(A) Allele-specific expression difference between B6 and BALB Usf1 mRNA revealed by DNA sequencing. We chose an SNP at the 3′-UTR of Usf1 as a marker to distinguish between the B6 and BALB alleles. The amplicon size from genomic DNA and cDNA are identical. We PCR-amplified the SNP-containing region in genomic DNA samples from B6, BALB, (B6 x BALB)F1 and three samples containing different mix ratios between B6 and BALB DNA (3:1, 1:1 and 1:3, respectively) as shown in the left panel. The panel on the right shows (B6 x BALB)F1 Usf1 cDNA sequence from hypothalamus (top three) and liver (lower three). The PCR products were sequenced by an ABI3700 machine to compare the fluorescent signal peak ratio at the SNP locus as an indicator of the copy number ratio of the two alleles. This clearly demonstrates that the expression difference of Usf1 between B6 and BALB is the result of polymorphisms in cis regulatory elements. (B) Analysis of allele-specific expression differences between B6 and BALB Usf1 mRNA by quantitative PCR. We amplified genomic DNA mixtures described in (A) with allele-specific PCR primers and created a standard curve for %BALB genomic DNA vs ΔCt (black data points). The blue data point is from F1 genomic DNA, which is naturally a 1:1 mixture of B6 and BALB. Red data points represent F1 cDNA from hypothalamus. Based on the standard curve generated from genomic DNA, 80% of the F1 cDNA contains the BALB Usf1 allele. (C) Analysis of allele-specific expression differences between B6 and BALB Usf1 mRNA by cloning PCR products. We amplified both F1 genomic DNA and cDNA using primers flanking an SNP in exon 10 (rs31093636) of Usf1. We generated four independent PCR products from a single F1 genomic DNA sample and four PCR products from four different F1 cDNA samples. The exon 10 SNP creates a restriction fragment length polymorphism such that only the BALB allele is cleaved by restriction enzyme TfiI. Each PCR product was cloned into a TA plasmid vector. From each transformation, we picked 24–48 colonies. We isolated 143 colonies containing F1 genomic DNA and 172 colonies containing F1 cDNA. This analysis, like that in (B), demonstrates that the F1 cDNA contains a higher percentage of the BALB Usf1 allele than expected. (D) Usf1 promoter analysis between B6 and BALB mouse strains. The putative Usf1 promoter sequence (∼2.3 kb upstream of exon 1) was cloned into the pGL4 luciferase reporter vector and 90 ng of either B6 or BALB construct was transfected into HEK293T cells. Promoter activity from the BALB clone is significantly higher than that from the B6 clone (p<10−6). Next, we swapped EcoRI-XhoI fragments between the B6 and BALB constructs. The B6 clone containing the BALB EcoRI-XhoI fragment has significantly higher activity than the original B6 clone (p<10−3). The activity is equivalent to the original BALB clone. On the other hand, the BALB clone containing the B6 EcoRI-XhoI fragment exhibits essentially similar activity as the B6 intact clone. The blue and green bars indicate B6 and BALB DNA fragments, respectively. Each data point represents mean ± SEM of 18–36 samples. (E) SNP distribution pattern among 16 mouse strains in the Usf1 promoter candidate region. We detected 14 polymorphisms among 16 mouse strains within the ∼1000 bp candidate region. The top eight strains are ClockΔ19 non-suppressor strains and the bottom eight are ClockΔ19 suppressor strains. There are only seven polymorphisms that perfectly match the phenotype distribution pattern. Blue indicates the B6 allele and green indicates the BALB allele. (F) Putative Usf1 promoter SNPs (containing ∼100 bp of flanking sequence of SNP1, SNP2, SNP3, SNP4, SNP5&6 and SNP7) from B6 and BALB were cloned into the pGL4 luciferase reporter vector. Only SNP3 and SNP7 show elevated promoter activity. In both cases, the BALB allele has significantly higher activity (p<0.001) than the B6 allele (p<0.01). In the NCBI RefSNP database, SNP3 corresponds to rs31538551 and SNP7 to rs31538547. Each data point represents the mean ± SEM of six samples. (G) We mutagenized the B6 promoter EcoRI-XhoI fragment to introduce the BALB allele at either SNP3 or SNP7. We observed a significant increase in the luciferase signal by SNP7 (p<0.05). Although we did not detect significant upregulation by the BALB allele at SNP3 (p=0.18), the level of luciferase was elevated with the BALB allele compared to the B6 allele. Each data point represents the mean ± SEM of 12 samples.