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Haploinsufficiency of hnRNP U Changes Activity Pattern and Metabolic Rhythms

  • Beibei Lai
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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  • Jianghuan Zou
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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  • Zhaoyu Lin
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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  • Zhipeng Qu
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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  • Anying Song
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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  • Ying Xu
    Correspondence
    Ying Xu, Ph.D., Medical College of Soochou University, 199 Renai Road, 215123, Suzhou, China.
    Affiliations
    Medical College of Soochou University, Suzhou, China
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  • Xiang Gao
    Correspondence
    Address correspondence to Xiang Gao, Ph.D., Model Animal Research Center, Nanjing University, 12 Xuefu Road, Pukou Area, 210061, Nanjing, China.
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China
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Open ArchivePublished:November 08, 2017DOI:https://doi.org/10.1016/j.ajpath.2017.09.017
      The neuropeptides arginine vasopressin (Avp) and vasoactive intestinal polypeptide (Vip) are critical for the communication and coupling of suprachiasmatic nucleus neurons, which organize daily rhythms of physiology and behavior in mammals. However, how these peptides are regulated remains uncharacterized. We found that heterogeneous nuclear ribonucleoprotein U (hnRNP U) is essential for the expression of Avp and Vip. Loss of one copy of the Hnrnpu gene resulted in fragmented locomotor activities and disrupted metabolic rhythms. Hnrnpu+/− mice were more active than wild-type mice in the daytime but more inactive at night. These phenotypes were partially rescued by microinfusion of Avp and Vip into free-moving animals. In addition, hnRNP U modulated Avp and Vip via directly binding to their promoters together with brain and muscle Arnt-like protein-1/circadian locomotor output cycles kaput heterodimers. Our work identifies hnRNP U as a novel regulator of the circadian pacemaker and provides new insights into the mechanism of rhythm output.
      Haploinsufficiency is often associated with severe neurologic abnormalities, such as sleep disorders and autism disease.
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      Heterogeneous nuclear ribonucleoprotein U (HnRNP U) is expressed in a circadian manner in the suprachiasmatic nucleus (SCN).
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      It is interesting to examine whether hnRNP U regulates Avp and Vip via a similar mechanism.
      We found that mice with a heterozygous loss-of-function mutation in Hnrnpu (Hnrnpu+/−) have significantly attenuated locomotor activity during the dark phase and increased activity during daytime. Correspondingly, Hnrnpu+/− mice reduced food intake during nighttime, which is a typical food intake period for nocturnal rodents. Moreover, hnRNP U regulates Avp and Vip gene expression together with Bmal1/Clock at their promoters. These studies suggested that hnRNP U haploinsufficiency changes activity and metabolic rhythms through the SCN-enriched neuropeptides Avp and Vip.

      Materials and Methods

       Animals

      Animals were housed in a specific pathogen-free facility accredited by Association for Assessment and Accreditation of Laboratory Animal Care International. Animal protocols and care were approved by the Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University. Mice were housed and bred under 12 hours of light and 12 hours of dark cycles at 25°C ± 1°C, with water and food available ad libitum. At 1 or 2 weeks before experiments, mice were transferred into cages in light-tight chambers equipped with independent daylight fluorescent lamps. Hnrnpu knockout mice (frame shifted by deleting exon 3 to exon 6) were generated at the Model Animal Research Center. Then, Hnrnpu+/− mice were continuously backcrossed to C57BL/6J strain for at least 10 generations. Genotyping primers are presented in Table 1. The PCR products were 330 and 394 bp for the wild-type and mutant alleles, respectively.
      Table 1Genotyping Primers
      Primer nameSequence
      Wild-type allele forward5′-GAGGCATTACATAGTTGCTGAG-3′
      Wild-type allele reverse5′-GCCTAACATGGAACCTTGGACA-3′
      Mutant allele forward5′-GAGGCATTACATAGTTGCTGAG-3′
      Mutant allele reverse5′-GGGTTCAAGCTGGCTTAGTGAC-3′

       Wheel-Running Activity Assay

      A locomotor activity assay was performed with 3- to 4-month–old males and females. Adult mice of both genotypes were individually maintained in wheel running cages placed in light-tight cabinets with independent illumination. Mice had free access to food and water. Daily locomotor activity was measured by connecting the wheel and detector to a computer running ClockLab software version 7.0 (Actimetrics, Evanston, IL). Activity in 6-minute bins was recorded after approximately 1 week of habituation and entrainment. The initial photoschedule was white light between 8:00 and 20:00 and darkness between 20:00 and 8:00 (LD). Approximately 1 week later, mice were transferred to constant darkness (DD) conditions. Locomotor activity periods and amplitudes were assessed by counting wheel revolutions. The time of onset, the distribution of activity, and the χ2 periodograms were analyzed using Clocklab software.

       Open-Field Test

      The open-field test was performed in 3- to 4-month–old male mice according to standard protocols at zeitgeber times (ZTs) 3 to 4 (11:00 to 12:00) and 15 to 16 (23:00 to 24:00). The open field consisted of a square with a length of 45 cm and a height of 35 cm (AES-LOC1; Anilab Software & Instruments Co. Ltd., Ningbo, China). Mice were placed in the field and then allowed to freely explore the field for 5 minutes. The total distance moved, the amount of time spent moving, and the mean speed were measured using video captured on infrared light video.

       Metabolic Rhythms Measurement

      Metabolic rhythms were continuously assessed and analyzed using the Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). After 2 days of acclimation in the instrument room, mice were placed in individual chambers and had free access to water and food. Another group of mice were intraperitoneally implanted with capsule sensors to detect real-time body core temperature (RPC-1; DSI, St. Paul, MN). Mice were detected for 24 hours in 10-second time bins. The data were then smoothed by averaging and transformed to ZT and double-plotted.

       Real-Time Quantitative PCR

      After entrainment, mice were sacrificed to collect tissues in 4-hour increments on the first day of DD. Livers and anterior ventral hypothalamus were collected, snap frozen in liquid nitrogen, and stored at −80°C. Total RNA was prepared using TRIzol Reagent (catalog number 10296028, Invitrogen, Carlsbad, CA) and reverse transcribed using a PrimeScript TR reagent kit (catalog number RR047A, TaKaRa, Dalian, China) according to the manufacturer's instructions. Real-time quantitative PCR (qPCR) was performed using the StepOnePlus Real-time PCR system (Applied Biosystems, Carlsbad, CA) with SYBR Premix Ex Taq reagents (catalog number RR820A, TaKaRa). Data were analyzed by StepOnePlus software version 2.1 (Applied Biosystems) using comparative Ct methods, and gene expression levels were calculated by normalizing to β-actin. Primers for detecting specific isoforms were designed by identification of the predicted alternative exon, and primers for detecting total RNA levels were designed via identification of the shared exon. The specific primers are provided in Table 2.
      Table 2Real-Time Quantitative PCR Primers
      Primer namePrimer sequence
      Neuropeptides
      Avp-F15′-ACTACGCTCTCCGCTTGTT-3′
      Avp-R15′-GCAGATGCTTGGTCCGAAG-3′
      Vip-F15′-CTTGGACAGCAGAGCACTAGC-3′
      Vip-R15′-GAAGAGTATCAGGAATGCCAGGAA-3′
      Central clock genes
      Clock QF5′-GGTTTGATCACAGCCCAACT-3′
      Clock QR5′-CCTCCGCTGTGTCATCTTTT-3′
      Cry1 QF5′-TCCCCTCCCCTTTCTCTTTA-3′
      Cry1 QR5′-TGAGTCATGATGGCGTCAAT-3′
      Cry2 QF5′-AAGCTGAATTCGCGTCTGTT-3′
      Cry2 QR5′-GTGGTTTCTGCCCATTCAGT-3′
      Bmal1 QF5′-TGGAGGGACTCCAGACATTC-3′
      Bmal1 QR5′-TGGGACTACTGGATCCTTGG-3′
      Per2 QF5′-CGCTTCATAGCCCGAGTGTA-3′
      Per2 QR5′-TTCTGCCGTGTCAGTGTTGG-3′
      Rev-erbα QF5′-GCTGCCATTGGAGTTGTCAC-3′
      Rev-erbα QR5′-GACATGACGACCCTGGACTC-3′
      Rev-erbβ QF5′-GAAGAGTGACCGCACAGATTG-3′
      Rev-erbβ QR5′-TCCGAAAGAAACCCTTACAGC-3′
      Rorα QF5′-CCCCTACTGTTCCTTCACCA-3′
      Rorα QR5′-AGCTGCCACATCACCTCTCT-3′
      Dbp QF5′-AAGAAGGCAAGGAAAGTCCAG-3′
      Dbp QR5′-ACCTCTTGGCTGCTTCATTG-3′
      Actβ QF5′-GTCCACCTTCCAGCAGATGT-3′
      Actβ QR5′-GAAAGGGTGTAAAACGCAGC-3′
      Alternative splicing
      Clock-EQF5′-AGCATGGTCCAGATTCCATC-3′
      Clock-EQR5′-CAGGAGCAGTCACTAATTTGGTC-3′
      Clock-GQF5′-CTTCCTGGTAACGCGAGAAAG-3′
      Clock-GQR5′-TCGAATCTCACTAGCATCTGACT-3′
      ChIP
      Avp-CHIPM-F5′-GGTGCTTCCTCTCATCCTATAC-3′
      Avp-CHIPM-R5′-GATTGGTGCTGTGCGATCA-3′
      Vip-CHIPM-F5′-CATCTTCTAGTTCATCACAGCTAAG-3′
      Vip-CHIPM-R5′-TTATAGGGCTTGTGCTTCGG-3′
      GapdhM-F5′-CTTGTGGCAAGAGGCTAGGG-3′
      GapdhM-R5′-CAGGGACGTGCTGACTGGC-3′
      Avp-CHIPR-F5′-GATGCTTCCTCTTCTCCTACAC-3′
      Avp-CHIPR-R5′-GATTGGTGCTGTGCAATCA-3′
      Vip-CHIPR-F5′-CGTCTTCCAGTTCATCACAACTAAG-3′
      Vip-CHIPR-R5′-TTATAGGGCTTGTGCTTAGG-3′
      GapdhR-F5′-GGCAGCCAAGGAAAGAGAGTC-3′
      GapdhR-R5′-TGAGTTGTACCGCCCAGGAT-3′
      ChIP, chromatin immunoprecipitation; CHIPM, CHIP–mouse; CHIPR, CHIP–rat; E, exon; F, forward; G, global; GapdhM, Gapdh–mouse; GapdhR, Gapdh–rat; Q, quantitative; R, reverse.

       Immunostaining

      Animals were anesthetized and perfused with 4% paraformaldehyde (PFA) at indicated time points. Brains were dissected carefully and fixed with 4% PFA for 4 hours at 4°C. After dehydrating in 30% sucrose, brains were embedded in optimal cutting temperature compound freeze medium at −80°C. Frozen brains were sliced into 10-μm-thick sections using a Leica CM1950 Cryostat (Leica Biosystems, Nussloch, German) and subjected to immunofluorescence staining. Briefly, the slides were blocked in 5% bovine serum albumin, incubated at 4°C in 1:200 hnRNP U antibody (ab180952; Abcam, Shanghai, China), washed with phosphate-buffered saline (PBS), and incubated in 1:200 goat anti-rabbit fluorescein isothiocyanate secondary antibody (F1262; Sigma, Shanghai, China). Nuclei were counterstained with 0.1% Hoechst 33,342 (Cell Signaling Technology, Shanghai, China). The images were obtained using a confocal microscope (Leica DM6000; Leica Biosystems) and analyzed using ImageJ software version 1.46 (NIH, Bethesda, MD; http://imagej.nih.gov/ij).

       In Situ Hybridization

      Animals were anesthetized and perfused with 4% PFA at indicated time points. Coronal serial sections (100-μm thick) through the SCN were cut on a Leica VT1000 S Vibrating blade microtome (Leica Biosystems). Given that the SCN region was not visible, 10 to 15 adjacent slides per mouse were prepared to ensure that sections that included the region were obtained. Digoxin-labeled Avp probe and Vip probe were synthesized with Promega reagents according to the manufacturer's instructions. Briefly, sections were subject to bleaching (6% H2O2, 40 minutes, room temperature), cells permeabilization (10 μg/mL of protease K, 10 minutes, room temperature), protease K inactivation (2 mg/mL of glycine, 5 minutes, room temperature), fixation (0.2% glutaraldehyde and 4% PFA, 20 minutes, room temperature), prehybridization (1 hour, 70°C), hybridization (specific probes, 12 hours, 70°C), RNase A treatment (10 μg/mL of RNase A, 2 hours, 37°C), blocking (1% heat inactivated sheep serum, 30 minutes, 4°C), hybridization (1:2000 alkaline phosphatase–linked DIG antibody, 11093274910; Roche, Shanghai, China,12 hours, 4°C), and staining (nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate, room temperature, in the dark). After staining in the dark, SCN regions were visible. Then, slices were selected, mounted, and photographed.

       Stereotaxic Microinjection

      Mice were entrained to LD cycles and then placed in DD conditions to detect circadian phases and periods. Unilateral cannulas (outer diameter = 0.21 mm, inner diameter = 0.11 mm) for infusion of peptides were implanted into the brain of anesthetized mice. Stereotaxic techniques were used to position the cannulas (bregma: anteroposterior, −0.46 mm; mediolateral, 0.12 mm; dorsoventral, −5.40 mm). The microsyringe was controlled at a speed of 250 nL/minute by a micromanipulator (RWD Life Science Co. Ltd, Shenzhen, China). Activity counts 1 week before and after injection were analyzed. Vip was purchased from Sigma-Aldrich (St. Louis, MO). Avp and a scrambled polypeptide were synthesized from GenScript (Nanjing, China).

       Cell Culture

      Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (26104-079; Gibco, Carlsbad, CA) and 60 μg/mL of penicillin and 50 μg/mL of streptomycin at 37°C in the presence of 5% carbon dioxide. Transient transfection of plasmid DNA using Lipofectamine 2000 (Invitrogen) was performed according to the manufacturer's instructions. The siRNAs were synthesized at Genepharma (Shanghai, China) and are presented in Table 3. Avp (−600 to +50 bp) and Vip (−550 to +30 bp) promoters were inserted into PGL3-Basic plasmid (pGL3-Avp and pGL3-Vip). pGL3 and siRNA and pRL-TK vector were cotransfected into HEK 293T cells. Luciferase activity was detected using a dual-reporter assay system following the manufacturer's manual (E1910; Promega, Beijing, China).
      Table 3siRNA Sequence
      siRNASequence
      hsiU1 (for HEK 293T cells)5′-GAUGAACACUUCGAUGACAdTdT-3′
      hsiU2 (for HEK 293T cells)5′-AGACCACGAGAAGAUCAUdTdT-3′
      rsiU1 (for SCN2.2 cell)5′-GACGAACACUUCGAUGACAdTdT-3′
      rsiU2 (for SCN2.2 cell)5′-AGACCCCGGGAAGAUCAUdTdT-3′
      siSCR (as negative control)5′-UUCUCCGAACGUGUCACGUdTdT-3′
      hsiU, human hnRNP U–specific siRNAs; rsiU, rat hnRNP U–specific siRNAs; siSCR, scrambled siRNA.

       Chromatin Immunoprecipitation

      HnRNP U (ab20666), Clock (ab3517), and Bmal1 (NB100-2288) antibodies were purchased from Abcam and NOVUS Biologicals (Littleton, CO). Protein G sepharose beads (GE Healthcare, Chicago, IL) were prepared by washing with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride added PBS (PBS++). Then, the beads were incubated with 1-mg antibodies at 4°C overnight.
      SCN2.2 cells and anterior ventral hypothalamus were crosslinked with 1% formaldehyde immediately. Then, 1.375 mol/L glycine was used to stop the linkage reaction, and the cells were washed in PBS++ twice. Cells were scraped and treated with ice-cold cell lysis buffer to collect nuclei. Then, the nuclei were treated with nuclear lysis buffer. Sonication was performed at a high-level grade (Bioruptor UCD-200; Diagenode, Liege, Belgium) for 15 minutes (5 minutes each three times). Samples were gently incubated with beads at 4°C overnight and then washed with high-salt washing buffer, low-salt washing buffer, LiCl washing buffer, and immunoprecipitation buffer twice for each. DNA was extracted after decrosslinking at 65°C and then subjected to qPCR using the primers listed in Table 2. Relative binding efficiency for a specific protein was normalized to normal IgG (nIgG).

       Statistical Analysis

      Data are presented as means ± SD. Analyses were performed using GraphPad Prism software version 5 (GraphPad Software, La Jolla, CA). Statistical significance between two groups was determined using an unpaired two-tailed t-test. Multigroup analysis was conducted using two-way analysis of variance. If significant differences were detected, the Bonferroni post hoc test was applied. P < 0.05 was considered statistically significant.

      Results

       Attenuated Locomotor Activity in Hnrnpu+/− Mice

      In a systematic phenotyping project, we monitored the wheel-running activity of Hnrnpu+/− mice on the C57BL/6J background because Hnrnpu−/− mice were embryonically lethal.
      • Roshon M.J.
      • Ruley H.E.
      Hypomorphic mutation in hnRNP U results in post-implantation lethality.
      Mice were placed in LD conditions (light from 8:00 to 20:00) for at least 1 week and then released into DD conditions according to standard protocols as reported in our previous studies.
      • Wang X.
      • Tang J.
      • Xing L.
      • Shi G.
      • Ruan H.
      • Gu X.
      • Liu Z.
      • Wu X.
      • Gao X.
      • Xu Y.
      Interaction of MAGED1 with nuclear receptors affects circadian clock function.
      • Qu Z.
      • Zhang H.
      • Huang M.
      • Shi G.
      • Liu Z.
      • Xie P.
      • Li H.
      • Wang W.
      • Xu G.
      • Zhang Y.
      • Yang L.
      • Huang G.
      • Takahashi J.S.
      • Zhang W.J.
      • Xu Y.
      Loss of ZBTB20 impairs circadian output and leads to unimodal behavioral rhythms.
      The representative double-plotted wheel-running actograms demonstrates that Hnrnpu+/+ mice had robust rest-activity rhythms, whereas 8 of 11 Hnrnpu+/− mice exhibited remarkably decreased activity during nighttime with increased activity during daytime. Moreover, 3/11 Hnrnpu+/− mice were completely arrhythmic in DD conditions (Figure 1A). Then, circadian period was estimated by χ2 periodograms. The endogenous dominant period lengths were not altered in Hnrnpu+/− mice compared with Hnrnpu+/+ mice during 2 weeks of DD conditions (Figure 1B).
      Figure thumbnail gr1
      Figure 1The locomotor activity patterns change in Hnrnpu+/− mice. A: Representative double-plotted actograms of wheel-running show disorganized activity rhythms in Hnrnpu+/− mice. Hnrnpu+/+ mice become active immediately after dark, but Hnrnpu+/− mice do not exhibit a discernible onset of activity. The onset line (the orange line) is ambiguous in eight Hnrnpu+/− mice (middle) and undetectable in the remaining three mice (right). B: Periods of locomotor activity in constant darkness (DD) are normal in Hnrnpu+/+ and Hnrnpu+/− mice. C and D: Distribution of locomotor activity shows that Hnrnpu+/− mice are relatively more active in the daytime but more inactive at night compared with wild-type mice. E and F: Batch analysis in white light between 8:00 and 20:00 and darkness between 20:00 and 8:00 (LD) and DD conditions were performed using χ2 periodograms. The circadian rhythm amplitudes of Hnrnpu+/− mice are reduced. The diagonal lines indicate the 99% CI. Data are presented as means ± SD (B–F). n = 7 Hnrnpu+/+ mice; n = 11 Hnrnpu+/− mice. ∗∗P < 0.01, ∗∗∗P < 0.001, unpaired two-tailed t-test. D, day; N, night; SD, subjective day; SN, subjective night.
      Quantification of wheel-running activity revealed that Hnrnpu+/− mice had considerably reduced locomotor activity compared with Hnrnpu+/+ mice at night. In contrast, the relative activity of Hnrnpu+/− mice in the daytime was considerably increased compared with wild-type controls. Therefore, Hnrnpu+/− mice tended to move in the daytime in LD or subjective day in DD (Figure 1, C and D).
      Rhythmic power in LD and DD conditions was measured by batch analysis using χ2 periodograms. The peaks in the corresponding χ2 periodograms indicated the dominant periods during days 1 to 7 (LD stage) and days 8 to 24 (DD stage). The decreased peaks demonstrated that Hnrnpu+/− mice exhibited significantly attenuated circadian rhythms and considerably reduced amplitudes compared with wild-type mice (Figure 1, E and F).
      To confirm this phenotype of abnormal activity, mice were subjected to an open-field test at ZT3 to ZT4 and ZT15 to ZT16, separately. Hnrnpu+/− mice were more active than Hnrnpu+/+ mice during daytime. The total moving distance, total moving duration, and mean speed were increased in Hnrnpu+/− mice compared with wild-type mice (Figure 2, A–C ). In contrast, Hnrnpu+/− mice exhibited reduced moving distance, moving duration, and speed at ZT15 to ZT16, indicating that Hnrnpu+/− mice were more inactive at night (Figure 2, D–F). In summary, the above data indicate that hnRNP U haploinsufficiency affects mouse activity pattern.
      Figure thumbnail gr2
      Figure 2Hnrnpu+/− mice are more active in the daytime compared with wild-type mice. A–C: Hnrnpu+/− mice explore more and move faster in the daytime compared with wild-type mice. D–F: Hnrnpu+/− mice explore less and move slower at night compared with wild-type mice. n = 6 Hnrnpu+/− mice; n = 11 Hnrnpu+/− mice. Data are expressed as means ± SD. P < 0.05, unpaired two-tailed t-test.

       Aberrant Metabolic Rhythms in Hnrnpu+/− Mice

      Because the Hnrnpu+/− mice displayed rest-activity abnormalities, it was assessed whether hnRNP U functioned specifically in animal behavior patterns or also physiologies or even the SCN pacemaker. To address this issue, we monitored mice using the Comprehensive Lab Animal Monitoring System. In LD conditions, Hnrnpu+/+ mice displayed overt circadian rhythms in oxygen consumption and carbon dioxide production. Consistent with the wheel-running test, Hnrnpu+/− mice displayed reduced oxygen and carbon dioxide peak values (Figure 3, A, B, D, and E ). Similarly, the peaks of food intake and the respiratory exchange ratio were largely decreased in Hnrnpu+/− mice at night (Figure 3, C, G, F, and J). The reduced activity, respiration, and ingestion at night resulted in a reduced peak of heat production (Figure 3, H and K). Furthermore, telemetric devices were implanted into the peritoneal cavity, and real-time core body temperature was measured. The body temperature of Hnrnpu+/− mice was increased compared with that of wild-type controls in the daytime but was lower at night (Figure 3, I and L). Altogether these observations suggest that hnRNP U haploinsufficiency specifically affects not only animal activity-rest patterns but also physiologic rhythms.
      Figure thumbnail gr3
      Figure 3Metabolic rhythm changes in Hnrnpu+/− mice. A, B, D, and E: Consecutive data were recorded and analyzed. Oxygen consumption and carbon dioxide production decrease significantly at night in Hnrnpu+/− mice. C, F, G, and J: Respiratory exchange ratio (RER) and food intake values decrease in Hnrnpu+/− mice at night. H and K: Thermogenesis decreases in Hnrnpu+/− mice at night. I and L: Core body temperature of Hnrnpu+/− mice is increased compared with wild-type controls in the daytime but reduces at night. Data are expressed as means ± SD. n = 7 Hnrnpu+/+ mice; n = 8 Hnrnpu+/− mice. Dashes over the curves indicate time points with significant differences. P < 0.05, ∗∗P < 0.01, unpaired two-tailed t-test.

       Intact Central Molecular Clock in Hnrnpu+/− Mice

      The aberrant patterns of locomotor activity and metabolic rhythms prompted us to measure the integrity of the molecular oscillator and SCN output pathway. The mRNA profiles of core clock genes were examined in the SCN and liver as described previously.
      • Wang X.
      • Tang J.
      • Xing L.
      • Shi G.
      • Ruan H.
      • Gu X.
      • Liu Z.
      • Wu X.
      • Gao X.
      • Xu Y.
      Interaction of MAGED1 with nuclear receptors affects circadian clock function.
      Mice were entrained to LD cycles 1 week before the experiments. Tissues were collected at 4-hour intervals during the first day in DD for 24 hours. Relative gene levels were detected with qPCR and normalized to β-actin. No significant difference was detected in Bmal1, Clock, Per2, Cry1, or Cry2 between Hnrnpu+/+ and Hnrnpu+/− SCN at any time points (Figure 4).
      Figure thumbnail gr4
      Figure 4Central clock genes are intact in the suprachiasmatic nucleus (SCN) of Hnrnpu+/− mice. A–E: Representative mRNA profiles of central clock genes were analyzed in the SCN. Tissues were collected at 4-hour intervals during the first day in constant darkness (DD). Relative gene levels were detected with real-time quantitative PCR and normalized to β-actin. No significant difference is detected in brain and muscle Arnt-like protein-1 (Bmal1), circadian locomotor output cycles kaput (Clock), period 2 (Per2), cryptochrome-1 (Cry1), or cryptochrome-2 (Cry2) between Hnrnpu+/+ and Hnrnpu+/− mice. Data are expressed as means ± SD (B–D, G, and H). Two-way analysis of variance followed by Bonferroni post hoc test, n = 21. CT, circadian time.
      Similarly, no differences in Bmal1, Clock, Per1, Per2, Cry1, Cry2, Dbp, Rev-erbα, Rev-erbβ, and Rorα mRNA levels were noted in the liver (Supplemental Figure S1, A–J). hnRNP U has been reported as a regulator of alternative splicing, so different splicing isoforms of the Clock gene in the liver were examined.
      • Ye J.
      • Beetz N.
      • O'Keeffe S.
      • Tapia J.C.
      • Macpherson L.
      • Chen W.V.
      • Bassel-Duby R.
      • Olson E.N.
      • Maniatis T.
      hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.
      • McGlincy N.J.
      • Valomon A.
      • Chesham J.E.
      • Maywood E.S.
      • Hastings M.H.
      • Ule J.
      Regulation of alternative splicing by the circadian clock and food related cues.
      A pair of specific qPCR primers were designed to evaluate the expression of some specific isoforms, and another pair were used to detect total mRNA levels. The relative ratio could be obtained by dividing the specific isoform by total levels. No difference was detected between Hnrnpu+/+ and Hnrnpu+/− mice (Supplemental Figure S1, K and L). These data excluded the possibility that hnRNP U affects the expression profiles of core Clock genes to a great extent.

       Reduced Levels of Avp and Vip in Hnrnpu+/− Mice

      Then, we hypothesized that hnRNP U regulates the SCN pacemaker output pathway. The expression of hnRNP U was determined in the SCN via immunostaining and found that the hnRNP U level was decreased significantly in Hnrnpu+/− mice. The optical density was measured with ImageJ (Figure 5, A and B ).
      Figure thumbnail gr5
      Figure 5Arginine vasopressin (Avp) and vasoactive intestinal polypeptide (Vip) transcription is suppressed in the Hnrnpu+/− suprachiasmatic nucleus (SCN). A and B: Immunofluorescence reveals that the expression levels are decreased in Hnrnpu+/− mice. C and D: Avp and Vip mRNA expression levels in the SCN are reduced in Hnrnpu+/− mice. E and F: In situ hybridization in the SCN exhibits less Avp and Vip in Hnrnpu+/− mice. G: Histogram revealing similar lengths from the rostral end to the caudal end of the SCN in both groups. H: The pGL3-Avp and pGL3-Vip luciferase activities are suppressed by heterogeneous nuclear ribonucleoprotein U (hnRNP U) RNA interference, whereas pGL3-Basic is not affected. Data are expressed as means ± SD (B–D, G, and H). n = 3 Hnrnpu+/− mice (A and B); n = 21 Hnrnpu+/− mice (C and D); n = 4 mice with Avp and 3 mice with Vip (E and F); n = 5 (G); n = 6 (H). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed t-test (B and G) and two-way analysis of variance followed by Bonferroni post hoc test (C, D, and H). Scale bars: 200 μm. CT, circadian time; SiSCR, scrambled siRNA.
      Avp and Vip are critical components of the circadian pacemaker. Mice defective in Avp and Vip signaling exhibit phenotypes similar to Hnrnpu+/− mice.
      • Hughes A.T.
      • Piggins H.D.
      Behavioral responses of Vipr2−/− mice to light.
      • Li J.D.
      • Burton K.J.
      • Zhang C.
      • Hu S.B.
      • Zhou Q.Y.
      Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei.
      • Colwell C.S.
      • Michel S.
      • Itri J.
      • Rodriguez W.
      • Tam J.
      • Lelievre V.
      • Hu Z.
      • Liu X.
      • Waschek J.A.
      Disrupted circadian rhythms in VIP- and PHI-deficient mice.
      • Aton S.J.
      • Colwell C.S.
      • Harmar A.J.
      • Waschek J.
      • Herzog E.D.
      Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons.
      • Cutler D.J.
      • Haraura M.
      • Reed H.E.
      • Shen S.
      • Sheward W.J.
      • Morrison C.F.
      • Marston H.M.
      • Harmar A.J.
      • Piggins H.D.
      The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro.
      • Harmar A.J.
      • Marston H.M.
      • Shen S.
      • Spratt C.
      • West K.M.
      • Sheward W.J.
      • Morrison C.F.
      • Dorin J.R.
      • Piggins H.D.
      • Reubi J.C.
      • Kelly J.S.
      • Maywood E.S.
      • Hastings M.H.
      The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei.
      • Hughes A.T.
      • Guilding C.
      • Lennox L.
      • Samuels R.E.
      • McMahon D.G.
      • Piggins H.D.
      Live imaging of altered period1 expression in the suprachiasmatic nuclei of Vipr2−/− mice.
      Thus, the Avp and Vip mRNA levels were examined in the anterior ventral hypothalamus using qPCR. Both Avp and Vip were expressed in a circadian manner.
      • Li J.D.
      • Burton K.J.
      • Zhang C.
      • Hu S.B.
      • Zhou Q.Y.
      Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei.
      • Harmar A.J.
      • Marston H.M.
      • Shen S.
      • Spratt C.
      • West K.M.
      • Sheward W.J.
      • Morrison C.F.
      • Dorin J.R.
      • Piggins H.D.
      • Reubi J.C.
      • Kelly J.S.
      • Maywood E.S.
      • Hastings M.H.
      The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei.
      • Georg B.
      • Hannibal J.
      • Fahrenkrug J.
      Lack of the PAC1 receptor alters the circadian expression of VIP mRNA in the suprachiasmatic nucleus of mice.
      • Dardente H.
      • Menet J.S.
      • Challet E.
      • Tournier B.B.
      • Pevet P.
      • Masson-Pevet M.
      Daily and circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal rodents.
      Distinct decreases in Avp and Vip were noted especially at the peak levels (Figure 5, C and D). To verify the above results, we performed in situ hybridization with Avp and Vip probes. Avp and Vip levels in Hnrnpu+/− mice were prominently reduced compared with wild-type controls (Figure 5, E and F). The SCN structures in Hnrnpu+/− mice were grossly normal (Figure 5, A, E, and F), and SCN sizes were normal in both genotypes (Figure 5G).
      To further examine whether hnRNP U directly regulated the transcription of these two neuropeptides, luciferase was used under the control of the Avp promoter and Vip promoter to evaluate the influence of hnRNP U as described in the Materials and Methods. Luciferase vectors were cotransfected with hnRNP U–specific siRNAs (sihnRNP U1 and sihnRNP U2) or scrambled siRNA (siSCR) into HEK 293T cells.
      • Xiao R.
      • Tang P.
      • Yang B.
      • Huang J.
      • Zhou Y.
      • Shao C.
      • Li H.
      • Sun H.
      • Zhang Y.
      • Fu X.D.
      Nuclear matrix factor hnRNP U/SAF-A exerts a global control of alternative splicing by regulating U2 snRNP maturation.
      • Ye J.
      • Beetz N.
      • O'Keeffe S.
      • Tapia J.C.
      • Macpherson L.
      • Chen W.V.
      • Bassel-Duby R.
      • Olson E.N.
      • Maniatis T.
      hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.
      The RNA interference (RNAi) efficiency was approximately 40% to 80% as detected by qPCR 24, 48, and 72 hours after transfection (Supplemental Figure S2). The transcriptional activity of both promoters was significantly inhibited by hnRNP U knockdown (Figure 5H), demonstrating that hnRNP U mediates Avp and Vip expression at the transcription level.

       Abnormal Activity Is Partially Restored by Avp and Vip

      To explore the causal link between these neuropeptides and abnormal activity in Hnrnpu+/− mice, suprachiasmatic microinjection was performed with synthetic Avp and Vip neuropeptides for a consecutive 7 days. Before injection, mice were entrained to LD cycles and then transferred to DD. After stable rhythms were observed, 0.5 μL of Avp (150 nmol), Vip (150 nmol), or scrambled peptide (150 nmol) was infused into the SCN region at CT4 to CT6 every day.
      • An S.
      • Harang R.
      • Meeker K.
      • Granados-Fuentes D.
      • Tsai C.A.
      • Mazuski C.
      • Kim J.
      • Doyle 3rd, F.J.
      • Petzold L.R.
      • Herzog E.D.
      A neuropeptide speeds circadian entrainment by reducing intercellular synchrony.
      • Shinohara K.
      • Honma S.
      • Katsuno Y.
      • Abe H.
      • Honma K.
      Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.
      • Piggins H.D.
      • Antle M.C.
      • Rusak B.
      Neuropeptides phase shift the mammalian circadian pacemaker.
      • Albers H.E.
      • Gillespie C.F.
      • Babagbemi T.O.
      • Huhman K.L.
      Analysis of the phase shifting effects of gastrin releasing peptide when microinjected into the suprachiasmatic region.
      The activity distribution was assessed before and after injection. The daytime activity was significantly decreased in Hnrnpu+/− mice after Avp or Vip injection, indicating that the activity pattern was partially rescued by these two neuropeptides (Figure 6). These data suggest that Avp and Vip are associated with hnRNP U–mediated circadian outputs.
      Figure thumbnail gr6
      Figure 6Suprachiasmatic microinjection partially rescues the activity patterns. A: Mice were entrained to white light between 8:00 and 20:00 and darkness between 20:00 and 8:00 (LD) cycles and then released into constant darkness (DD). Arginine vasopressin (Avp) (‡), vasoactive intestinal polypeptide (), and a scrambled peptide (†) were injected into the suprachiasmatic nucleus. The locomotor activity patterns 1 week before and after injection are presented in the red and yellow rhombi. B: Hnrnpu+/− mice activity in the daytime is not reduced after control injection. C: Hnrnpu+/− mice activity in the daytime significantly reduces after Avp injection. D: Hnrnpu+/− mice activity in the daytime reduces after Vip injection. Data are expressed as means ± SD (B–D). n = 3. P < 0.05, unpaired two-tailed t-test. Ctl, control.

       HnRNP U Bound to the Avp and Vip Promoters

      Because hnRNP U RNAi inhibited Avp-promoter and Vip-promoter activity in the luciferase assay, we hypothesized that hnRNP U regulated Avp and Vip expression by binding to their promoters directly. Chromatin immunoprecipitation was performed using anti-hnRNP U antibody and nIgG. The binding efficiency was detected in mouse anterior ventral hypothalamus. The efficiency was reduced in Hnrnpu+/− mice compared with littermate controls (Figure 7A). To further explore the molecular mechanism, their interaction was examined in the SCN2.2 cell line.
      • Earnest D.J.
      • Liang F.Q.
      • Ratcliff M.
      • Cassone V.M.
      Immortal time: circadian clock properties of rat suprachiasmatic cell lines.
      hnRNP U bound to Avp and Vip promoters in SCN2.2 cells (Figure 7B).
      Figure thumbnail gr7
      Figure 7Heterogeneous nuclear ribonucleoprotein U (hnRNP U) bound to arginine vasopressin (Avp) and vasoactive intestinal polypeptide (Vip) promoters. A: hnRNP U bound to Avp and Vip promoters in the Hnrnpu+/+ hypothalamus but not Hnrnpu+/− hypothalamus. B: hnRNP U bound to Avp and Vip promoters in the SCN2.2 cells. C: hnRNP U knockdown disrupts brain and muscle Arnt-like protein-1 (Bmal1) binding to Avp and Vip promoters in suprachiasmatic nucleus (SCN) 2.2 cells. D: hnRNP U knockdown disrupts circadian locomotor output cycles kaput (Clock) binding to Avp and Vip promoters in SCN2.2 cells. Data are normalized to normal IgG (nIgG) and presented as means ± SD. n = 5 Hnrnpu+/+ mice; n = 3 Hnrnpu+/− mice; and n = 8 nIgG mice (A); n = 3 (B–D). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, two-way analysis of variance followed by Bonferroni post hoc test (A, C and D) and unpaired two-tailed t-test (B). αBmal1, anti-Bmal1; αClock, anti-clock; αhnRNP U, anti-hnRNP U; αnIgG, anti-normal IgG; ChIP, chromatin immunoprecipitation.
      HnRNP U affected some transcription factor binding to specific promoters.
      • Vizlin-Hodzic D.
      • Johansson H.
      • Ryme J.
      • Simonsson T.
      • Simonsson S.
      SAF-A has a role in transcriptional regulation of Oct4 in ES cells through promoter binding.
      • Vizlin-Hodzic D.
      • Runnberg R.
      • Ryme J.
      • Simonsson S.
      • Simonsson T.
      SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.
      Avp and Vip are two well-established clock-controlled genes, and Bmal1 and Clock transcribed these genes periodically by binding to their promoters.
      • Reppert S.M.
      • Weaver D.R.
      Molecular analysis of mammalian circadian rhythms.
      • Hurst W.J.
      • Mitchell J.W.
      • Gillette M.U.
      Synchronization and phase-resetting by glutamate of an immortalized SCN cell line.
      Thus, we hypothesized that hnRNP U might affect the binding between Bmal1/Clock heterodimers and Avp and Vip promoters in a similar manner. The binding efficiency was separately examined in SCN2.2 cells transfected with siSCR or small interfering hnRNP U1. hnRNP U RNAi strongly suppressed Bmal1 and Clock binding to the Avp and Vip promoters (Figure 7, C and D). These results demonstrate that hnRNP U is indispensable for Bmal1 and Clock binding to Avp and Vip promoters.

      Discussion

      Circadian expression of hnRNP U in rodents has been known for years. Patients with one copy deletion of Hnrnpu experience sleep disorders.
      • Thierry G.
      • Beneteau C.
      • Pichon O.
      • Flori E.
      • Isidor B.
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      • Thuresson A.C.
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      • Arveiler B.
      • de Vries B.B.
      • Jonveaux P.
      • David A.
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      Molecular characterization of 1q44 microdeletion in 11 patients reveals three candidate genes for intellectual disability and seizures.
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      Up to half of elderly individuals have chronic sleep disturbances, and one-quarter to a half have several kinds of nocturnal restlessness at some stage of diseases.
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      In this report, we found that Hnrnpu haploinsufficiency in mice led to abnormal rest-activity cycles with obviously increased activity in the daytime and reduced activity at nighttime. Defective expression of Avp and Vip participate in mediating these irregular activity patterns and abnormal metabolic rhythms.
      Avp and Vip are crucial for the pacemaker to synchronize with the clock in peripheral tissues. The phenotypes of Hnrnpu+/− mice were similar to Vip−/− mice or V1a−/− mice with defects in central and peripheral clock coupling.
      • Li J.D.
      • Burton K.J.
      • Zhang C.
      • Hu S.B.
      • Zhou Q.Y.
      Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei.
      • Colwell C.S.
      • Michel S.
      • Itri J.
      • Rodriguez W.
      • Tam J.
      • Lelievre V.
      • Hu Z.
      • Liu X.
      • Waschek J.A.
      Disrupted circadian rhythms in VIP- and PHI-deficient mice.
      • Aton S.J.
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      • Herzog E.D.
      Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons.
      All these mice expressed fragmented locomotor activities and ambiguous activity onset lines. Hnrnpu+/− mice were similar to LIM homeodomain transcription factor 1 (Lhx1) knockout mice given that Lhx1 is a regulator of SCN-enriched neuropeptides. Without Lhx1, mice were deficient in Avp and Vip.
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      Lhx1 controls terminal differentiation and circadian function of the suprachiasmatic nucleus.
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      Lhx1 maintains synchrony among circadian oscillator neurons of the SCN.
      Thus, it is reasonable to speculate that HnRNP U regulates circadian rhythm outputs through Avp and Vip.
      Several studies have indicated that hnRNP U bound directly to DNA and regulated gene expression.
      • Onishi Y.
      • Hanai S.
      • Ohno T.
      • Hara Y.
      • Ishida N.
      Rhythmic SAF-A binding underlies circadian transcription of the Bmal1 gene.
      • Matsuoka Y.
      • Uehara N.
      • Tsubura A.
      hnRNP U interacts with the c-Myc-Max complex on the E-box promoter region inducing the ornithine decarboxylase gene.
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      SAF-A has a role in transcriptional regulation of Oct4 in ES cells through promoter binding.
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      SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.
      In most cases, hnRNP U bound to DNA regions named matrix associated regions in a nonspecific manner, whereas data in this paper and other papers have indicated that hnRNP U could bind to specific DNA regions with some other specific transcription factors and regulators. Our previous work suggested that hnRNP U participated in the long-distance regulation of Shh by binding to a cis-element 1 Mb away from the transcription starting point.
      • Zhao J.
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      In embryonic stem cells, hnRNP U bound to oct4 proximal promoter and recruited RNA polymerase II.
      • Vizlin-Hodzic D.
      • Johansson H.
      • Ryme J.
      • Simonsson T.
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      SAF-A has a role in transcriptional regulation of Oct4 in ES cells through promoter binding.
      Moreover, hnRNP U bound to E-box regions of the clock-controlled gene Odc and activated its transcription together with the c-Myc-Max complex.
      • Matsuoka Y.
      • Uehara N.
      • Tsubura A.
      hnRNP U interacts with the c-Myc-Max complex on the E-box promoter region inducing the ornithine decarboxylase gene.
      Both Avp and Vip are well-established clock-controlled genes that contain E-boxes in their promoters.
      • Reppert S.M.
      • Weaver D.R.
      Molecular analysis of mammalian circadian rhythms.
      • Hurst W.J.
      • Mitchell J.W.
      • Gillette M.U.
      Synchronization and phase-resetting by glutamate of an immortalized SCN cell line.
      hnRNP U interacted with Avp and Vip promoters together with Bmal1 and Clock. In addition, hnRNP U might not bind to all E-boxes because the expression of Per2, Cry1, and Dbp was not altered in Hnrnpu+/− mice. Other potential cis-elements and trans-acting factors that regulate the expression of these genes might exist.
      Interestingly, changes in the Bmal1 transcript were not observed in mutant mice, although it has been reported that hnRNP U regulates Bmal1 expression in NIH 3T3 cells.
      • Onishi Y.
      • Hanai S.
      • Ohno T.
      • Hara Y.
      • Ishida N.
      Rhythmic SAF-A binding underlies circadian transcription of the Bmal1 gene.
      We hypothesized that the residual hnRNP U in heterozygous mice might be sufficient to sustain typical Bmal1 expression, whereas Avp and Vip promoters are more sensitive to hnRNP U dosage.
      Significantly, mammals maintain circadian rhythms not only in an appropriate period and phase but also in adequate robustness and amplitude. For diurnal animals, like humans, most activity should occur in the daytime, whereas for nocturnal animals, like mice, most activity should be at night. Logically, there are two main possible factors related to circadian expression of activity: the stability of the oscillator per se and the extent of coupling between the pacemaker and peripheral oscillators.
      • Satinoff E.
      • Li H.
      • Tcheng T.K.
      • Liu C.
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      • Gillette M.U.
      Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?.
      The data presented here underline the significance of the latter factor.
      In summary, our work identifies distinct transcriptional networks of Avp, Vip, Bmal1, Clock, and hnRNP U. hnRNP U mediates Bmal1/Clock, transcribing these neuropeptides periodically. These results not only provide new insights into the highly complicated regulation of SCN but also aid understanding of the pathogenesis of human 1q44 deletion syndrome.
      • Thierry G.
      • Beneteau C.
      • Pichon O.
      • Flori E.
      • Isidor B.
      • Popelard F.
      • Delrue M.A.
      • Duboscq-Bidot L.
      • Thuresson A.C.
      • van Bon B.W.
      • Cailley D.
      • Rooryck C.
      • Paubel A.
      • Metay C.
      • Dusser A.
      • Pasquier L.
      • Beri M.
      • Bonnet C.
      • Jaillard S.
      • Dubourg C.
      • Tou B.
      • Quere M.P.
      • Soussi-Zander C.
      • Toutain A.
      • Lacombe D.
      • Arveiler B.
      • de Vries B.B.
      • Jonveaux P.
      • David A.
      • Le Caignec C.
      Molecular characterization of 1q44 microdeletion in 11 patients reveals three candidate genes for intellectual disability and seizures.

      Acknowledgments

      All authors contributed intellectually; X.G., Y.X., and B.L. conceptualized and designed the research; B.L. and J.Z. performed most experiments and analyzed data; B.L. drafted the manuscript; Z.L. revised the manuscript; X.G. approved the final version of the manuscript; Z.Q. aided in the wheel running test; and A.S. assisted with molecular experiments.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Central clock genes are intact in the liver of Hnrnpu+/− mice. A–J: Representative mRNA profiles of central clock genes were analyzed in Hnrnpu+/+ and Hnrnpu+/− livers. Livers were collected at 4-hour intervals during the first day in constant darkness (DD) for a total of 24 hours. Relative gene levels were detected with real-time quantitative PCR and normalized to β-actin. No significant difference is detected in Bmal1, Clock, Per1, Per2, Cry1, Cry2, Dbp, Rev-erbα, Rev-erbβ, and Rorα between Hnrnpu+/+ and Hnrnpu+/−. K and L: Alternative splicing of clock mRNA is detected by measuring isoform and total mRNA levels. No significant differences are detected in the specific isoform and the splicing ratio. Data are expressed as means ± SD. n = 21. P < 0.05, two-way analysis of variance followed by Bonferroni post hoc test.
      Figure thumbnail figs2
      Supplemental Figure S2Heterogeneous nuclear ribonucleoprotein U (hnRNP U) RNA interference (RNAi) efficiency. Two hnRNP U–specific siRNA (siU1 and siU2) and a scrambled siRNA (siSCR) were transfected into cells. The RNAi efficiency was detected by real-time quantitative PCR. Data are expressed as means ± SD. n = 2. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, two-way analysis of variance followed by Bonferroni post hoc test.

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