An ENU mutagenesis screen identifies the first mouse mutants of a novel epigenetic modifier, Rearranged L-Myc Fusion (Rlf). S. K. Harten1, L. Bourke1, V. Bharti1, H. Oey2, N. Whitelaw1, L. Daxinger2, E. Whitelaw1,2 1) Genetics & Population Health, QIMR, Brisbane, QLD, Australia; 2) La Trobe Institute of Molecular Science, Melbourne, VIC, Australia.

   An ENU mutagenesis screen was established to identify novel genes involved in epigenetic reprogramming. The screen utilizes a transgenic GFP reporter gene, which is expressed in a variegated manner in erythrocytes and is highly sensitive to epigenetic changes. A gene discovery pipeline involving SNP arrays and whole exome sequencing has enabled identification of causative mutations in ~40 mutant lines. To date the screen has produced mouse mutants of both known modifiers of epigenetic state, such as Dnmt1 and Smarca5, and genes not previously implicated as epigenetic 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 is conserved between human and mouse and contains multiple widely-spaced zinc finger domains, however little else is known. Our studies revealed reduced body-weight and postnatal lethality in Rlf homozygous null mutants, indicating that Rlf is critical for proper development. Histological analysis of mid-gestation Rlf mutant embryos revealed 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 both within and outside of CpG islands. The functional effects of non-CpG island methylation, both on gene expression and development are also being investigated.

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