Wang Y et al. (2001), Rhythmic expression of Nocturnin mRNA in multip...

XB-ART-8933

BMC Dev Biol 2001 Jan 01;1:9. doi: 10.1186/1471-213x-1-9.

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Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse.

Wang Y , Osterbur DL , Megaw PL , Tosini G , Fukuhara C , Green CB , Besharse JC .

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BACKGROUND: Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. RESULTS: cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. CONCLUSION: The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

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Species referenced: Xenopus laevis
Genes referenced: actl6a ccr4 clock hhip noct pir sri

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	Figure 1. Comparison of the conceptual amino acid sequences of nocturnin homologues from chicken (CNOC), cow (BNOC), rat, (RNOC), Xenopus (XNOC), mouse (MNOC), human (hNoc) and Drosophila (DNOC). Sequences were analyzed using the Clustal W Alignment procedure. Gaps indicated with dots are inserted to achieve optimum alignment. Dark gray and light gray highlights indicate amino acid identities and similarities respectively. The horizontal bar marks the position of the heptad leucine repeat in Xenopus and the asterisks indicate the position of the leucines. The positions of introns 1 and 2 based on the Xenopus gene [36] are indicated by arrows. Note that both mouse [39] and human (from data in public databases of the National Library of Medicine) appear to have a similar gene structure based on 3 exons. The XNOC sequence is from GenBank accession number U74761, HNOC is from NP036250.1 [39], and DNOC is from AAF54601.1 [40]. Our complete mNoc cDNA from retina (accession number AF199491) has the same coding sequence as that reported earlier from liver [38,39]. The cNoc (AF199498), bNoc (AF199497), and rNoc (AF199495) partial sequences are from PCR amplified DNA segments.
	Figure 2. mNoc mRNA is expressed in multiple tissues of the C3H/He mouse. Tissues for RNA extraction were collected at Zeitgeber Time (ZT) 12 (time of normal light offset) except for that from embryo and thymus, which were purchased from Ambion (Austin, TX); the latter samples were from Swiss mice. Ten μg of total RNA was loaded in each lane except for liver where only 5 μg was loaded. The lower panel shows methylene blue staining of the 28 and 18s ribosomal RNA bands after blotting.
	Figure 3. mNoc mRNA is expressed rhythmically in C3H/He mouse retina (A), heart (B), spleen (C), and kidney (D) in a light dark cycle (LD). Tissues for RNA extraction were collected at Zeitgeber Times (ZT) 0 (24), 6, 12 and 18 with lights on at ZT 0 and off at ZT12. A through D are typical blots of mNoc for each tissue, and the lower panel is a hybridization of the same membrane with a β-actin probe. These blots are representative of three replicate experiments. In E phosphor imaging was used for quantitation of changes in mNoc mRNA level seen in A-D, standardized to β-actin. The minimum for each plot is one and the Y-axis shows the fold change.
	Figure 4. mNoc mRNA in liver exhibits a high amplitude rhythm with peak expression at ZT12 in both LD (A and C) and DD (B and D). In LD samples were taken at 6 hour intervals as in Figure 4. Samples in DD were taken at Zeitgeber Times (ZT) 0 (24), 4, 8, 12, 16 and 20 referenced to the LD cycle immediately before DD treatment. Mice were in DD for 36 hours before beginning collections. The rhythmic changes illustrated are representative of three replicates for LD and two for DD. Phosphor imaging was used as in Figure 4E to quantitate mNoc mRNA levels (C and D). Note that the amplitudes of the rhythms are much higher in liver than for other tissues.
	Figure 5. mNoc mRNA is expressed rhythmically in C3H/He mouse retina (A), heart (B), spleen (C), and kidney (D) in constant darkness (DD). Tissues for RNA extraction were taken in DD at Zeitgeber Times (ZT) 0 (24), 4, 8, 12, 16 and 20 referenced to the LD cycle immediately before DD treatment. Mice were in DD for 36 hours before beginning collections. Note that for most tissues the RNA yield was low at ZT16 for technical reasons; the lower actin signal is taken into account in the quantitation. The rhythmic changes illustrated are representative of two replicate experiments. In E, phosphor imaging was used for quantitation of the changes seen in A-D, standardized to β-actin; the minimum for each plot is one and the Y-axis shows the fold change.
	Figure 6. C3H/He and 129/SV mice lack an intracisternal A-particle (IAP) insert in the first intron of the nocturnin/CCR4 gene. A. Diagram showing the results of partial sequencing of a nocturnin genomic clone derived from a 129/SV bacterial artificial chromosome (BAC) library. Green boxes identify regions of medium repetitive sequences in the mouse genome. The blue arrows represent mNoc coding regions corresponding to Xenopus exons II and III separated by an intron. The IAP element identified in BALB/c mice (bar above) was expected between bp 6583 and 6584 in our BAC sequence based on published data [39] but was lacking in this BAC sequence. F1, F2 and R1 are the positions of forward and reverse primers used for genomic PCR. Note that there are two potential polyadenylation sites in the 3' UTR 650 base pairs apart. The 3' UTR probe used in our northern analysis lies between the two potential polyadenylation sites, and hybridizes to the same mRNA band as the probe derived from Exon II. This suggests that only the most 3' site is used. B. Genomic PCR with primers from the BAC clone flanking the IAP site (labeled as F1 and R1 in A) resulted in a 107 bp band in C3H/He and 129SV mice that was lacking in BALB/c mice. Pairing the R1 primer from the BAC clone with primer F2 derived from the IAP sequence (see A) resulted in a 1500 bp band in BALB/c mice but not 129SV or C3H/He. DNA sequencing revealed the expected sequence from the BAC clone for the 107 bp band and the expected sequence from the IAP insert for the 1500 bp band. The lanes labeled 412D6 are control PCR reactions using the 129/SV BAC as template. Note that the absence of ≅ 5 Kb band for BALB/c mice with primers F1 and R1 is due to the inefficiency of Taq polymerase for large products; we have separately obtained the full IAP insert in BALB/c mice using a long PCR procedure. DNA size markers are included in the two outer lanes.
	Figure 7. mNoc mRNA is expressed rhythmically in 8 week old BALB/c mouse liver (A) and kidney (B) in a light dark cycle (LD). Tissues for RNA extraction were collected at Zeitgeber Times (ZT) 0 (24), 6, 12 and 18 with lights on at ZT 0 and off at ZT12. Images of the methylene blue stained 28S and 18S rRNA bands on the same blot are shown below as loading controls.
	Figure 8. In situ hybridization of coronal sections through the mouse brain shows that mNoc is expressed in multiple brain regions. A. Section showing intense hybridization of an antisense probe in the suprachiasmatic nucleus (SCN). B. Section showing hybridization in the arcuate nucleus (ARC), piriform cortex (Pir), and hippocampus (Hip). C. Section showing hybridization in the cerebellum.D. Section showing intense hybridization in the olfactory bulbs. E. Section through the olfactory bulbs showing lack of hybridization of a sense probe.

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Adams, The genome sequence of Drosophila melanogaster. 2000, Pubmed