Alternative splicing yields novel BMAL2 variants: tissue distribution and functional characterization

John A. Schoenhard1, Mesut Eren1, Carl H. Johnson3, and Douglas E. Vaughan1,2

1 Division of Cardiovascular Medicine, Departments of Medicine and Pharmacology, Vanderbilt University and 2 Veterans Affairs Medical Centers, Nashville 37232; and 3 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The BMAL2 gene encodes a member of the basic helix-loop-helix PER-ARNT-SIM family of transcription factors, which control diverse physiological processes including circadian rhythms. We identified four novel human BMAL2 transcripts that differ by alternative splicing within their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing for transcriptional activation by CLOCK:BMAL2 heterodimers were assessed using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mouse period1, mouse vasopressin, and human plasminogen activator inhibitor-1 genes. These studies revealed that alternative splicing generates BMAL2 isoforms possessing high, medium, low, or no transcriptional activity. Similar results were obtained with each promoter, suggesting that alternative splicing may influence the amplitudes of both central and peripheral oscillators. Indeed, alternative splicing of BMAL2 may provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities to accommodate varied metabolic demands and physiological roles.

circadian clock; cryptochrome; period; plasminogen activator inhibitor-1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CIRCADIAN CLOCKS CONTROL daily oscillations in physiology and behavior. In mammals, a master circadian pacemaker resides in the suprachiasmatic nuclei of the anterior hypothalamus, while additional circadian oscillators are present in most peripheral tissues (32). Both central and peripheral clocks are cell-autonomous oscillators that coordinate circadian outputs to regulate overt circadian rhythms (24, 39). These oscillators are entrained by periodic environmental cues, with the central clock most potently affected by photic stimulation of the retinohypothalamic tract (7) and peripheral clocks synchronized by humoral and neuronal signals under either direct or indirect control of the central clock (2, 27, 33).

At the molecular level, both central and peripheral clocks are thought to mark time according to a common autoregulatory feedback loop with positive and negative limbs (32). The positive limb of this feedback loop consists of four basic helix-loop-helix (bHLH) proteins containing PER-ARNT-SIM (PAS) domains, namely, two class I proteins, BMAL1 and BMAL2, and two class II proteins, CLOCK and NPAS2.1 Heterodimers formed between these class I and class II proteins bind E-box enhancer elements to activate transcription of genes encoding the negative limb of this feedback loop, as well as genes encoding outputs from the clock. Components of the negative limb of this feedback loop are encoded by the period genes (Per1, Per2, and possibly Per3) and the cryptochrome genes (Cry1 and Cry2). After translation of PER and CRY proteins, PER:CRY oligomers translocate to the nucleus, where they inhibit gene expression driven by class I:class II heterodimers of the positive limb. Upon degradation of these PER and CRY proteins, such inhibition is relieved, and transcription of clock-related genes is renewed. Oscillations in gene expression resulting from this feedback loop have period lengths of ~24 h and thus give rise to overt circadian rhythms.

Although a limited number of genes that encode components of this core clock mechanism have been identified so far (5), an expanded repertoire of activators and inhibitors derived from these genes may participate at the protein level due to both alternative splicing and alternative translation initiation site usage. Indeed, multiple splice variants and translation initiation sites have been identified in the human, mouse, and rat for both BMAL1 (12, 14, 15, 36, 40, 41) and BMAL2 (13, 16, 25, 28, 29). However, the functional consequences of these posttranscriptional variations have yet to be reported.

The aim of this study was to compare the abilities of human BMAL2 splice and start site variants to activate gene transcription. In this work, we identified four novel human BMAL2 transcripts that differed by alternative splicing in their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing and alternative translation initiation site usage were assessed using luciferase reporter gene constructs that contained one of three promoters, namely, those of the mouse period1 (mPer1), mouse vasopressin (mAVP), and human plasminogen activator inhibitor-1 (hPAI-1) genes. These promoters were selected based on their relevance in diverse physiological processes controlled by circadian clocks. While PER1 is a core component of the circadian autoregulatory feedback loop, AVP and PAI-1 are rhythmic outputs from central and peripheral clocks, respectively. Vasopressinergic efferent projections from the suprachiasmatic nuclei to the paraventricular and dorsomedial nuclei of the hypothalamus inhibit corticosterone release in a circadian manner (3, 18). Diurnal variation in circulating PAI-1 levels may underlie both increased incidence of acute myocardial infarction and decreased efficacy of thrombolytic therapy in the morning (1, 6, 22). Our results establish alternative splicing as a potentially important regulatory mechanism capable of influencing the amplitudes of both central and peripheral clocks.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Cloning of human BMAL2 splice and start site variants. Full-length human BMAL2 (hBMAL2) cDNAs were cloned by RT-PCR from primary cultures of human umbilical vein endothelial cells (HUVECs) using hBMAL2 forward primer 1 (5'-TGAGAATTCGACCAAGTGGCTCCTGCGATG-3') and hBMAL2 reverse primer 1 (5'-CAAGGATCCGAGGGTCCACTGGATGTCACT-3'). Resulting amplicons were digested with EcoR I and BamH I and cloned into pcDNA3.1/Myc-His(-)A (Invitrogen). This placed an in-frame COOH-terminal Myc-His tag on each protein except hBMAL2f and hBMAL2b W309*.

To place this tag in-frame for hBMAL2f protein, hBMAL2f cDNA was amplified by PCR using a forward primer complementary to the T7 site on pcDNA3.1/Myc-His(-)A and hBMAL2 reverse primer 2 (5'-TGGTGGAATTCGATTCTTTTCAGTTTGGTATGA-3'), digested with EcoR I, and religated into pcDNA3.1/Myc-His(-)A. To generate hBMAL2bDelta N, -gDelta N, and -iDelta N expression plasmids, hBMAL2b,g,i cDNAs were amplified by PCR using hBMAL2 forward primer 2 (5'-GCGTGAATTCAGGGACAAGACCAACAGCTATG-3') and hBMAL2 reverse primer 1, digested with EcoR I and BamH I, and religated into pcDNA3.1/Myc-His(-)A. To generate hBMAL2gDelta C and -iDelta C expression plasmids, Bgl II fragments encoding the NH2 termini of hBMAL2g,i were obtained from our hBMAL2g,i expression plasmids and ligated into a Bgl II fragment encoding the COOH terminus of hBMAL2b W309* obtained from our hBMAL2b W309* expression plasmid. Expression plasmids encoding hBMAL2g (i.e., hCLIF), mCLOCK, mPER1, mPER2, mCRY1, and mCRY2 have been described previously (9, 21, 25). All constructs were verified by chain termination sequencing.

Comparison of hBMAL1 and hBMAL2 expression by RT-PCR. First-strand cDNA preparations from 16 human tissues were obtained from Clontech. Total RNA was extracted from human aortic smooth muscle and endothelial cells (Clonetics), human coronary artery endothelial cells derived from hypertrophied human hearts explanted during heart transplantation, and HUVECs derived from recently birthed human umbilical cords, using a RNeasy Mini Kit (Promega). All tissues were collected in accordance with protocols approved by the Vanderbilt University Institutional Review Board. From these templates, cDNA encoding the variable NH2-terminal region of hBMAL2 was amplified by (RT)-PCR using either an Advantage 2 PCR kit (Clontech) or a Titanium 1-Step RT-PCR kit (Clontech) and the combination of hBMAL2 forward primer 1 and hBMAL2 reverse primer 3 (5'-CTTTTCAGTTTGGCTATGAGCTTCT-3'). hBMAL1 was amplified using primers 5'-GAACGGGGAAATCAGGGTGAAATCT-3' and 5'-TTGGTCCAAGGGTTCATGAAACTGA-3'. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using primers 5'-GATGACATCAAGAAGGTGGTGAAGC-3' and 5'-TTCGTTGTCATACCAGGAAATGAGC-3'. The identities of all resulting amplicons were confirmed by restriction digest as well as by direct cloning and chain termination sequencing.

Transfection studies/luciferase reporter assays. Human embryonic kidney 293 (HEK 293) cells and bovine aortic endothelial cells (BAECs) were plated in 12-well dishes and transfected with 100 ng luciferase reporter gene construct and 400 ng each cytomegalovirus promoter-containing expression construct using Lipofectamine Plus (Invitrogen) according to the manufacturer's instructions. The total amount of expression plasmid transfected in each experiment was kept constant by addition of varying amounts of empty vector. To correct for potential variation in transfection efficiency, we cotransfected 50 ng pRL-CMV (Promega) in all experiments. Reporter constructs were as follows: mPer1-Luc contained three E-boxes in a 2.0-kb fragment of the mPer1 promoter (9), mAVP-Luc contained one E-box in a 200-bp fragment of the mAVP promoter (17), and hPAI-1-Luc contained two E-boxes in an 800-bp fragment of the hPAI-1 promoter (38). The mPer1 and mAVP promoters were studied in HEK 293 cells, while the hPAI-1 promoter was studied in BAECs. Cells were deprived of serum at 24 h after transfection and lysed with passive lysis buffer (Promega) at 48 h after transfection. Firefly and Renilla luciferase activities were sequentially assayed with a dual-luciferase reporter assay kit (Promega). The ratio of firefly to Renilla luciferase activity then served as a measure of normalized luciferase activity for each sample. This ratio was both cell passage dependent and batch dependent. For this reason, we expressed the data from each experiment in terms of mean (±SE) fold induction of normalized luciferase activity, setting those samples cotransfected only with empty expression vector and reporter plasmids as the baseline. Each experiment was transfected on at least three different occasions, and each such transfection was completed at least in quadruplicate. Statistical analyses were performed using Student's t-test, with significance defined as P < 0.05 for a two-tailed distribution. Equal expression of each transfected hBMAL2 isoform was verified by Western blot using an anti-Myc horseradish peroxidase-conjugated antibody (Invitrogen) and chemiluminescent detection using ECL Plus reagents (Amersham Pharmacia).

Immunohistochemistry. BAECs were plated in two-well chamber slides and transfected with hBMAL2 and mCLOCK expression plasmids as described above. Forty-eight hours after transfection, cells were washed twice with phosphate-buffered saline (PBS), fixed with 10% formalin (20 min), washed with PBS/0.1% Triton X-100 (PBST), and blocked with 10% fetal bovine serum in PBST (20 min). Mouse anti-Myc IgG (kindly provided by Lee Limbird) was applied in blocking buffer for 1 h. Cells were washed with PBST (3 × 10 min) and incubated in the dark (30 min) with Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes). Cells were washed again, their nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes), and the cells were mounted for fluorescence microscopy. The entirety of each slide was scanned. Representative cells are presented. At least three independently transfected slides were considered per experiment.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of hBMAL2 splice and start site variants. We cloned hBMAL2 cDNAs from primary cultures of HUVECs via RT-PCR using primers complementary to sequences flanking the translation initiation site of exon 1 and the termination site of exon 18. By this method, five wild-type hBMAL2 cDNAs were identified. These clones encoded four novel splice variants, as well as the splice variant previously reported as hBMAL2b (29). Addition of these four novel splice variants to the existing catalog of hBMAL2 isoforms increased the total number of known hBMAL2 splice variants to nine.

To facilitate classification of both novel and known hBMAL2 splice and start-site variants, a standardized nomenclature was developed based on that proposed by Okano et al. (29). Consistent with this terminology, isoforms previously reported as hBMAL2, hMOP9, or hCLIF (13, 16, 25, 29), as well as those first identified in this work, were ordered by length and designated hBMAL2a-i (Fig. 1). Alignment of these sequences with a human genomic database allowed for localization of the exon-intron boundaries of the hBmal2 gene on chromosome 12p2.2 (Table 1).


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Fig. 1.   The primary structures of novel and known human (h) BMAL2 splice variants. A: splicing choices of hBMAL2a-i. Top: the variable NH2-terminal, basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS)-A, and PAS-B domains of hBMAL2 are indicated, as well as the boundaries of exons 1-5. A bar above these exon 1-5 labels indicates sequences that are aligned in B. Splice junctions defining exons 1-Short and 3-Short are shown as zig-zag lines, while those defining exons 1-Long, 2, 3-Long, 4, and 5 are shown as straight lines. The premature stop codon of hBMAL2b W309* is indicated with an asterisk. Bottom: sequences inserted by hBMAL2a-i are illustrated as horizontal lines, while sequences deleted by hBMAL2a-i are illustrated by carets. To aid visualization, the scale of exons 1-5 is twice that of exons 6-18. Left: hBMAL2a-i nomenclature used in this paper. Right: previous terminology used to describe exon 1 vs. exon 2 translation start site variants. B: alignment of the NH2-terminal regions of hBMAL2a-i, through the first 11 basic amino acids of exon 6. Exon boundaries are indicated by long arrows. The alternative translation start site of exon 2 is indicated by a short arrow. Alignment gaps are indicated by dashes. The in-frame stop codon of hBMAL2f is indicated with an asterisk.


                              
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Table 1.   hBmal2 exons and splice junction sequences

Splice variants of hBMAL2 are each composed of a variable NH2-terminal region (exons 1-5) followed by 13 invariant exons (exons 6-18) that, with the exception of hBMAL2f, encode the defining bHLH, PAS-A, PAS-B, and COOH-terminal domains of hBMAL2. Within the variable NH2-terminal region of hBMAL2, alternative splicing controls the length of exon 1 via selection of one of two possible exon 1/intron 1 splice junctions and determines the sequence between exons 2 and 6 via deletion or insertion of exons 3-5. To better understand the results of these splicing events, the NH2-terminal region of each hBMAL2 isoform is briefly described below.

Defining the longest of hBMAL2 isoforms, the NH2-terminal region of hBMAL2a is encoded by exons 1-Short, 2, 3-Long, and 5. In contrast, the NH2-terminal regions of all other hBMAL2 isoforms are encoded by either one or none of exons 3-5. Specifically, hBMAL2b,c,d,e insert only exon 3; hBMAL2f inserts only exon 4; hBMAL2g inserts only exon 5; and hBMAL2h,i insert none of exons 3-5.

Although the 3' boundaries of exons 1-Long and 1-Short agree with reported splice junction consensus sequences, the 3' boundaries of exons 3-Long and 3-Short are defined by consensus and nonconsensus splice junctions, respectively (42). With the use of only consensus splice junctions, hBMAL2b and hBMAL2e transcripts insert either short or long forms of exon 1, respectively, followed by exons 2 and 3-Long. hBMAL2b was cloned from HUVECs in this work and from HEK 293 cells by Okano et al. (29). hBMAL2e was cloned for the first time in this study. hMOP9-Long, with identical exon 3-5 splicing as these clones but an alternative translation initiation site, was cloned from human brain by Hogenesch et al. (13).

The NH2-terminal regions of hBMAL2c and hBMAL2d are similarly derived from long vs. short forms of exon 1, respectively. However, their transcripts each use an early nonconsensus exon 3/intron 3 splice junction to generate a truncated form of exon 3; that is, hBMAL2c inserts exons 1-Long, 2, and 3-Short, while hBMAL2d inserts exons 1-Short, 2, and 3-Short. Both were cloned by Okano et al. (29).

The novel splice variant hBMAL2f encodes an abbreviated protein of just 89 amino acids. This results from insertion of a novel exon 4 that frameshifts exons 6-18 and thereby introduces a premature stop codon in place of the bHLH domain common to all other hBMAL2 isoforms. Introduction of a frameshift and premature stop codon by means of alternative splicing is not without precedent. Indeed, alternative splicing at the border of the first PAS domain of mouse BMAL1 has been shown to introduce a frameshift and subsequent premature stop codon such that mBMAL1g' lacks both PAS-A and PAS-B domains (41).

Two newly identified transcripts encoding hBMAL2h and hBMAL2i delete exons 3-5 and instead differ according to exon 1 length. Corresponding isoforms termed hMOP9-Short and hBMAL2 (the latter having no alphabetic suffix in this case), which also delete exons 3-5 but use an alternative translation initiation site, were cloned previously from human brain by Ikeda et al. (16) and Hogenesch et al. (13).

As indicated above, two translation initiation sites have been described for hBMAL2. Located in exons 1 and 2, these competing initiation sites each share 7 of 10 bp with the Kozak consensus sequence, including the requisite purine at position -3 bp (exon 1, CCTGCG ATGG; exon 2, ACAGCT ATGG; consensus, GCCRCC ATGG, R = A/G; Ref. 20). In the present study, the translation initiation site of exon 1 was selected as the standard for comparison. Variants of hBMAL2b,e and hBMAL2h,i that utilize the translation initiation site of exon 2 have been reported previously as hMOP9-Long and hMOP9-Short/hBMAL2, respectively (13, 16).

In addition, a novel point mutation was identified in one of many hBMAL2b clones derived from our pooled multiple-donor primary cultures of HUVECs and named hBMAL2b W309*. In this clone, substitution of guanine for adenine at the wobble position of codon 309 introduced a premature termination signal between the PAS-A and PAS-B domains of hBMAL2b. Similar truncations of the PAS-B and COOH-terminal domains of human BMAL1 (e.g., hBMAL1c; Ref. 15) and mouse BMAL2 (e.g., mBMAL2b; Ref. 28) have been reported and shown to result from alternative splicing. Although the point mutation in hBMAL2b W309* most likely resulted from genetic variation, the possibility of a mismatch in the RT-PCR cannot be excluded. Because this mutation has not been reported previously in any public database, its prevalence in the human population in not yet known.

Tissue distribution of hBMAL2 splice variants. Differential expression of the variable NH2-terminal region of hBMAL2 and an invariant region of hBMAL1 was assessed in human tissues by RT-PCR. Constitutive expression of hGAPDH was assessed as a positive control. For amplification of hBMAL2 splice variants, forward and reverse primers complemented the proximal 5'-untranslated region (UTR) and translation initiation site of exon 1 or the first 25 bp of exon 6, respectively. As shown in Fig. 2A, RT-PCR yielded four major bands corresponding to hBMAL2e (369 bp), hBMAL2b (336 bp), hBMAL2h (267 bp), and hBMAL2i (234 bp). Amplification of hBMAL1 and hGAPDH yielded single bands of 299 and 186 bp, respectively. The identities of these amplicons were confirmed by restriction digest, as well as by direct cloning and sequencing.


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Fig. 2.   Tissue distribution of hBMAL1 and hBMAL2 splice variants. A: expression of the variable NH2-terminal region of hBMAL2 (top), an invariant region of hBMAL1 (middle), and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) (bottom) in 16 different human tissues, as assessed by RT-PCR. For amplification of hBMAL2 splice variants, forward and reverse primers complemented the proximal 5'-untranslated region (UTR) and translation initiation site of exon 1 and the first 25 bp of exon 6, respectively. Resulting amplicons were specific for hBMAL2e (369 bp), hBMAL2b (336 bp), hBMAL2h (267 bp), and hBMAL2i (234 bp). Also as described in METHODS, amplification of hBMAL1 and hGAPDH sequences yielded bands of 299 and 186 bp, respectively. B: expression of the variable NH2-terminal region of hBMAL2 (lanes labeled 2), an invariant region of hBMAL1 (lanes labeled 1), and hGAPDH (lanes labeled G) in primary cultures of human aortic smooth muscle cells (HASMs), human aortic endothelial cells (HAECs), human coronary artery endothelial cells (HCAECs), and human umbilical vein endothelial cells (HUVECs), as assessed by RT-PCR. Similar amplicons were obtained as in A.

Although nine splice variants of hBMAL2 have been described, these results indicated that four splice variants of hBMAL2 predominate in vivo. These four splice variants resulted from differential utilization of early vs. late exon 1/intron 1 splice junctions and either deletion or insertion of exon 3. In general, splice variants containing exon 1-Short were more prevalent than those containing exon 1-Long, while deletion was more common than insertion of exon 3; that is, the relative intensities of observed hBMAL2 amplicons roughly followed the series hBMAL2i > hBMAL2b > hBMAL2e > hBMAL2h. Splice variants containing exons 4 or 5 were not detected in human tissues by this method.

Expression of hBMAL2 was observed in 15 of 16 human tissues examined. However, both the total concentrations of hBMAL2 and the relative concentrations of specific hBMAL2 splice variants differed among tissues. For example, hBMAL2i was expressed most intensely in brain, heart, lung, placenta, and liver, while hBMAL2b expression varied greatly in these tissues with strong expression in placenta, moderate expression in lung and liver, and weak expression in brain and heart. hBMAL2e was expressed strongly in placenta and weakly in brain, lung, liver, spleen, colon, and skeletal muscle. hBMAL2h was expressed very weakly exclusively in brain, heart, lung, placenta, liver, pancreas, and ovary.

Expression of hBMAL1 was observed in all human tissues examined, with strong expression in brain, heart, lung, placenta, liver, kidney, spleen, and pancreas; moderate expression in thymus, leukocytes, and skeletal muscle; and weak expression in digestive and reproductive organs. Expression of hGAPDH was invariant.

To further identify potential molecular components of a vascular circadian clock (25, 27), we assessed expression of the variable NH2-terminal region of hBMAL2 and an invariant region of hBMAL1 in primary cultures of human aortic smooth muscle cells, human aortic endothelial cells, human coronary artery endothelial cells, and HUVECs by RT-PCR. Constitutive expression of hGAPDH was assessed as a positive control. As shown in Fig. 2B, similar results were obtained for each cell type. Amplification of the variable NH2-terminal region of hBMAL2 yielded four major bands corresponding to hBMAL2b,e,h,i. Consistent with other human tissues, the relative intensities of observed hBMAL2 amplicons again followed the series hBMAL2i > hBMAL2b > hBMAL2e > hBMAL2h. Splice variants containing exons 4 or 5 were not detected. Strong expression of hBMAL1 was observed in each vascular cell type.

Subcellular distribution of hBMAL2 splice variants. The variable NH2-terminal region and initial 12 amino acids of exon 6 have been shown to be required for nuclear localization of hBMAL2 (16). Indeed, this region of hBMAL2 contains two clusters of basic amino acids (in exon 2, RKRK; in exon 6, KRRR) that may function as nuclear localization signals. We assessed the subcellular distributions of hBMAL2 splice variants composed of exon 1-Short and either one or none of exons 3-5. As illustrated in Fig. 3, BAECs cotransfected with mCLOCK and Myc-tagged hBMAL2b, hBMAL2g, or hBMAL2i showed anti-Myc immunoreactivity predominantly in their nuclei, while BAECs cotransfected with mCLOCK and Myc-tagged hBMAL2f showed diffuse anti-Myc immunoreactivity extending throughout both cytoplasm and nuclei. The locations of nuclei were confirmed by DAPI staining of dsDNA (data not shown). These results indicated that alternative splicing of exons 3 and 5 does not affect nuclear localization of hBMAL2, while frameshift and truncation of hBMAL2f by insertion of exon 4 prevents subcellular compartmentalization.


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Fig. 3.   Subcellular distribution of hBMAL2 splice variants. Bovine aortic endothelial cells were transfected with mCLOCK and Myc-tagged hBMAL2b, -f, -g, or -i, fixed 48 h later, and immunostained with anti-Myc antibodies. The resulting antibody-antigen complex was detected with Alexa Fluor 488-conjugated anti-mouse IgG (green).

Activation capacities of hBMAL2 splice and start site variants. The relative abilities of hBMAL2 splice and start site variants to activate gene transcription were compared using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mPer1, mAVP, and hPAI-1 genes. First we assessed the functional consequences of alternative splicing of exons 3-5 for hBMAL2 isoforms derived from transcripts that insert exon 1-Short and utilize its early translation initiation site (Fig. 4). In the absence of mCLOCK overexpression, hBMAL2b, -g, or -i overexpression mildly induced luciferase reporter gene transcription by 2.6-, 2.1-, or 1.8-fold, respectively (P < 0. 001 for each isoform). In the presence of mCLOCK overexpression, however, distinct activation capacities were revealed for each mCLOCK:hBMAL2b, -g, or -i heterodimer. Although hBMAL2i lacked exons 3-5, overexpression of the mCLOCK:hBMAL2i heterodimer induced luciferase reporter gene transcription up to 15-fold (P < 0. 001). Incrementally greater inductions up to 36- or 66-fold were obtained by pairing mCLOCK with exon 3-containing hBMAL2b or exon 5-containing hBMAL2g, respectively (P < 0. 001 for each heterodimer). In contrast, overexpression of exon 4-containing hBMAL2f failed to induce gene transcription in either the absence or presence of mCLOCK. Qualitatively similar results were obtained for each promoter studied. Taken together, these results indicated that alternative splicing of exons 3-5 generates functionally distinct hBMAL2 isoforms possessing high, medium, low, or no activity.


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Fig. 4.   Effects of alternative splicing of exons 3-5 on the activation capacity of hBMAL2. For each panel, the abscissa indicates overexpression of hBMAL2 splice variants either in the absence of mCLOCK overexpression (open bars) or in the presence of mCLOCK overexpression (solid bars). Ordinates display mean (±SE) fold induction of luciferase activity over control (no mCLOCK, no hBMAL2) levels as a reporter of mouse period 1 (mPer1; A), mouse arginine vasopressin (mAVP; B), or human plasminogen activator inhibitor-1 (hPAI-1; C) promoter activity.

This conclusion was affirmed when we assessed the effects of alternative translation start site selection on the activation capacities of hBMAL2b, -g, and -i. Deletion of the first 37 amino acids from our hBMAL2b, -g, and -i clones placed the translation initiation sites of our hBMAL2bDelta N, -gDelta N, and -iDelta N expression plasmids at the putative translation initiation site of exon 2 (Fig. 5A). This yielded hBMAL2bDelta N and -iDelta N proteins identical to those previously reported as hMOP9-Long and hBMAL2 (no alphabetic suffix), respectively (13, 16). Paired overexpression of mCLOCK with each of these combined splice and start site variants revealed that alternative translation initiation site usage does not significantly affect the activation capacities of mCLOCK:hBMAL2b, -g, or -i heterodimers (Fig. 5, B-D); that is, similar magnitudes of luciferase reporter gene transcription were observed for hBMAL2Delta N proteins as for corresponding full-length hBMAL2 proteins, independent of the promoter studied. Furthermore, amino acid sequences encoded by exon 1 and the first 80 bp of exon 2 (i.e., sequences untranslated when the translation initiation site of exon 2 is utilized) were not required for maximal hBMAL2 function. Although a 4-kDa size difference between the Myc-tagged hBMAL2 and hBMAL2Delta N proteins overexpressed in this work was confirmed by Western blot (data not shown), the true translation initiation sites of native hBMAL2 splice variants in vivo remain unknown.


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Fig. 5.   Effects of alternative translation initiation site utilization on the activation capacities of hBMAL2 splice variants. A: exon 1 vs. exon 2 translation initiation sites of hBMAL2 vs. hBMAL2Delta N clones, respectively. As in Fig. 1A, exons 1-5 are shown by straight and zig-zag lines at twice the scale of exons 6-18. B-D: comparison of the transcriptional activation capacities of exon 1-initiated hBMAL2 splice variants (open bars) with those of exon 2-initiated hBMAL2Delta N splice variants (solid bars). Ordinates display mean (±SE) fold induction of luciferase activity over control (no mCLOCK, no hBMAL2) levels as a reporter of mPer1 (B), mAVP (C), or hPAI-1 (D) promoter activity.

Deletion of the COOH terminus of hBMAL2. The transcriptional activation domains of hARNT, mARNT2, and mARNT3/mBMAL1 have been mapped to the COOH termini of these class I bHLH-PAS proteins (10, 34-36). To better characterize the functional domains of hBMAL2, we exploited the point mutation first identified in the clone hBMAL2b W309*. Introduction of this point mutation into wild-type hBMAL2g and hBMAL2i cDNAs yielded hBMAL2gDelta C and hBMAL2iDelta C proteins complementary to hBMAL2b W309*/hBMAL2bDelta C (Fig. 6A). Paired overexpression of mCLOCK with each truncated splice variant revealed a distinct activation capacity for each resulting mCLOCK:hBMAL2Delta C heterodimer that averaged from one-third to one-half that of the corresponding full-length mCLOCK:hBMAL2 heterodimer (Fig. 6, B-D). Although transcriptional activation was reduced in the absence of the PAS-B and COOH-terminal domains of hBMAL2, these results indicated that the variable NH2-terminal, bHLH, and PAS-A domains of hBMAL2 together are sufficient to significantly induce luciferase reporter gene transcription in the presence of mCLOCK (P < 0.001 for each heterodimer).


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Fig. 6.   Effects of COOH-terminal deletion on the activation capacities of hBMAL2 splice variants. A: lengths of hBMAL2 and hBMAL2Delta C clones. B-D: comparison of the transcriptional activation capacities of full-length hBMAL2 splice variants (open bars) with those of truncated hBMAL2Delta C splice variants (solid bars). Ordinates display mean (±SE) fold induction of luciferase activity over control (no mCLOCK, no hBMAL2) levels as a reporter of mPer1 (B), mAVP (C), or hPAI-1 (D) promoter activity.

Transcriptional activation in the presence of multiple hBMAL2 splice variants. Alternative splicing has been identified as a mechanism by which both active and dominant negative transcription factors may be generated from the same gene (8). Thus we investigated the possibility that less active hBMAL2 isoforms may interact with more active hBMAL2 isoforms in a dominant negative fashion (Fig. 7). Cotransfection of a constant, nonlimiting mass of mCLOCK plasmid with varying quantities of hBMAL2b, -g, or -i plasmid revealed dose-dependent luciferase reporter gene transcription, thereby demonstrating strong correlation between mCLOCK:hBMAL2 heterodimer availability and hPAI-1 promoter activity. Cotransfection with binary mixtures of hBMAL2b, -g, or -i plasmids, each with constant total mass of hBMAL2 plasmid but varied stoichiometry of component hBMAL2 splice variants, revealed the activities of hBMAL2 splice variants to be additive; that is, the extent to which luciferase reporter gene transcription was induced by mCLOCK in the presence of two different hBMAL2 splice variants was approximately equal to the sum of the transcriptional activation capacities of the resulting mixture's component mCLOCK:hBMAL2b, -g, or -i heterodimers. Similar results were obtained for mixtures that included hBMAL2b W309* (data not shown), indicating that deletion of the PAS-B and COOH-terminal domains of hBMAL2 via point mutation does not yield a dominant negative protein analogous to the mutant mCLOCK-Delta 19 (9, 19). In addition, hBMAL2f failed to inhibit gene transcription mediated by other mCLOCK:hBMAL2 heterodimers (data not shown).


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Fig. 7.   Coordinate regulation of hPAI-1 promoter activity by hBMAL2 splice variants. A: mean (±SE) inductions of hPAI-1 promoter activity resulting from transfection of increasing concentrations of hBMAL2b plasmid from 0.0 to 0.4 µg/well (), decreasing concentrations of hBMAL2g plasmid from 0.4 to 0.0 µg/well (open circle ), or binary mixtures of hBMAL2b,g plasmid, each with a constant total mass of hBMAL2 plasmid at 0.4 µg/well and an increasing ratio of hBMAL2b to hBMAL2g from left (L) to right (R) (). B: mean (±SE) inductions of hPAI-1 promoter activity resulting from transfection of increasing concentrations of hBMAL2b plasmid from 0.0 to 0.4 µg/well (), decreasing concentrations of hBMAL2i plasmid from 0.4 to 0.0 µg/well (open circle ), or binary mixtures of hBMAL2b,i plasmid, each with a constant total mass of hBMAL2 plasmid at 0.4 µg/well and an increasing ratio of hBMAL2b to hBMAL2i from L to R (). C: mean (±SE) inductions of hPAI-1 promoter activity resulting from transfection of increasing concentrations of hBMAL2g plasmid from 0.0 to 0.4 µg/well (), decreasing concentrations of hBMAL2i plasmid from 0.4 to 0.0 µg/well (open circle ), or binary mixtures of hBMAL2g,i plasmid, each with a constant total mass of hBMAL2 plasmid at 0.4 µg/well and an increasing ratio of hBMAL2g to hBMAL2i from L to R ().

Inhibition of mCLOCK:hBMAL2 heterodimers by PER and CRY proteins. Finally, we investigated the effects of alternative splicing of hBMAL2 on the abilities of mPER1, mPER2, mCRY1, and mCRY2 to inhibit mCLOCK:hBMAL2-mediated induction of hPAI-1 promoter activity (Fig. 8). In fact, alternative splicing of hBMAL2 did not affect the functions of these inhibitors. Consistent with previous reports (21, 25), mCRY1 and mCRY2 exhibited greater inhibitory activities than mPER1 and mPER2. On average, mCLOCK:hBMAL2 heterodimers were inhibited with mCRY1 by 91%, with mCRY2 by 86%, with mPER1 by 53%, and with mPER2 by 78% (P < 0.001 for each inhibitory interaction). Similar results were obtained for hBMAL2b W309* (data not shown), indicating that the PAS-B and COOH-terminal domains of hBMAL2 are not required for inhibition by PER and CRY proteins. Also illustrated in Fig. 8, neither mPER1,2 nor mCRY1,2 significantly affected hPAI-1 promoter activity in the absence of mCLOCK:hBMAL2 heterodimer overexpression.


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Fig. 8.   Inhibition of mCLOCK:hBMAL2 heterodimers by mPER1,2 and mCRY1,2 proteins. The ordinate displays mean (±SE) fold induction of luciferase activity over control (no mCLOCK, no hBMAL2) levels as a reporter of hPAI-1 promoter activity. At left, mPER1,2 or mCRY1,2 were overexpressed in the absence of mCLOCK:hBMAL2 heterodimers. At right, mCLOCK and hBMAL2b, -g, -i plasmids were cotransfected in either the absence or presence of mPER1,2 or mCRY1,2 plasmids.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this work, we identified four novel human BMAL2 transcripts that differed by alternative splicing within their NH2-terminal regions. Addition of these transcripts to the existing catalog of hBMAL2 isoforms increased the total number of known hBMAL2 splice variants to nine. Considered together, hBMAL2 splice variants were each composed of a variable NH2-terminal region (exons 1-5) followed by 13 invariant exons (exons 6-18) that, with the exception of hBMAL2f, encoded the defining bHLH, PAS-A, PAS-B, and COOH-terminal domains of hBMAL2. Although the relative levels of native hBMAL2a-i proteins in vivo remain unknown, RT-PCR analysis revealed the predominant hBMAL2 transcripts in human tissues and primary cultures of human vascular smooth muscle and endothelial cells to be those encoding hBMAL2b, -e, -h, and -i. These most common hBMAL2 transcripts differed from each other either by exon 1 length or by deletion vs. insertion of exon 3 immediately before the bHLH domain encoded by exon 6.

Alternative splicing is not unique to hBMAL2. As an example from the circadian clock field, Drosophila clockwork is modulated by a thermosensitive splicing event in the 3'-untranslated region of Period that ultimately enables flies to maintain daytime activity at low temperatures (26).

Alternative splicing also determines the sequences that immediately precede the bHLH domains of many class I bHLH-PAS proteins in addition to hBMAL2. Human and mouse ARNT1, as well as zebrafish ARNT2, are subject to deletion vs. insertion of an exon encoding 15 amino acids adjacent to their bHLH domains (11, 31, 37). Mouse BMAL1 is subject to deletion vs. insertion of an exon encoding seven amino acids adjacent to its bHLH domain (36, 41). Rat BMAL2 is subject to splicing of an alternative exon orthologous to exon 3 of hBMAL2 (28).

Given such conservation of alternative splicing immediately preceding the bHLH domains of divergent species and duplicated genes, it is important to consider the functional consequences of this regulatory process. We have demonstrated that alternative splicing of exons 3-5 determines the relative abilities of resulting hBMAL2 isoforms to activate gene transcription in cooperation with mCLOCK, while alternative translation initiation site usage does not. In comparative studies, isoform-dependent differences in hBMAL2 activation capacity were similar for each of three different diurnally regulated promoters, indicating that alternative splicing of hBMAL2 may influence both central and peripheral circadian rhythms.

We have shown that individual human tissues coincidently express multiple hBMAL2 splice variants. As both the total concentrations of hBMAL2 and the relative concentrations of specific hBMAL2 splice variants differ among tissues, it is reasonable for us to speculate that hBMAL2-dependent transcriptional activation also differs among tissues according to both total hBMAL2 expression and alternative splicing of expressed hBMAL2 transcripts. As we have demonstrated, coexpression of multiple hBMAL2 splice variants has an additive effect on the transcriptional activation of responsive genes. Although hBMAL2 splice variants with high, medium, low, or no activity neither synergize nor directly inhibit each other, their coincident formation in tissue-specific ratios may allow different tissues to regulate CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities.

Although light-responsive circadian oscillations in BMAL2 transcription have been reported for the zebrafish and chick (4, 29, 30), no such oscillations have been observed yet for mouse BMAL2 (25, 28). Because repeated sampling of human tissues was beyond the scope of this study, we cannot conclude at this time whether or not temporal variation exists among either total hBMAL2 levels or specific hBMAL2 isoform ratios in response to either the "ticking" of circadian clocks or other environmental stimuli. Nonetheless, alternative splicing is an intriguing mechanism by which different tissues may control the amplitudes of their circadian oscillators to accommodate their varied metabolic demands and physiological roles.

This work has also used alternative splicing as a means to study the functional domains of hBMAL2. As indicated by the effects of alternative splicing of exons 3-5 on hBMAL2 function, amino acid sequences immediately preceding the bHLH domain of hBMAL2 strongly influence the transcriptional activation capacities of mCLOCK:hBMAL2 heterodimers. On the other hand, as indicated by the negligible impact of alternative translation start site selection on hBMAL2 function, amino acid sequences encoded by exon 1 and the first 80 bp of exon 2 are not required for maximal mCLOCK:hBMAL2-mediated transcriptional activation. While a previous report has shown the variable NH2-terminal region and initial basic amino acids of exon 6 to be required for nuclear localization of hBMAL2 (16), our experiences with the abbreviated splice variant hBMAL2f have revealed the NH2-terminal region of this isoform to be incapable of mediating either nuclear localization or transcriptional activation.

Nonetheless, the importance of the variable NH2-terminal region of hBMAL2 for transcriptional activation was affirmed by our COOH-terminal truncations of hBMAL2. Although the transcriptional activation domains of hARNT, mARNT2, and mARNT3/mBMAL1 have been mapped to the COOH termini of these class I bHLH-PAS proteins (10, 34-36), our deletions of the PAS-B and COOH-terminal domains of hBMAL2 reduced, but did not completely abrogate, hBMAL2-dependent transcriptional activation. In addition, deletion vs. insertion of exons 3-5 modulated the activation potentials of these truncated hBMAL2 isoforms in parallel with full-length hBMAL2 isoforms, indicating that amino acid sequences immediately preceding the bHLH domain of hBMAL2 determine the relative activation capacities of mCLOCK:hBMAL2 heterodimers independent of a potential hBMAL2 COOH-terminal transcriptional activation domain. These results were consistent with those demonstrating that COOH-terminal truncation of the final one-third of zebrafish BMAL2 fails to inhibit its association with CLOCK (4). Comparisons may also be drawn between these results and those demonstrating that deletion of the COOH-terminal transcriptional activation domain of mARNT reduces, but does not completely abrogate, induction of phosphoglycerate kinase 1 gene expression in response to hypoxia (23). Indeed, our results are fully consistent with a hypothesis stating that although a transcriptional activation domain located in the COOH terminus of BMAL2 is required for maximal CLOCK:BMAL2-mediated transcriptional activation, this domain is not required for up to half-maximal transcriptional activation, since alternative mechanisms of CLOCK:BMAL2-mediated transcriptional activation both exist and are modulated by alternative splicing of exons 3-5 of BMAL2.

By what mechanism might alternative splicing of exons 3-5 modulate CLOCK:BMAL2-mediated transcriptional activation? For hBMAL2f, the frameshift induced by exon 4 truncates the hBMAL2f protein and thereby prevents its nuclear localization and transcriptional activity. Otherwise, alternative splicing of exons 3-5 does not appear to affect either nuclear localization or inhibition by PER or CRY proteins. Rather, alternative splicing of BMAL2 likely affects either promoter binding or protein-protein interactions with necessary coactivators such as CLOCK. Although our results allow for either of these possibilities, we favor the latter hypothesis based on the results presented in a recent report on alternative splicing of zebrafish ARNT2 (37). According to this paper, alternative splicing immediately preceding the bHLH domain of zARNT2 does not affect DNA binding, even though deletion of the alternatively spliced exon obviates transcriptional activation mediated by the zAHR:zARNT2 heterodimer. Meanwhile, transcriptional activation mediated by the zEPAS-1:zARNT2 heterodimer is unaffected by alternative splicing. Given this analogy, we suggest that alternative splicing of exons 3-5 may regulate hBMAL2 by modulating its interactions with selected coactivators, rather than by altering its DNA binding. Furthermore, alternative splicing may provide for tissue-specific regulation of CLOCK:BMAL2 heterodimer activity without affecting the functions of oligomers formed between BMAL2 and other class II bHLH-PAS proteins.

Taken together, the results presented in this study not only contribute to our understanding of the functional domains of hBMAL2 but also establish alternative splicing of hBMAL2 as a potentially important regulatory mechanism controlling both central and peripheral circadian rhythms.


    ACKNOWLEDGEMENTS

We are grateful to Mark A. Perrella, Steven M. Reppert, and Charles J. Weitz for their generous gifts of plasmids. We thank Corrie A. Painter for skillful technical assistance. We also thank Guo Huang for help during the initial phase of this project.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-51387 and HL-65192 (to D. E. Vaughan), MH-43836 (to C. H. Johnson), and GM-07347 (to the Vanderbilt University Medical Scientist Training Program) and by a Merit Award from the Veterans Administration Research Service (to D. E. Vaughan).

Address for reprint requests and other correspondence: D. E. Vaughan, Div. of Cardiovascular Medicine, Dept. of Medicine, Vanderbilt University Medical Center, 2220 Pierce Ave., 383 PRB, Nashville, TN 37232 (E-mail: doug.vaughan{at}mcmail.vanderbilt.edu).

1 BMAL1 is also termed MOP3 in humans (12, 15), ARNT3 in the mouse (36, 41), and TIC in the rat (14, 40). BMAL2 is also termed MOP9 or CLIF in humans (13, 16, 25, 29) but remains BMAL2 in the mouse and rat (28). NPAS2 is also termed MOP4 (12, 43).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 13, 2002;10.1152/ajpcell.00541.2001

Received 13 November 2001; accepted in final form 10 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angleton, P, Chandler WL, and Schmer G. Diurnal variation of tissue-type plasminogen activator and its rapid inhibitor (PAI-1). Circulation 79: 101-106, 1989[Abstract].

2.   Balsalobre, A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, Schutz G, and Schibler U. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289: 2344-2347, 2000[Abstract/Free Full Text].

3.   Carter, DA, and Murphy D. Nuclear mechanisms mediate rhythmic changes in vasopressin mRNA expression in the rat suprachiasmatic nucleus. Brain Res 12: 315-321, 1992.

4.   Cermakian, N, Whitmore D, Foulkes NS, and Sassone-Corsi P. Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function. Proc Natl Acad Sci USA 97: 4339-4344, 2000[Abstract/Free Full Text].

5.   Clayton, JD, Kyriacou CP, and Reppert SM. Keeping time with the human genome. Nature 409: 829-831, 2001[ISI][Medline].

6.   Cohen, MC, Rohtla KM, Lavery CE, Muller JE, and Mittleman MA. Meta-analysis of the morning excess of acute myocardial infarction and sudden cardiac death. Am J Cardiol 79: 1512-1516, 1997[ISI][Medline].

7.   Foster, RG. Shedding light on the biological clock. Neuron 20: 829-832, 1998[ISI][Medline].

8.   Foulkes, NS, and Sassone-Corsi P. More is better: activators and repressors from the same gene. Cell 68: 411-414, 1992[ISI][Medline].

9.   Gekakis, N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, and Weitz CJ. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280: 1564-1569, 1998[Abstract/Free Full Text].

10.   Hirose, K, Morita M, Ema M, Mimura J, Hamada H, Fujii H, Saijo Y, Gotoh O, Sogawa K, and Fujii-Kuriyama Y. cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt). Mol Cell Biol 16: 1706-1713, 1996[Abstract].

11.   Hoffman, EC, Reyes H, Chu FF, Sander F, Conley LH, Brooks BA, and Hankinson O. Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252: 954-958, 1991[ISI][Medline].

12.   Hogenesch, JB, Chan WK, Jackiw VH, Brown RC, Gu YZ, Pray-Grant M, Perdew GH, and Bradfield CA. Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway. J Biol Chem 272: 8581-8593, 1997[Abstract/Free Full Text].

13.   Hogenesch, JB, Gu YZ, Moran SM, Shimomura K, Radcliffe LA, Takahashi JS, and Bradfield CA. The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors. J Neurosci 20: RC83, 2000[Medline].

14.   Honma, S, Ikeda M, Abe H, Tanahashi Y, Namihira M, Honma K, and Nomura M. Circadian oscillation of BMAL1, a partner of a mammalian clock gene Clock, in rat suprachiasmatic nucleus. Biochem Biophys Res Commun 250: 83-87, 1998[ISI][Medline].

15.   Ikeda, M, and Nomura M. cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage. Biochem Biophys Res Commun 233: 258-264, 1997[ISI][Medline].

16.   Ikeda, M, Yu W, Hirai M, Ebisawa T, Honma S, Yoshimura K, Honma KI, and Nomura M. cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2: its mRNA expression, subcellular distribution, and chromosomal localization. Biochem Biophys Res Commun 275: 493-502, 2000[ISI][Medline].

17.   Jin, X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, and Reppert SM. A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96: 57-68, 1999[ISI][Medline].

18.   Kalsbeek, A, Buijs RM, Engelmann M, Wotjak CT, and Landgraf R. In vivo measurement of a diurnal variation in vasopressin release in the rat suprachiasmatic nucleus. Brain Res 682: 75-82, 1995[ISI][Medline].

19.   King, DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, and Takahashi JS. Positional cloning of the mouse circadian clock gene. Cell 89: 641-653, 1997[ISI][Medline].

20.   Kozak, M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res 12: 857-872, 1984[Abstract].

21.   Kume, K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH, and Reppert SM. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98: 193-205, 1999[ISI][Medline].

22.   Kurnik, PB. Circadian variation in the efficacy of tissue-type plasminogen activator. Circulation 91: 1341-1346, 1995[Abstract/Free Full Text].

23.   Li, H, Ko HP, and Whitlock JP. Induction of phosphoglycerate kinase 1 gene expression by hypoxia. Roles of Arnt and HIF1alpha. J Biol Chem 271: 21262-21267, 1996[Abstract/Free Full Text].

24.   Liu, C, Weaver DR, Strogatz SH, and Reppert SM. Cellular construction of a circadian clock: period determination in the suprachiasmatic nuclei. Cell 91: 855-860, 1997[ISI][Medline].

25.   Maemura, K, de la Monte SM, Chin MT, Layne MD, Hsieh CM, Yet SF, Perrella MA, and Lee ME. CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression. J Biol Chem 275: 36847-37851, 2000[Abstract/Free Full Text].

26.   Majercak, J, Sidote D, Hardin PE, and Edery I. How a circadian clock adapts to seasonal decreases in temperature and day length. Neuron 24: 219-230, 1999[ISI][Medline].

27.   Nonaka, H, Emoto N, Ikeda K, Fukuya H, Rohman MS, Raharjo SB, Yagita K, Okamura H, and Yokoyama M. Angiotensin II induces circadian gene expression of clock genes in cultured vascular smooth muscle cells. Circulation 104: 1746-1748, 2001[Abstract/Free Full Text].

28.   Okano, T, Sasaki M, and Fukada Y. Cloning of mouse BMAL2 and its daily expression profile in the suprachiasmatic nucleus: a remarkable acceleration of Bmal2 sequence divergence after Bmal gene duplication. Neurosci Lett 300: 111-114, 2001[ISI][Medline].

29.   Okano, T, Yamamoto K, Okano K, Hirota T, Kasahara T, Sasaki M, Takanaka Y, and Fukada Y. Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation. Genes Cells 6: 825-836, 2001[Abstract/Free Full Text].

30.   Pando, MP, Pinchak AB, Cermakian N, and Sassone-Corsi P. A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock. Proc Natl Acad Sci USA 98: 10178-10183, 2001[Abstract/Free Full Text].

31.   Reisz-Porszasz, S, Probst MR, Fukunaga BN, and Hankinson O. Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Mol Cell Biol 14: 6075-6086, 1994[Abstract].

32.   Reppert, SM, and Weaver DR. Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63: 647-676, 2001[ISI][Medline].

33.   Sakamoto, K, Nagase T, Fukui H, Horikawa K, Okada T, Tanaka H, Sato K, Miyake Y, Ohara O, Kako K, and Ishida N. Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J Biol Chem 273: 27039-27042, 1998[Abstract/Free Full Text].

34.   Sogawa, K, Iwabuchi K, Abe H, and Fujii-Kuriyama Y. Transcriptional activation domains of the Ah receptor and Ah receptor nuclear translocator. J Cancer Res Clin Oncol 121: 612-620, 1995[ISI][Medline].

35.   Takahata, S, Ozaki T, Mimura J, Kikuchi Y, Sogawa K, and Fujii-Kuriyama Y. Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3. Genes Cells 5: 739-747, 2000[Abstract/Free Full Text].

36.   Takahata, S, Sogawa K, Kobayashi A, Ema M, Mimura J, Ozaki N, and Fujii-Kuriyama Y. Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock. Biochem Biophys Res Commun 248: 789-794, 1998[ISI][Medline].

37.   Tanguay, RL, Andreasen E, Heideman W, and Peterson RE. Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. Biochim Biophys Acta 1494: 117-128, 2000[ISI][Medline].

38.   Van Zonneveld, AJ, Curriden SA, and Loskutoff DJ. Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter. Proc Natl Acad Sci USA 85: 5525-5529, 1988[Abstract].

39.   Welsh, DK, Logothetis DE, Meister M, and Reppert SM. Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14: 697-706, 1995[ISI][Medline].

40.   Wolting, CD, and McGlade CJ. Cloning and chromosomal localization of a new member of the bHLH/PAS transcription factor family. Mamm Genome 9: 463-468, 1998[ISI][Medline].

41.   Yu, W, Ikeda M, Abe H, Honma S, Ebisawa T, Yamauchi T, Honma K, and Nomura M. Characterization of three splice variants and genomic organization of the mouse BMAL1 gene. Biochem Biophys Res Commun 260: 760-767, 1999[ISI][Medline].

42.   Zhang, MQ. Statistical features of human exons and their flanking regions. Hum Mol Genet 7: 919-932, 1998[Abstract/Free Full Text].

43.   Zhou, YD, Barnard M, Tian H, Li X, Ring HZ, Francke U, Shelton J, Richardson J, Russell DW, and McKnight SL. Molecular characterization of two mammalian bHLH-PAS domain proteins selectively expressed in the central nervous system. Proc Natl Acad Sci USA 94: 713-718, 1997[Abstract/Free Full Text].


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