Supplementary Table 4: Description of the 34 Positional Candidate Genes.
Fn1: Fibronectin (Fn) (-2.91)* found in Q1.1 is a high molecular weight extracellular matrix protein that binds to
cell membrane receptor integrins and extracellular matrix (ECM) components including fibrin, collagen and
syndecans. It plays an important role in wound healing by forming clots, allowing fibroblasts to adhere, migrate
and proliferate, supporting wound contraction. A reduced level of Fn seen in LG/J should lead to reduced fibrosis
(Hernnas et al., 1992) and be permissive for regeneration. Fn has only synonymous coding SNPs, 5 highly
conserved non-coding intronic SNPs and 1 in the 2500 bp proximal to the gene.
Smarcal1: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-like protein in
Q1.1 is involved in fork stability during DNA replication.
Xrcc5: This molecule (Q1.1) is a subunit of the Ku protein and is involved in DNA repair and recombination and
has a single non-synonymous coding SNP.
Lgr6: Lgr6 is found in the Q1.2 locus and is a transmembrane receptor that is a stem cell marker found in all stem
cell lineages of the skin (Snippert et al., 2010). It has been implicated in the repair and regeneration of skin and
hair follicles (Blanpain, 2010).
Lgr6 is not differentially expressed between strains and contains a single
nonsynonymous coding SNP in exon 1. It also has 8 highly conserved non-coding intronic SNPs (see Figure 3B).
Ptprv: Ptprv (2.47) in the Q1.2 locus, is a tyrosine phosphatase that has increased expression in LG/J. Ptprv
functions as a mediator of the p53-induced cell cycle exit and has been described as a potent inhibitor of cell
proliferation (Doumont et al., 2005).
It also appears as an early marker for mesenchymal potential during
osteogenic and chondrogenic differentiation (Kawaguchi et al., 2005). Even though Ptprv is a strong positional
candidate gene it contains no highly conserved SNPs and only a single non-synonymous coding variant.
Ptprc: Ptprc (-1.73) is a related protein tyrosine phosphatase in the Q1.3 locus. It has reduced expression in LG/J
and has 4 MHC non-coding SNPs including the only 5’UTR highly conserved SNP found in all 34 genes.
Fan1: FANCD2/FANCI-associated nuclease 1 is involved in DNA repair and recombination
Ndn: Necdin (1.22) an imprinted gene found in Q7.1 is differentially expressed between LG/J and SM/J but have
little sequence variation.
Blm: This molecule in Q7.1, involved in fork stability, has 4 non-synonymous coding SNPs all in exon 7. This
exon is known to carry Bloom Syndrome mutations (Kaneko and Kondo, 2004). The protein domains carrying
these variants are just proximal to the helicase domains and interact with MLH, a DNA mismatch repair protein,
SUMO-1 localization affecting cell-cycle progression, PML protein localization affecting cell proliferation, and
WRN (Werner Syndrome) which is involved in DNA repair.
Mesp2: This molecule (1.12) is found in the Q7.3 locus, is up-regulated in LG/J, is a notch signaling component,
and is involved in epithelial-mesenchymal transitions (Theiry et al., 2009). Patients lacking this protein suffer from
spondylocostal dysostosis (Dunwoodie, 2009). It has no SNPs in the region surveyed but expression levels could
be affected by more distant sequence variants.
Kif23: (3.38) See Discussion
Coro2b: Coro2b (1.96) has no coding SNPs but has highly conserved SNPs in the introns (5) and 3’UTR (3) and
involved in cell migration.
Atr: is a DNA damage checkpoint gene and has a single non-synonymous coding SNP. The Atr SNP is in exon
10 and is predicted to be potentially damaging by the PolyPhen-2 algorithm.
Rnf7: (-53.98) See Discussion
Cdk2: (-1.77) This cyclin-dependent kinase is involved in cell cycle regulation and is differentially expressed.
Cdk2 codes for a Ser/Thr protein kinase and, in complex with cyclin A or E, acts in G1 to promote S phase
transition, phosphorylates Rb, rendering E2F transcriptionally active and inducing proliferation genes.
molecule is particularly interesting since p21
Cip1/Waf1
This
is an inhibitor of the cyclin/CDK2 complex. Cdk2 has no
coding SNPs and only four MHC non-coding SNPs, one in the 2500 bp proximal to the gene, one in the 2500 kb
distal to the gene, and two in introns.
Cs: (5.51) See Discussion
Itga7: The integrin, Itga7, (3.14) affects both morphogenesis and wound healing. It resides at the Q10.1 locus, is
up-regulated 3-fold in LG/J, and is a laminin receptor that has splice variants that are differentially expressed
during muscle cell regeneration (Kaarianen et al., 2002). Also, Itga7 is involved in motor axon regeneration in
mice (Ekstrom et al., 2003).
This gene has only two highly conserved non-coding SNPs that are intronic.
However, it has 17 synonymous coding SNPs and two nonsynonymous coding SNPs, one of which is predicted to
be potentially damaging by PolyPhen.
Myl6: Myosin light chain 6 (-1.51) found in Q10.1 and down-regulated in LG/J is involved in actin-myosin
interactions, cytoskeletal remodeling, and thus cell adhesion and cell migration. Reduced myosin light chain
expression could lead to a functionally reduced Rho kinase pathway and reduced fibrosis in LG (van Beuge et al.,
2011; Melton et al., 2007). Myl6 has a non-synonymous coding SNP in exon 5 and one highly conserved noncoding SNP each in the 2500 bp proximal and distal to the gene, in the introns, and in the 3’UTR.
Obfc2b: Hssb1 or Nabp2, found in Q10.1, is a DNA damage checkpoint gene and is also differentially expressed.
Obfc2b has only two MHC non-coding SNPs, one each in the 2500 bp proximal and distal to the gene.
Smarcc2, Hist3ha (H2A) and Hist3h2ba (H2Ba): These are genes involved in chromatin remodeling.
Wnt3a: (1.96) See Discussion
Krt15: This keratin is differentially expressed between LG/J and SM/J but have little sequence variation. Keratins
have been shown to be important in regenerating mice (Cheng et al, 2013). )
Becn1: Beclin 1(-2.52), found in the Q11.3 locus, is an orthologue of the yeast ATG6 protein which is involved
in autophagy (Funderburk et al., 2010). In the LG/J mouse ear, this gene is down-regulated suggesting that
autophagy is reduced in the healing ears. Autophagy-inducing drugs have also recently been shown to suppress
wound healing in the endothelium (Hayashi et al., 2009). Becn1 has only synonomous coding SNPs and 3 highly
conserved noncoding SNPs in the introns, one in the 3’UTR, and 5 in the 2500 bp downstream from the gene.
Brca1: This is a DNA damage checkpoint gene in Q11.2 and has 5 non-synonymous coding SNPs, three of
which are highly conserved in mammals. Four of these SNPs are in exon 9 and one in exon 13. Exon 9 carries
mutations resulting in breast cancer (Judkins et al., 2012) and the germline duplication of exon 13 can also lead to
cancer (Cerutti et al., 2010).
Rdm1: This molecule is found in Q11.2 and is involved in DNA repair and recombination.
Adamts16: Adamts16 (1.75) found in Q13.1 is a disintegrin-like and metallopeptidase which has 27 coding SNPs,
10 of which are non-synonymous including one predicted to make an important functional difference by the LRT
algorithm. There is only 1 highly conserved non-coding SNP, however, there are two SNPs with conservation
higher than 0.70 adjacent to intron-exon boundaries that could affect splicing.
Adamts6: Adamts6 is not differentially expressed between the strains and has a single non-synonymous coding
SNP in exon 4. It has a very large number of highly conserved non-coding SNPs including one at an intron-exon
boundary and three in the 3’UTR.
Atg14: Atg14, another autophagy gene, is in Q14.1 but is not differentially expressed and has no coding or highly
conserved non-coding SNPs differing between the strains.
Diap1: This gene (-1.86) is involved in cell polarity and cell migration. Actin polymerization is necessary for cell
migration and the candidate gene Diap1 found in Q18.3 is involved in actin recruitment and assembly, mediates
Rho GTPase activity, and microtubule dynamics (Yamana et al., 2006).
Reduced polarity has been shown
previously in MRL ear fibroblasts and may be important in maintaining a more stem-like state (Naviaux et al.,
2009). Reduced Diap1 expression in LG/J supports this finding. This gene has 7 highly conserved non-coding
SNPs, three in the 2500 bp proximal to the gene and four in the introns. In addition, it has one non-synonymous
coding SNP in exon 15.
Fgf1: Fgf1 (-1.65) in Q18.1 is down-regulated in LG/J and should reduce the level of fibroblast proliferation and
fibrotic response (Yu et al., 2003). Fgf1 has 4 highly conserved non-coding SNPs, 3 of them in the 3’UTR.
Hdac3: This histone deacetylase (1.16) is involved in chromatin remodeling and is differentially expressed
between LG/J and SM/J but have little sequence variation.
Mcc: This molecule (--1.32) is involved in cell cycle regulation and differentially expressed. Mcc is a very long
gene with abundant MHC non-coding SNPs in the introns (19), 3’UTR (3), and the 2500 bp downstream (1) of the
gene. It also has a single nonsynonymous coding SNP in exon 2 (Ser:53:Asn).
Notes:
*Number in parentheses is the LG/J/SM/J gene expression ratio.
Genes associated with given functions include Metabolism (Cs ,Rnf7, Becn1); Cell Cycle Regulation and DNA
Damage, Replication, and Repair (Cdk2, Kif23, Brca1, Obfc2b (Hssb1) Atr , Blm, Smarca,1 Rdm1, Fan1, Xrcc5,
Hdac3, H2a, H2ba, and Smarcc2); Cell migration and adhesion (Kif23, Diap1, Itga7, Coro2b, Myl6, and Adamts6
and 16), Fibrosis (Fn, Myl6, Fgf1); and Development-related genes (Wnt3a, Lgr6, Mesp2,and Fgf1).