Genetically regulated epigenetic transcriptional activation of retrotransposon insertion confers mouse dactylaplasia phenotype

Abstract

Dactylaplasia, characterized by missing central digital rays, is an inherited mouse limb malformation that depends on two genetic loci. The first locus, Dac, is an insertional mutation around the dactylin gene that is inherited as a semidominant trait. The second locus is an unlinked modifier, mdac/Mdac, that is polymorphic among inbred strains. Mdac dominantly suppresses the dactylaplasia phenotype in mice carrying Dac. However, little is known about either locus or the nature of their interaction. Here we show that Dac is a LTR retrotransposon insertion caused by the type D mouse endogenous provirus element (MusD). This insertion exhibits different epigenetic states and spatiotemporally expresses depending on the mdac/Mdac modifier background. In dactylaplasia mutants (Dac/؉ mdac/mdac), the LTRs of the insertion contained unmethylated CpGs and active chromatin. Furthermore, MusD elements expressed ectopically at the apical ectodermal ridge of limb buds, accompanying the dactylaplasia phenotype. On the other hand, in Dac mutants carrying Mdac (Dac/؉ Mdac/mdac), the 5 LTR of the insertion was heavily methylated and enriched with inactive chromatin, correlating with inhibition of the dactylaplasia phenotype. Ectopic expression was not observed in the presence of Mdac, which we refined to a 9.4-Mb region on mouse chromosome 13. We report a pathogenic mutation caused by MusD. Our findings indicate that ectopic expression from the MusD insertion correlates with the dactylaplasia phenotype and that Mdac acts as a defensive factor to protect the host genome from pathogenic MusD insertions.dactylin ͉ ectrodactyly ͉ LTR ͉ split hand/split foot malformation ͉ methylation D actylaplasia is an inherited mouse limb malformation that is characterized by missing central digital rays. The Dac mutation is inherited as a semidominant trait, evidenced by missing central digits in the fore-and hindlimbs of heterozygous mice and monodactyly in homozygous mice (1, 2). The Dac locus has been mapped to the distal end of chromosome 19. Two independent Dac mutant alleles, Dac 1J and Dac 2J , arose spontaneously in breeding colonies. Both are insertions residing within the same locus: Dac 1J is located in the region upstream of the dactylin gene, and Dac 2J is located in intron 5 of dactylin (3). Southern blot analysis indicated that both insertions are larger than 4.5 kb; however, ''jumping PCR'' identified only the LTR portion of the insertion (3). Partial PCR products terminating in the 5Ј and 3Ј LTRs cross-prime each other, resulting in amplified products that lack any of the internal sequence between LTRs. Therefore, these insertions were thought to be caused by an early transposon (ETn) element, which is a common mutagen in mice (4, 5).Split hand/split foot malformation (SHFM) in humans is a congenital limb malformation that has an ectrodactyly phenotype analogous to that of the dactylaplasia mouse. SHFM is genetically heterogeneous; to date, five different loci, SHFM1 to SHFM5, have been mapped. Dactylaplasia is a mouse model of S...

Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3

“…Mice heterozygous for a loss of function of Fgf8 +/-have normal limbs (Moon and Capecchi 2000). Furthermore, the complete loss of expression of Fgf8 in the central part of Dac heterozygous animals cannot be explained by a cis effect as proposed by Kano et al (2007) since the Fgf8 copy on the wild-type chromosome should be expressed normally. Instead it indicates a progressive loss of the apical ectodermal ridge (AER), even though we cannot rule out that a reduction of Fgf8 could contribute to the phenotype.…”

“…We demonstrate here that neither Dac 1j nor Dac 2j mice carry similar duplications, suggesting that gene dosage increase is not primarily responsible for the limb malformations. Accordingly, in a recent report Kano et al (2007) failed to detect any change in expression of the genes from the SHFM3 interval in Dac mutant embryos. The positions of both the MusD insertions and most of the distal duplication breakpoints as well as reported expression changes have highlighted Fbxw4 as a potential candidate (Basel et al 2003;Sidow et al 1999).…”

“…However, Fbxw4 transcript size or dosage is not affected in Dac 1j (Sidow et al 1999), and, more importantly, in a Mdac background where Dac 2j animals have normal limbs, the Dac 2j MusD insertion still leads to downregulation of Fbxw4, suggesting that Fbxw4 may not be directly involved in the ectrodactyly. Kano et al (2007) showed that the inserted MusD elements harbor different epigenetic modifications in Dac animals depending on their Mdac genotype. These observations provide important insights into the possible function of the Mdac gene and how it might control the consequences of the MusD insertions, but the nature of these consequences and their contribution to the Dactylaplasia phenotype are yet unknown.…”

Retroelements: molecular features and implications for disease

“…Their tissue-specific promoter activity is also affected by DNA methylation status (Reiss et al, 2007;Huh et al, 2008;Stengel et al, 2010;Cohen et al, 2011). Also, in genic regions, the correlation with intronic ERVs and DNA methylation status regulate gene expression (Kano et al, 2007;Li et al, 2012).…”

“…Several eukaryotes use these epigenetic mechanisms to inhibit the activity of retroelements. The correlation between retroelements and epigenetic modifications was first identified in plants and fungi (Slotkin and Martienssen, 2007;Suzuki and Bird, 2008), and later in mammals (Kano et al, 2007; (Onno et al, 1992) Recombination AZFa HERV (Hughes and Coffin, 2004) BRCA1,2 SINE APC LINE, SINE (Mayer et al, 2005) Retroposition FIX, NF1 SINE (Hutchinson et al, 1993) Function Promoter PTN HERV (Mager et al, 1999) KIAA1797, CLCN5, SLCO1A2 LINE (Bird, 2002) Enhancer ESR SINE (Norris et al, 1995) Suppressor BRCA SINE (Slotkin and Martienssen, 2007) Polyadenylation HHLA2, HHLA3 HERV (Kolomietz et al, 2002) Genomic features of retroelements and diseases Cruickshanks and Tufarelli, 2009;Richards et al, 2009;Ichiyanagi et al, 2011). In somatic and mature germ cells, retrotransposition and transcriptional activity of retroelements is predominantly suppressed by methylation and chromatin structure (Bird, 2002).…”