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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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*.
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.
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RESULTS |
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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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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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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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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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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 hBMAL2gC
and hBMAL2i
C proteins complementary to hBMAL2b W309*/hBMAL2b
C
(Fig. 6A). Paired
overexpression of mCLOCK with each truncated splice variant revealed a
distinct activation capacity for each resulting mCLOCK:hBMAL2
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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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-19 (9, 19). In addition, hBMAL2f failed to
inhibit gene transcription mediated by other mCLOCK:hBMAL2 heterodimers
(data not shown).
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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