A promoter polymorphism in the Per3 gene is associated with alcohol and stress response.

Cis-modulation of Per3 expression by B and D alleles in the hippocampus. (a) Expression of Per3 was measured by six Affymetrix M430v2 probe sets. Probe sets 1421087_at and 1460662_at target different portions of the 3′ UTR and probe set 1421086_at targets the 3′ UTR and the last coding exons of Per3. Approximate positions of these probe sets and the Per3 isoforms are shown. The reference gene model is based on RefSeq gene NM_016831 (UCSC genome Browser mm9, build 37). (b) Expression QTLs for the three probe sets (1421087_at, 1460662_at and 1421086_at) show significant cis-regulation of Per3 expression. The y axis denotes likelihood ratio statistic (LRS) scores and the x axis denotes physical position (Mb) on chromosome 4. The peak LRS is near the location of the Per3 gene (gray triangle). The horizontal lines indicate the genome-wide suggestive (gray) and significant (red) thresholds.

Analysis of PER3 protein level in mouse hippocampus. (a) Per3 has significant expression variation in the BXDs. The wide expression range is shown by the PC1 of the Per3 probe sets (1421087_at, 1460662_at and 1421086_at). The extreme strains (red bars) are BXD2 and BXD22 with the lowest transcript level, and BXD28 and BXD42 with the highest transcript level. Strains are indicated along the x axis. (b) PER3 protein level in the hippocampus of the two high and two low strains was analyzed by western blot. PER3 protein bands are detected as doublets ranging from 131 to 150 kDa. (c) Densitometric analysis shows a significant expression difference between the strains that is consistent with transcript level. The difference was determined using Student's t test (*=P<0.01).

Functional characterization of the indel in the STRE promoter element. (a) The protein binding properties of the core STRE element in B6 and D2 was evaluated by EMSA. Double-stranded probe sequences were designed for the B6 (49-mer oligonucleotide on the left; lanes 1–6) and D2 (50-mer oligonucleotide on the right; lanes 7–12) alleles. In all, 100 fmol and 40 fmol of these probes were used to assay protein extracted from the brain tissue of B6 and D2. DNA–protein complexes are indicated by arrows. The B allele, which has the deletion, does not show DNA–protein complexes, whereas the D allele formed two DNA–protein complexes with protein from both B6 and D2 when 100 fmol of probes was used (lane 7 and 9). Control lanes 5 and 11 have 0 fmol probes. Lanes 6 and 12 are free oligonucleotides without protein. The upper three bands in lanes 1–5 and 7–11 are non-specific DNA–protein complexes. (b) Supershift assay using antibody against ATF3 shows no binding of the STRE-protein complex by ATF3. The arrowhead indicates the location of the STRE-protein complex. (c) Supershift assay with antibody against NRF3 reveal a slow migrating STRE-protein complex that contains NRF2. The arrowhead (complex) indicates the location of the STRE-DNA complex, and the arrow indicates the location of the NRF2 supershifted complex.

Correlation network graph of Per3 with behavioral phenotypes. The PC1 of Per3 expression (1421087_at, 1460662_at and 1421086_at) is highly correlated with ethanol-related and stress-related traits (P<0.05). The correlation coefficient, P-value and GeneNetwork's Trait IDs are shown in Supplementary Table S4. The thickness of line denotes the absolute correlation coefficient.

Downstream genes regulated by Per3. 48 transcripts map as trans-QTLs to Per3 (chromosome 4, 150–151 Mb) at likelihood ratio statistic >10 (P<0.01) in the neocortex dataset. These include genes associated with circadian rhythm (highlighted yellow), stress response (green), alcoholism (blue), schizophrenia (gray) and synaptic functions (turquoise). Seven of these genes are differentially expressed between Per3 KO and wildtype (red). Nodes highlighted in two colors are genes that are differentially expressed between Per3 KO and wildtype as well as associated with stress, alcoholism or schizophrenia.

Effects of stress and ethanol on the Per3 expression. (a) Nested ANOVA found significant stress × ethanol effect on Per3 expression in the BXDs. Solid line denotes expression of Per3 under ethanol followed by stress treatments; dotted line denotes expression of Per3 under saline followed by stress treatments. (b) Expression of Per3 in 146 BXD strains under stress and non-stress treatments are depicted as box plots. The horizontal lines indicate the median values, the boxes represent the inter-quartile range between 25th and 75th percentiles; the upper line extending from the box indicates the largest value between the 75th percentiles and 1.5 times the inter-quartile range. The lower line extending from the box indicates the smallest point between the 25th percentiles and 1.5 times the inter-quartile range. The open circle represents values that lie outside these ranges.