Structural variation at the ZNF558 locus controls a gene regulatory network in human brain development

The human forebrain has expanded in size and complexitycompared to that of chimpanzeedespite limited changesinprotein-coding genes, suggesting that gene regulation is an importantdriver of brain evolution. Here we identify a KRAB-ZFPtranscription factor, ZNF558, that isexpressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved asa suppressor of LINE-1 transposons but has been co-opted to regulate the mitophagy geneSPATA18, supporting a link between mitochondrial homeostasis andcorticalexpansion. Theunusual on-off switchforZNF558expression resides in a downstream variable number tandemrepeat (VNTR) that is contracted in humans relative to chimpanzee.Our data reveal the brain-specific co-option of a transposon-controllingKRAB-ZFPand how a human-specificregulatory network is established by acis-acting structural genome variation. Thisrepresentsapreviouslyundescribedgenetic mechanisminthe evolution of the human brain. Overall design: In order to silence the transcription of ZNF558 we used the catalytically inactive Cas9 (deadCas9) fused to the transcriptional repressor KRAB. Single guide sequences were designed to recognize DNA regions just down-stream of the transcription start site (TSS) according to the GPP Portal (Broad Institute). The guides were inserted into a deadCas9-KRAB-T2A-GFP lentiviral backbone containing both the guide RNA under the U6 promoter and dead-Cas9-KRAB and GFP under the Ubiquitin C promoter (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP, a gift from Charles Gersbach, Addgene plasmid #71237 RRID:Addgene_71237). The guides were inserted into the backbone using annealed oligos and the BsmBI cloning site. Lentiviruses were produced as described below yielding titers between 4.9E+08 and 9.3E+09. Control virus with a gRNA sequence not present in the human genome (LacZ) was also produced and used in all experiments. All lentiviral vectors were used with an MOI between 5 and 20. (GSM5525367-GSM5525378) H6 and H48 are different cell lines. They are HS1 and HS2 (mentioned in the paper), respectively. bulk and single-cell RNA-seq and CUT&RUN