Divergence in KRAB zinc finger proteins is associated with pluripotency spectrum in mESCs

Noncoding regulatory elements coordinate cell type specific gene expression programs. The majority of disease-associated variants are concentrated in noncoding regulatory elements; however how genetic variation impacts regulatory variation is poorly understood. To begin to address this we developed a cellular systems genetics approach using mouse embryonic stem cells (mESCs). We derived a mapping panel of mESCs from 33 BXD recombinant inbred lines originating from a cross between C57BL/6J (B6) and DBA/2J (D2) mouse strains; which differ in their ability to acquire and maintain naïve pluripotency. Measuring chromatin accessibility (ca) and gene expression (e) in in this mapping population maintained in naïve pluripotency, we identified large-scale cis and trans quantitative trait loci (QTL). Many eQTL and caQTL map to six major distal loci, indicating a common regulatory system driving changes in chromatin and gene expression in trans. One prominent distal QTL on chromosome 13 (Chr 13) has previously been associated with multiple developmental and disease-related phenotypes including auto-immune disorders, craniofacial abnormalities, and limb development. In mESCs, the Chr 13 locus distally controls differential expression of Obox6 (Chr 7), a gene that supports pluripotency, resulting in high expression when B6 at the QTL and repressed when D2. Using (B6xD2) F1 hybrids and a mouse strain made congenic for the Chr 13 QTL, we confirmed the D2 allele represses distal targets, including Obox6. Interestingly, the Chr 13 QTL contains a cluster of genes encoding KRAB zinc finger proteins (KZFPs), which function to repress chromatin. TRIM28, a KZFP co-factor, preferentially binds chromatin at the Chr 13 QTL target sites in D2, including an Obox6 enhancer, and importantly, lacks binding in B6. This newly emergent Chr 13 KZFP cluster is hyper variable across common inbred strains suggesting rapid evolution. We find that B6, D2, and C57BL/6NJ (NJ) genomes have a ~1Mb structural variant within the QTL. A deletion in NJ and duplication in D2 encompasses a KZFP identified by mediation analysis as the best candidate for Obox6 regulation. Subsequently, chromatin accessibility at an upstream enhancer and expression of Obox6 are consistent with an allelic series of copy number variation at Chr 13. Current efforts are focused on confirming the candidate KZFP, determining the role of Chr 13 on 3D genome organization, and testing a functional role of Chr 13 QTL on cell state transitions.