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Human Molecular Genetics Advance Access originally published online on August 7, 2006
Human Molecular Genetics 2006 15(18):2732-2742; doi:10.1093/hmg/ddl209
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Identification of the transcription factor ARNTL2 as a candidate gene for the type 1 diabetes locus Idd6

Ming-Shiu Hung{dagger}, Philip Avner and Ute Christine Rogner*

Unité de Génétique Moléculaire Murine CNRS URA 2578, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France

* To whom correspondence should be addressed. Tel: +33 145688602; Fax: +33 145688656; Email: urogner{at}pasteur.fr

Received July 3, 2006; Accepted July 31, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
The Idd6 murine type 1 diabetes locus has been shown to control diabetes by regulating the protective activity of the peripheral immune system, as demonstrated by diabetes transfer assays using splenocytes. The analysis of three novel subcongenic (NOD.C3H nonobese. C3H) diabetes strains has confirmed the presence of at least two diabetes-related genes within the 5.8 Mb Idd6 interval with the disease protection conferred by splenocyte co-transfer being located to the 700 kb Idd6.3 subregion. This subinterval contains the circadian rhythm-related transcription factor Arntl2 (Bmal2), a homologue of the type 2 diabetes-associated ARNT (HIF1ß) gene. Arntl2 exhibited a six-fold upregulation in spleens of the NOD.C3H 6.VIII congenic strain compared with the NOD control strain, strain-specific splice variants and a large number of polymorphisms in both coding and non-coding regions. Arntl2 upregulation was not associated with changes in the expression levels of other circadian genes in the spleen, but did correlate with the upregulation of the ARNT-binding motif containing Pla2g4a gene, which has recently been described as being protective for the progression of insulitis and autoimmune diabetes in the NOD mouse via regulation of the tumour necrosis factor-alpha pathway. Our studies strongly suggest that the HIFß-homologous Arntl2 gene is involved in the control of type 1 diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Type 1 or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by the progressive destruction of insulin-producing ß-cells of the islets of Langerhans by infiltrating lymphocytes (1). The disease, which affects ~0.3% of the Caucasian population, is both multifactorial and polygenic, with the major histocompatibility complex (MHC) class II locus and the insulin locus being the two best studied genetic loci (2,3).

The nonobese diabetes (NOD) mouse (4,5) is a well-characterized animal model of IDDM. More than 20 murine insulin-dependent diabetes susceptibility loci (Idd) have been genetically identified (6), but little information has been obtained about the nature of these non-MHC Idd genes. Construction of congenic strains, differing from the NOD receiver strain by only a selected genetic region derived from a non-diabetes-prone parental donor strain (7,8), is a widely used approach allowing the definition of disease-related candidate regions. A promising strategy for candidate gene identification is to combine a variety of phenotypic studies of congenic mice with expression profiling, haplotype and mutational analysis (9–12).

Several Idd loci have been identified on mouse chromosome 6 (13–15). The IDD-associated loci Idd6, Idd19 and Idd20 on distal chromosome 6 have been further defined by the analysis of a series of congenic strains, carrying C3H/HeJ genomic material for distal chromosome 6 introgressed onto the NOD/Lt genetic background, with their candidate regions being refined, respectively, to 4.5, 7 and 4 cM (16).

NOD/Lt alleles at the Idd6 locus on distal mouse chromosome 6 confer susceptibility to IDDM, whereas C57BL/6, C57BL/10 and C3H/HeJ alleles all confer resistance to diabetes (13,16,17). The NOD.C3H congenic strain described in this study carries NOD alleles at both the natural killer gene complex (17) and the candidate region for the islet-specific BDC-6.9 autoantigen gene (18) (K. Haskins, personal communication; our unpublished data), which excludes both these loci as responsible for the disease resistance. The Idd6 candidate region does however overlap with the candidate region for the resistance of immature T-cells to dexamethasone (19–21). Idd6 has also been suggested to control low rates of proliferation in immature NOD thymocytes (22).

Recently, we have undertaken a detailed phenotypic analysis of the Idd6 locus containing congenic strain NOD.C3H 6.VIII (16), which shows resistance to the spontaneous development of diabetes. We have shown that this resistance is not ascribable to the resistance of islet ß-cells to immune destruction or to a default in pathogenic T cells. Protection of the congenic strain likely involves changes in the proportions of the various leukocyte subsets infiltrating the pancreatic islet and, in particular, that of CD4+ T cells. Critical to our understanding of the reduced diabetes susceptibility of the Idd6 congenic mice has been our finding that their splenocytes conferred enhanced disease protection in diabetes transfer assays (23).

The present study describes the transcriptional profiling of some 81 identified transcripts falling within the Idd6 interval. A total of six transcripts distributed throughout the interval were found to have strongly altered expression profiles when comparing splenic tissues of the disease-protected congenic NOD.C3H 6.VIII and a NOD control strain. Analysis of newly created subcongenic strains showed the presence of at least three diabetes-related subloci within the Idd6 locus. The recently identified control of disease protection-mediated splenocytes was mapped to a 700 kb interval, which contains the aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2, Bmal2) encoding gene. This candidate gene was strongly upregulated in the NOD.C3H 6.VIII congenic strain and exhibited a large number of sequence polymorphisms and alternative splice variants. Arntl2 upregulation correlated with the upregulation of the ARNT-binding site containing Pla2g4a gene that has recently been shown to be required for protection against insulitis progression and autoimmune diabetes development. We suggest that Arntl2 and its downstream targets play a major role in controlling type 1 diabetes resistance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Refinement of the Idd6 interval by analysis of subcongenic strains
The original microsatellite-based genotyping of the diabetes-resistant Idd6 congenic strain NOD.C3H 6.VIII (strain 6.VIII) indicated that the C3H introgressed donor sequence was located at the end of chromosome 6, distal to the microsatellite marker D6Mit113 (16,24). Random sampling of potential SNPs listed in the genomic databases identified four polymorphisms located between base pairs 144 874 468 and 144 874 516 on mouse chromosome 6. These SNPs included a SNP at base pair position 144 874 516 associated with a silent amino acid exchange in the Sox5 gene, located distal to D6Mit113 (Ensembl mouse database, http://www.ensembl.org/ Mus_musculus) (Supplementary Material, Table S1). The mapping of these newly identified SNPs allowed the size of the Idd6 interval to be estimated as between 5.3 Mb (Sox5 to the telomere at 150.2 Mb) and 5.8 Mb (D6Mit113 to telomere).

In order to further refine the type 1 diabetes-associated Idd6 candidate region, we constructed a series of subcongenic strains by intercrossing the Idd6 congenic NOD.C3H 6.VIII strain (6.VIII) and the NOD control congenic (CO) strain, which were originally derived from crosses between the C3H/HeJ and NOD/Lt mouse strains. Heterozygous male mice resulting from the intercross were then again backcrossed to the CO strain, and recombinants were selected among the offspring using the polymorphic markers D6Mit14, D6Mit15, D6Mit294 and D6Mit304. Out of the 200 BC1 animals that were tested, three were found to be recombinants. The corresponding subcongenic intervals were fixed by further backcrossing to the CO strain and by intercrossing of the heterozygous recombinant animals. Genotyping using a large marker panel for distal chromosome 6 allowed the estimation of the size of the C3H-derived intervals. We noticed that all three breakpoints for recombination were located between the markers D6Mit294 and D6Mit373, suggesting that this region may recombine more frequently than others within the Idd6 interval (Fig. 1).


Figure 2091
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  		Figure 1. Map showing the C3H-derived intervals (grey bars) on distal chromosome 6 contained in the original NOD.C3H 6.VIII and new subcongenic mouse strains 6.VIIIa, 6.VIIIb and 6.VIIIc. The localization of the newly defined candidate region for the splenocyte subphenotype (Idd6.3) is indicated by dotted lines. Based on NCBI Human version 35.1 June 2004 and Mouse version 34.1 Feb 28, 2005.

 
We tested the diabetes incidence weekly for all three subcongenic strains in parallel to the parental strains over a period of 30 weeks (Fig. 2A). In female mice, all the newly created strains were significantly protected compared with the CO strain, although each strain was slightly less protected than the 6.VIII strain. Data were similar for male mice, although male mice of each strain developed less diabetes than female mice (data not shown). This result led us to conclude that at least two intervals (Idd6.1 and Idd6.2) and several genes in the Idd6 region contribute to the overall diabetes protection of the 6.VIII strain (Fig. 1, Table 1).


Figure 2092
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  		Figure 2. (A) Cumulative diabetes incidences of different mouse strains and (B) diabetes protection in splenocyte co-transfer. P-values are <0.0001 for 6.VIII, <0.0001 for 6.VIIIa, 0.01 for 6.VIIIb and 0.026 for 6.VIIIc against CO in diabetes incidence; 0.012 for 6.VIII, 0.568 for 6.VIIIa, 0.339 for 6.VIIIb and 0.048 for 6.VIIIc against CO, <0.0001 for CO against diabetogenic splenocytes (Db) in diabetes transfer assay. F, females; R, NOD/scid recipients.

 


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  		Table 1. Genes that are significantly differentially expressed in the diabetes-resistant strain 6.VIII compared with the diabetes-sensitive NOD mice at 6–7 weeks of age

 
Analysis of inhibition of diabetes transfer
We have previously shown that Idd6 modifies suppression of diabetes in co-transfer assays when using splenocytes (23). We tested whether this splenocyte subphenotype segregated with one or other of the newly derived C3H-derived subintervals. A total of 2x107 splenocytes from 7-week-old mice were injected into NOD/Scid recipient mice, together with 107 total splenocytes from diabetic mice. As expected, injection of the diabetogenic cells alone resulted in the rapid induction of diabetes in the NOD/Scid recipient. Co-transfer of splenocytes inhibited significantly the diabetes transfer in all the groups tested (Fig. 2B). As previously described, stronger protection was found with the 6.VIII splenocytes than with the CO splenocytes. Similar significant protection was found for strain 6.VIIIc, but not for either strain 6.VIIIa or 6.VIIIb. It should be noted that strain 6.VIIIc differs from strain 6.VIIIa by only a 700 kb C3H-derived interval (Idd6.3). Strain 6.VIIIc carries C3H alleles at markers D6Mit373 (147.9 Mb) and D6Mit15 (147.3 Mb) but NOD alleles at D6Mit294 (147.2 Mb). Strain 6.VIIIa carries C3H alleles at D6Mit373 and NOD alleles at both D6Mit15 and D6Mit294 (Fig. 1).

Transcriptional profiling of genes in the Idd6 interval
Diabetes-associated genes are expected to be either functional coding sequence variants or to show differential regulation between diabetes sensitive and a diabetes-resistant strains. Detection of functional coding variants would require extensive sequencing efforts throughout the entire Idd6 candidate interval and would likely throw up a very large number of sequence variants for evaluation that need to be functionally addressed, as even synonymous and non-exonic variations can have significant effects on gene function and expression.

We turned therefore first to expression analysis for the identification of potential candidate genes responsible for the susceptibility to IDDM. A total of 123 potential transcripts within Idd6 were identified from a combined manual analysis of the Celera and public database annotations, with additional information being obtained by examination of the syntenic region to Idd6 in the human genome, which maps to the 12p11–p12.2 chromosomal region (NCBI version 35.1, Fig. 1). We were able to confirm 81 of the 123 putative transcripts by RT–PCR as being expressed in spleen, thymus, brain and/or testis. As our previous results had indicated that splenocytes contribute to the disease regulation mediated by Idd6, those transcripts that were not expressed in spleen were eliminated from further consideration. All genes that we found to be expressed in the spleen, leaving a total of 70, were then analysed by real-time RT–PCR for differential expression in the diabetes-sensitive NOD mice and the diabetic-resistant congenic strain 6.VIII. Spleen samples from both 4-week-old and 6–7-week-old mice were chosen in order to capture genes showing differences during the primary stages of disease progression before the onset of overt diabetes. Six transcripts were found to have such differential expression in spleen. These genes were Bcat1, Csac1 (Las1), Arntl2 (Bmal2), a gene of unknown function represented by two EST clones BE647206 [GenBank] and AW120472 [GenBank] , Mlstd1 (Msl2) and the predicted transcript mCG1027210 (Celera database) (Table 1).

The 700 kb Idd6.3 interval contains a total of 10 genes (Fig. 1, Table 2) with seven transcripts, 4933424B01Rik, Tm7sf3, Stk381, LOC232534, 1700023A16Rik, Ppfib1 and 2210417D09Rik, being unlikely candidates for IDDM either because of their known role or inappropriate expression pattern. Two others, Surb7, a suppressor of RNA polymerase B homologue, and the FGFR1 oncogene partner 2 gene (Fgrf1op2), are ubiquitously expressed. Sequence analysis of the coding regions of these genes failed to reveal any polymorphism between NOD and C3H for Fgfr1op2, whereas a single polymorphism in Surb7 (6.VIII: A, NOD CO: G at base pair 173 of NM_025315 [GenBank] .1) was identified, which is however only associated with a synonymous amino acid change. None of the nine genes showed differential expression between the 6.VIII and CO strains, unlike the remaining candidate gene Arntl2 (Bmal2, brain-muscle-ARNT-like protein 2), which was found to be six-fold overexpressed in spleens of the 6.VIII strain. The localization of Arntl2 within the 700 kb Idd6.3 interval was confirmed by direct mapping of a length polymorphism lying within its 3'-UTR (discussed subsequently). In addition, the Arntl2 overexpression segregated exclusively with the C3H-derived interval of strain 6.VIIIc, but not with the stain 6.VIIIa or 6.VIIIb C3H-derived intervals (data not shown). We therefore turned into a more detailed analysis of the Arntl2 candidate gene.


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  		Table 2. Candidate genes in the Idd6.3 candidate region

 
Expression and transcript analysis of the Arntl2 candidate gene
The Arntl2 gene encodes a basic helix–loop–helix-Per-Arnt-Sim (bHLH-PAS) transcription factor and has been functionally linked to circadian clock-mediated activities and to the regulation of cell proliferation (25). The Arntl2 gene was expressed in significantly higher amounts in spleen samples obtained from either 4-week-old or 6–7-week-old diabetes-resistant strain 6.VIII animals than from diabetes-sensitive NOD mice. Thymi obtained from the same groups of animals showed a similar tendency, although with only two-fold difference, between the strain 6.VIII and NOD mice (Fig. 3A). Lymphocyte subsets, including B cells, CD4+ T cells, CD8+ T cells and CD4+CD25+ regulatory T cells, showed similar expression differences to the whole tissue preparations (data not shown). Further expression profiling of spleens showed that differential expression of Arntl2 was independent of the age of the animals and maintained from 2 to 12 weeks of age and in diabetic animals (Fig. 3B). These results suggest that the differential expression of Arntl2 in the two mouse strains is independent of disease progression.


Figure 2093
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  		Figure 3. The relative expression of the Arntl2 (Bmal2) gene in strain 6.VIII (spleen: white bars; thymus: white-shaded bars) and NOD CO mice (spleen: black bars; thymus: black-shaded bars) analysed by quantitative RT–PCR is indicated as arbitrary units±standard deviation. RNA was pooled from spleen and thymus of four pre-diabetic female mice (A), from spleens of four pre-diabetic females at different ages and 15-week-old diabetic mice (B). P-values for the expression differences in spleen are all <0.005 and in thymus <0.04.

 
The transcript pattern of Arntl2 in multiple tissues of strain 6.VIII and NOD mice was examined in detail (Fig. 4). For most of the organs examined such as brain or lung, the major transcripts identified were 9 or 0.6 kb in size. The transcript profiles of the spleen and thymus were however surprisingly varied compared with those of other organs, and additional 3.9 and 1.6 kb transcripts were found in both spleen and thymus. A 1.4 kb transcript was exclusively found in thymus. The 0.6 kb transcript, which corresponds to the 3' end of the Arntl2 coding region (including exon 17, data not shown), was present in most of the organs but not in the thymus (Fig. 4A). Such complex transcription profiles with transcripts specific to the spleen and thymus may indicate a role for specific splice forms of the Arntl2 gene in the immune system.


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  		Figure 4. Transcription profile and architecture of Arntl2. (A) Transcript profiles of Arntl2 in NOD and 6.VIII mice were identical as shown by Northern blotting. 1, thymus; 2, testis; 3, spleen; 4, skeletal muscle; 5, lung; 6, liver; 7, kidney; 8, heart; 9, brain; the 18S RNA band is shown as loading control. (B) Transcripts corresponding to the isoforms Arntl2a and Arntl2b, and the putative isoform Arntl2c of 579 199 and 355 amino acids length, were present in the spleen of both strains. (C) Partial sequences identified in the spleen show the presence of strain-specific isoforms, with 6.VIII forms skipping exons 12 and 13 and NOD forms skipping exon 14.

 
Prior studies have identified two protein products of Arntl2, Arntl2a and Arntl2b (26) containing, respectively, 579 and 199 amino acids. While examining the transcripts expressed in spleen of 6.VIII and NOD CO strains, we were able to identify both transcripts. A third putative alternative spliced variant, Arntl2c, was identified in a 5'-RACE experiment. This transcript initiated within an intron, 100 nucleotides upstream of the start of exon 7 in the consensus mRNA (AY005163 [GenBank] , Arntl2a mRNA). This transcript encodes a 355 amino acid protein containing only the C-terminal half of the full-length protein and was missing both the bHLH and PAS-A domains (Fig. 4B). However, Northern hybridization experiments have not allowed the identification of a specific band corresponding to this transcript. Although the Northern or RACE identified transcripts were identical between 6.VIII and NOD spleen, the analysis of partial cDNA sequences amplified from nested primers indicated the existence of transcripts specific for strains 6.VIII and NOD, generated by differential exon use (Fig. 4C). Exons 12 and 13 were skipped in strain 6.VIII transcripts, whereas exon 14 was missing in the NOD strain transcripts.

Sequence polymorphisms in the Arntl2 gene
To validate Arntl2 as a candidate gene, we analysed the sequence of its coding, 3'-UTR and 5'-UTR for polymorphisms between strain 6.VIII and NOD CO mice. Within exonic regions, one synonymous polymorphism at the wobble position of amino acid 94 (6.VIII: A and NOD mice: G), five non-synonymous polymorphisms and one insertion/deletion were identified (Fig. 5A). The leucine residue located within helix I of the HLH region at amino acid position 71 in strain 6.VIII has been replaced by a methionine in NOD mice. This leucine residue, which is highly conserved in the bHLH family, serves as an important contact site by interacting with residues in helix II in the formation of the helix structure and is also involved in protein dimerization (27,28). The other polymorphisms identified were all located between the PAS-B domain and the C-terminus. Three serine residues in the strain 6.VIII at amino acid positions 425, 426 and 455 were all replaced by glycine residues in the NOD mice. In NOD mice, the valine residue at position 450 in strain 6.VIII was replaced by an isoleucine, and the glutamic acid at position 483 was deleted.


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  		Figure 5. C3H versus NOD polymorphisms within Arntl2. (A) Within the coding sequence of Arntl2, six codons at positions 71, 425, 426, 450, 455 and 483 and one synonymous mutation corresponding to codon 94 differed between the 6.VIII and NOD strains. (B) The alignment of partial 3'-UTR of the Arntl2 gene, corresponding to Ensembl chromosome 6 sequence positions 147 759 731–147 760 164, from strain 6.VIII (upper sequence), and NOD displays significant sequence variation. The length of the total 3'-UTR in 6.VIII is 123 bases shorter than that in NOD mice.

 
The 3'-UTR sequences of the Arntl2 gene also showed striking variation. Multiple long insertion/deletions lying between the sequence position 147 759 748, located some 90 nucleotides distal to the stop codon of Arntl2, and the position 147 760 154 on mouse chromosome 6 resulted in major variation of the lengths of the 3'-UTR in 6.VIII (513 bp) and in NOD (636 bp) strains. Numerous base substitutions were also identified (Fig. 5B).

Analysis of the EST clone BY242187 [GenBank] allowed the identification of the upstream 5'-UTR sequence of Arntl2. Two additional exons were identified between positions 147 716 951 and 147 717 036 (E1') and between positions 147 723 827 and 147 723 947 with the E1' exon locating ~9.4 kb upstream of the ATG. The position of the splice donor of the initial exon of AY005163 [GenBank] was found at position 147 726 370, some 95 nucleotides upstream of the ATG. No polymorphisms were identified within the 5'-UTR. However, numerous SNPs were identified adjacent to the E1' exon (data not shown).

Circadian regulation of the Arntl2 gene and the circadian genes
The circadian expression of Arntl2 (Bmal2) in spleen was examined in mice housed under a cycle of 14 h artificial light and 10 h obscurity. These settings were identical to that used when diabetes incidence has been monitored. Although splenic expression of Arntl2 oscillated moderately during the day, the differences in transcript level between strain 6.VIII and the NOD control were maintained over the whole 24 h period (Fig. 6). The strain difference in Arntl2 transcript levels suggested a possible alteration in the expression of other circadian genes regulated by Arntl2. Arntl1 (Bmal1), a master circadian gene and a close homolog of Arntl2, oscillated with a cycle, which showed lowest expression close to mid-day and highest during the dark phase (Fig. 6). No significant strain-specific differences were found for Arntl1. Per1, which is negatively regulated by Arntl1, showed significant differences only at one single time point (13.5 h, P<0.005, t-test) (Fig. 6). Similar results were obtained for other circadian genes involved in the autoregulatory feedback loop such as Per2, Per3 and Dec1 (data not shown). In addition, the plasminogen activator inhibitor 1 (PAI-1) gene, a downstream circadian output gene regulated by Arntl2 in vitro (29), did not display strain-specific differences in its transcription level (Fig. 6). Hypoxia-inducible factor-1{alpha} (Hif-1{alpha}), a protein capable of heterodimerizing with Arntl2 in vitro (30), mediates expression of Adrenomedullin, which is in turn involved in T cell survival (31,32). Analysis of Adrenomedullin expression failed to reveal differences between the 6.VIII strain and the NOD control spleens (data not shown). We conclude that the upregulation of Arntl2 in strain 6.VIII does not lead to a general alteration in transcription levels in the spleen of other circadian and hypoxia-induced genes.


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  		Figure 6. Circadian transcription profiles of Arntl2, Arntl1, Per1, and PAI-1 expression in spleens of single eight weeks old 6.VIII (white circles) and NOD (black triangles) mice housed under 14 hour light (blank bar) and 10 hour dark cycle (filled bar) are shown as arbitrary units. All P-values for Arntl2 are<0.012; for Arntl1>0.362, for PAI-I>0.111, for Per1 0.046 at ZT 1.5 hours, 0.005 at ZT 13.5 hours, and>0.181 for all other time points. ZT= zeitgeber time.

 
Cytosolic phospholipase A2 is a potential downstream target of Arntl2
From microarray experiments using pooled spleen samples from four 8-week-old pre-diabetic females, we estimated that the replacement of the 5.8 Mb Idd6 interval by C3H alleles resulted in the deregulation of ~2% of the transcriptome in 6.VIII mice compared with CO mice (Rogner et al., manuscript in preparation). We selected seven downregulated and 14 upregulated transcripts with known or potential immune function to test whether their expression difference in spleen of 6.VIII mice would correlate with that of Arntl2. Real-time RT–PCR using pooled RNA from 6–7-week-old females confirmed the microarray results for two of the downregulated and nine of the upregulated genes that showed fold changes in excess of 1.5. Highest upregulation in the 6.VIII mice was found for the chitinase 3-like 1 (Chi3l1) (5.6-fold), the myeloperoxidase (Mpo) (2.1-fold) and cytosolic phospholipase A2 (Pla2g4a) (1.7-fold) genes. When the genes were subject to detailed transcriptional analysis using spleen samples from mice of different ages, the hypoxia-involved Pla2g4a gene (33,34) showed a particular interesting expression pattern, as it was, like Arntl2, upregulated in strain 6.VIII at all ages (Fig. 7A). A two-fold upregulation was also measured when spleen samples from strain 6.VIIIc were compared with 6.VIIIa samples, confirming that the upregulation was, at least to some extent, related to factors lying within Idd6.3 (data not shown). A potential ARNT binding site (TGCGTG) was identified at +101 to +106 of its transcription start site, which indicated that Pla2g4a might be a direct target of Arntl2. Similar to Arntl2, Pla2g4a expression was upregulated in different splenic cell population, including CD4+ T cells, CD8+ T cell, B cells and macrophages. We analysed its circadian profile and showed that although the expression of Pla2g4a oscillated mildly, the variation between strain 6.VIII and CO mice was maintained throughout the day (Fig. 7B). Interestingly, the circadian profile of Pla2g4a, although very similar to that of Arntl1 (Bmal1) and Arntl2 (Bmal2), was clearly different from those of Per1 and PAI-1. This suggests that Pla2g4a circadian expression correlates with that of the Arntl1 and Arntl2 and that it may be regulated by these transcription factors.


Figure 2097
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  		Figure 7. Relative expression in arbitrary units of Pla2g4a (A) in the spleens of different aged mice of 6.VIII (white bars) and NOD (black bars) strains. Data were pooled from four pre-diabetic female mice (P<0.05 for all ages). (B) Circadian profile of Pla2g4a in spleen samples of 8-week-old 6.VIII (white circles) and NOD (black triangles) mice kept under 14 h of light (blank bar) and 10 h of dark (filled bar). P-values are all <0.005, except for ZT 21.5 h (P=0.439). ZT, zeitgeber time.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Both others and we have previously shown that the immune system, notably spleen and thymus, is required for Idd6-mediated disease susceptibility in the NOD mouse. In the present manuscript, we describe a systematic transcriptional profiling approach to genes located within the candidate region for the murine type 1 diabetes locus Idd6. In a comparison of the disease-protected NOD.C3H congenic stain 6.VIII with its NOD control strain, six genes were found to be differentially expressed in the spleen. We mapped the subphenotype of diabetes disease protection in splenocyte co-transfer assays to a restricted interval of 700 kb by analysis of three newly created NOD.C3H congenic strains. Although this region (Idd6.3) contains 10 transcripts, only the bHLH-PAS transcription factor superfamily member Arntl2 (Bmal2), a component of the circadian clock pathway, was found to be differentially expressed in the disease-protected 6.VIII strain. Arntl2 contains a large number of NOD/C3H polymorphisms within the 5'-UTR, exonic and 3'-UTR sequences of its transcript. Several of the polymorphisms in Arnt2 will lead to changes in its functional domains and could be expected to influence either its dimerization, transcriptional activity and/or specificity. In addition, putative alternative strain-specific splice forms were identified. It has previously been suggested that such alternative splicing of ARNTL2 (BMAL2) might provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities and that this might be important in accommodating a variety of metabolic demands and physiological roles (35).

Our studies have shown that changes in Arntl2 transcript levels are not associated with widespread generalized changes in the expression levels of other circadian and hypoxia-induced genes in spleen. The BMAL-CLOCK heterodimers are however known to activate E-box element-dependent transcription (26), and our microarray and quantitative RT–PCR analyses on spleen samples have revealed the cytosolic phospholipase A(2)alpha [cPLA(2) alpha], which contains an ARNT-binding motif, as a potential downstream target of Arntl2. The cytosolic phospholipase A(2)alpha [cPLA(2)alpha, Pla2g4a] gene is known to play an important role in arachidonate pathway. NOD mice deficient in cPLA(2)alpha show severe insulitis and an increased incidence of diabetes. In the macrophages of these knockout mice, prostaglandin E(2) production is decreased and tumour necrosis factor (TNF)-alpha production is increased. Overall, the results suggest that cPLA(2)alpha plays a protective role in the progression of insulitis and the development of autoimmune diabetes via suppression of TNF-alpha production from macrophages (36). This observation correlates with our finding that peritoneal macrophages of pre-diabetic 6.VIII mice show a 2.8-fold decrease in Tnf-alpha expression (unpublished data); data that could suggest that Arntl2 may be involved in the control of the Tnf-alpha pathway in macrophages.

A more precise understanding of how the upregulation and polymorphisms of the widely expressed Arntl2 gene in the 6.VIII strain interact in the regulation of different aspects of the immune system will certainly require additional studies, in particular, as it can be expected that the role of Arntl2 may vary from tissue to tissue and between cell types. In relation with previously described phenotypes for Idd6 whose alleles appear to be involved in the regulation of proliferation and apoptosis in the thymus (21,22), it is important to note that Arntl2 downregulation was found to enhance cell proliferation (25). Another study has identified Arntl2 as being differentially expressed in various CD4+CD25+ regulatory T cell subpopulations (37). This finding is of interest because CD4+CD25+ T cell activity has been found to be modulated by Idd6 alleles (23).

Recent data showed that a homologue of Arntl2, and ARNT (HIF1ß), is associated with type 2 diabetes in both human and mouse and as being essential for normal pancreatic beta cell function and insulin production (38,39). ARNT, also known as the hypoxia-inducible factor 1, heterodimerizes with both ARNTL1 (BMAL1) and ARNTL2 (BMAL2) to regulate gene transcription. These and our data implicating Arntl2 in type 1 diabetes in the mouse suggest that ARNT-like genes may set the clock for mechanisms of disease protection.

Ongoing studies into the functional inactivation of the Arntl2 gene by a lentivirus-based RNAi transgenesis approach in the NOD mouse, as well as detailed studies on the effect of nucleotide variation, should provide further insight into the role of this gene in immune system function and give the definitive functional proof for Arntl2 and its potential downstream targets including Pla2g4a as being involved in the development of type 1 diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 References
 
RNA preparation, cDNA synthesis and microarray analysis
Total RNA was prepared using RNABle (Eurobio). Random cDNA synthesis was carried out on 6 µg DNase I treated total RNA using SuperScriptTM II reverse transcriptase (Invitrogen), according to the manufacturer's conditions. For microarray experiments, RNA quality was examined using an Agilent 2100 Bioanalyser (Agilent). DNA microarrays (8K mouse cDNA, Agilent) were hybridized using 10 µg of total RNA transcribed in the presence of Cy3-dCTP or Cy5-dCTP. Data were obtained from four individual experiments, each including a dye swap. Data were analyzed using feature extraction (P<0.05) and Rosetta Resolver software, evaluated by two-tailed t-tests (P<0.05) and annotated using SOURCE software (provided by the Genetics Department, Stanford University).

Northern blot and RACE experiments
Total RNA of various tissues from the 6.VIII and NOD control strains was separated in TBE on 1% agarose gels containing 1% formaldehyde and transferred on Hybond N+ membranes (Amersham). Northern blots were hybridized using a 3' NOD cDNA fragment amplified with the Arntl2-specific primers AY-555F 5'-AGGCAACACCAGAGCACTGA-3' and AY334R 071-334R 5'-GCCAGGATTACAAAGTGTGCAC-3'. 5'- and 3'-RACE experiments were performed using both total spleen RNA extracted from NOD CO and 6.VIII strain and the GeneRacer Kit (Invitrogen).

Quantitative PCR
Quantitative PCR was performed on an ABIPRISM 7700 Sequence detector using the SYBR Green PCR Master Mix (PE Biosystems), according to the manufacturer's conditions. Primers were designed using PrimerExpress software and used at optimal concentration. Quantification of the amplification product was carried out using the standard curve method. For the circadian rhythm analysis, we used the {Delta}CT method and the Gapdh gene expression as reporter.

Sequences of the oligonucleotides used were as follows:


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Sequence analysis
DNA fragments were amplified and sequenced from genomic DNA or cDNA of the NOD.C3H strain 6.VIII and the NOD control mice. Polymorphisms were identified by sequence alignment using Megalign (DNASTAR Inc.). Potential transcription factor binding sites were identified using the MatInspector program (http://www.genomatix.de) (40).

Construction of mouse strains
The subcongenic strains were constructed by intercrossing the Idd6 congenic NOD.C3H 6.VIII strain (6.VIII) and the NOD CO strain, both originally derived from crosses between C3H/HeJ and NOD/Lt mice (16). Male mice heterozygous for the Idd6 interval were then backcrossed to the CO strain. Recombinant offspring were selected using the polymorphic markers D6Mit14, D6Mit15, D6Mit294 and D6Mit304. The corresponding subcongenic intervals were fixed by intercrossing of the heterozygous offspring resulting from a backcross to the CO strain.

Diabetes assessment and transfer assays
Spontaneous diabetes incidence was monitored weekly from 10 to 30 weeks of age by assessment of glucosuria (Diabur test, Roche). Splenocyte co-transfer was performed by transferring 107 splenocytes from diabetic NOD mice together with 2x107 splenocytes from 7-week-old mice of various mouse strains onto 5-week-old NOD/Scid mice. Cumulative diabetes incidence was monitored weekly throughout 10 weeks post-transfer.

Statistical analysis
Statistics were performed by Kaplan–Meier estimation and log-rank test for group comparison. Pooled data from quantitative RT–PCR were compared as mean±standard deviation and statistical analysis was performend using unpaired Student's t-test.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
References
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are grateful to Christina Grimm for primer design and microarray hybridization. We thank Joëlle Morin and Corinne Veron for excellent technical help on the phenotypic analysis of the mouse strains, and Cécile Julier and Christian Boitard for critical reading of the manuscript. This work was supported by grants from the Juvenile Diabetes Research Foundation International (1-2000-600) and recurrent funding from the CNRS and the Pasteur Institute. M.-S.H. was recipient of fellowships from the CNRS, the Association Française des Diabétiques (ALFEDIAM) and Manlio Cantarini (Institut Pasteur).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli 350, Taiwan. Back


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 References
 

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