An ENU mutagenesis screen to identify novel epigenetic modifiers was established in mice carrying a GFP transgene, which is expressed in a variegated manner in erythrocytes and is highly sensitive to epigenetic silencing1,2 . A gene discovery pipeline involving SNP arrays and whole exome sequencing allowed the rapid identification of causative mutations in ~40 mutant lines. The screen has produced mouse mutants of both known modifiers of epigenetic state, such as Dnmt1 and Smarca52 , and novel modifiers, such as Re-arranged L-Myc fusion (Rlf).
Here we report three independent lines with mutations in Rlf. Each line shows a reduced percentage of GFP expressing cells compared to wild-types. Homozygous Rlf mutants show increased methylation at the transgene and haploinsufficiency for Rlf alters transcriptional silencing at Agouti Viable Yellow (Avy), an independent endogenous epiallele. Taken together, these findings suggest that Rlf is a modifier of epigenetic state. Rlf encodes a protein containing multiple widely-spaced zinc finger domains, about which very little is known. Homozygous Rlf null mutants weigh less than wild-types, and show postnatal lethality, indicating that Rlf is critical for proper development. Initial histology suggests the presence of a heart defect. Analysis of RNA-Seq data, comparing RNA from wild-type and Rlf mutant fetal livers, showed differential expression of genes involved in metabolism. Levels of 4-hydroxyphenylpyruvic acid dioxygenase (HPD), an enzyme involved in tyrosine catabolism, were markedly reduced. Genetic deficiency of HPD underlies Tyrosinemia type 3 in humans and is associated with mental retardation and ataxia. Genome-wide bisulphite sequencing studies are underway to examine the role of Rlf in the control of DNA methylation.