction with transcription factors of the SOX and FOX families. MEIS1 harbors the strongest risk factor for restless legs syndrome (RLS). Our data suggest that MEIS1 can directly regulate the RLS-associated genes NTNG1, MDGA1 and DACH1, constituting new approaches to study the elusive pathomechanism or RLS. To investigate individual target genes of MEIS1 and MEIS2, we performed separate CRISPR-activation an CRISPR/Cas9 knockout of MEIS1 and MEIS2 in cultured human neural stem cells (hNSC). RNA extraction from cryopreserved cells was performed with the RNeasy Kit (Qiagen). RNAs with a RIN value > 7 were selected for mRNA sequencing. The libraries were prepared using the TruSeq stranded mRNA Sample Preparation kit (Illumina), following the kit’s instructions. After a final QC, the libraries were sequenced in paired-end mode (2 × 100 bases) on the Novaseq6000 sequencer (Illumina) with a depth of ≥ 10 Million reads per sample. Reads were aligned to the human genome (hg19) using the STAR (v 2.4.2a) and were counted using HTSeq v0.6.0115. DESeq2 v1.34.0 was used for differential gene expression analyses.