Biochem. J. (2014) 461, 403–412 (Printed in Great Britain)
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doi:10.1042/BJ20131618
*School of Molecular and Biomedical Science (Biochemistry) and Centre for Molecular Pathology, University of Adelaide, Adelaide 5005, South Australia, Australia †CNRS-UMR8199, Lille Pasteur Institute, 59010 Lille, France ‡Lille Nord de France University, 59044 Lille, France §Department of Genomics of Common Disease, School of Public Health, Imperial College London, Hammersmith Hospital, London W12 ONN, U.K. University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, U.K.
The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Lowactivity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization
defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hotspot’ in SIM1 that is critical for protein function.
INTRODUCTION
within the SIM1 coding region in individuals with severe earlyonset obesity. Many of these variants cause a decrease in SIM1 protein activity [8,9]. However, the molecular mechanisms by which SIM1 variants impair function have not been explored to date. SIM1 forms a functional transcription factor by dimerizing with a partner bHLH PAS protein, specifically one of either ARNT (aryl hydrocarbon nuclear translocator) or ARNT2, via the bHLH and PAS repeat domains. Although SIM1–ARNT and SIM1–ARNT2 dimers behave similarly in cell-based assays [10], ARNT2 is likely to be the obligate partner of SIM1 in vivo as ARNT2 expression is mostly restricted to neurons and Arnt2-null mice appear to phenocopy Sim1-null mice [11]. In the present study we characterize the molecular basis of impaired function in selected human SIM1 variants, focusing on poor dimerization with ARNT2 and anomalous intracellular localization. To determine whether the effects of mutation are unique to the function and dimerization interface of SIM1, comparison is made with a series of equivalent variants in the coding region of the highly conserved paralogue SIM2 (singleminded 2). These data are then used to predict that specific SNVs listed in the 1000 Genomes and dbSNP human genomic databases will also be deficient in activity. Finally, the cumulative data is used to make observations about the specific interfacing of SIM1 and ARNT2 PAS domains, and define a ‘hot-spot’ in SIM1 and SIM2 which spans protein residues 290–326 and is
SIM1 (single-minded 1) is a transcription factor of the bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) family, a set of mammalian proteins which typically function in early development and stress-response pathways [1]. SIM1 is expressed most prominently in neurons of distinct regions in the hypothalamus, including the SON (supraoptic nucleus) and the PVN (paraventricular nucleus). Sim1 − / − mice die perinatally, presumably due to hypodevelopment of these nuclei as a result of impaired neuronal migration and differentiation [2]. SIM1 haploinsufficient mice (Sim1 − / + ) demonstrate hyperphagia and develop diet-related obesity without a change in energy expenditure, which is consistent with the role of the PVN in the regulation of energy homoeostasis and the feeding response [3,4]. Obesity is also observed in mice with a postnatally conditional knockout allele of Sim1 [5], whereas Agouti yellow (Ay ) mice, which are an established mouse obesity model, show a decrease in food intake when overexpressing ectopic SIM1 [6]. This indicates that SIM1 is also critical for post-developmental function of the PVN. SIM1 is also strongly associated with obesity in humans: a chromosomal translocation affecting the SIM1 gene was observed in a patient with juvenile-onset obesity and hyperphagia [7]. In addition, two recent studies have identified multiple cases of rare non-synonymous SNVs (single nucleotide variants) occurring
Key words: aryl hydrocarbon nuclear translocator 2 (ARNT2), obesity, Per/Arnt/Sim (PAS), single-minded (SIM), single nucleotide variant (SNV).
Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon nuclear translocator; bHLH, basic helix–loop–helix; BMAL1, brain and muscle ARNT-like 1; CLOCK, circadian locomotor output cycles kaput; CME, central midline element; DMOG, dimethyloxalylglycine; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; HRE, hypoxic-response element; NLS, nuclear localization sequence; PAS, Per/Arnt/Sim; PVN, paraventricular nucleus; SIM, single-minded; SNV, single nucleotide variant; Trh, Trachealess; WT, wild-type. 1 Present address: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LE, U.K. 2 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2014 Biochemical Society
Biochemical Journal
Adrienne E. SULLIVAN*, Anne RAIMONDO*1 , Tanja A. SCHWAB*, John B. BRUNING*, Philippe FROGUEL†‡§, I. Sadaf FAROOQI, Daniel J. PEET* and Murray L. WHITELAW*2
www.biochemj.org
Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2
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critical for function. The work described in the present study is of particular interest when considering the contributions of SIM1 and SIM2 activity to the development of obesity and select cancers respectively in human patients.
assays, each sample had triplicate wells assayed using the DualLuciferase Assay System (Promega), in at least three independent experiments. Firefly luciferase units were normalized to Renilla luciferase units for each well to give relative luciferase units, and the mean was calculated for each triplicate.
MATERIALS AND METHODS Plasmid construction
pcDNA5-FRT/TO-hSIM1-2Myc has been described previously [9]. V290E and V326F mutations were introduced via Gibson Assembly using Phusion High-Fidelity DNA Polymerase to pEFhSIM1-2Myc-IRES-Puro, and then subcloned into pcDNA5FRT/TO-hSIM1-2Myc. The SIM2s-2myc cassette from pEFhSIM2s-2Myc-IRES-Puro (described previously [12]) was removed with NotI/PmeI, and ligated into pENTR1A (Invitrogen) cut with DraI/NotI. The 2Myc sequence was removed and replaced with a 3FLAG tag sequence. Mutations and diagnostic silent restriction enzyme digestion sites were introduced to pENTR1A-hSIM2s-3FLAG using overlap extension PCR. The SIM2s-3FLAG cassette was then subcloned into pcDNA5FRT/TO-Gateway using LR recombination (Invitrogen). SIM2s truncation constructs were generated by PCR amplification and subcloning into pEF-2Myc-IRES-Puro cut with NheI/EcoRV. The details of all primers used are available in Supplementary Table S1 (at http://www.biochemj.org/bj/461/bj4610403add.htm). Plasmids pEF-hARNT-IRES-Neo and pEF-hARNT2-IRES-Neo [9] have been described previously. pML-6CME was a gift from Dr J. Pelletier (Department of Biochemistry, McGill University, Montreal, Canada). Generation and maintenance of cell lines
Doxycycline-inducible stable cell lines were generated using pcDNA5-FRT/TO-based plasmids and the 293 Flp-In TRex system (Invitrogen) according to the manufacturer’s instructions. All 293 Flp-In TRex and HEK (human embryonic kidney)293T cell lines were maintained in DMEM (Dulbecco’s modified Eagle’s medium; Gibco) supplemented with 10 % FBS (Gibco), 2 mM GlutaMAX (Gibco), 10 000 units/ml penicillin and 10 mg/ml streptomycin (Invitrogen) at 37 ◦ C with 5 % CO2 . Dual-luciferase assays
Cells were seeded in a 24-well tray the day before and transfected with 400 ng of pML-6CME luciferase reporter construct, 0.5 ng of pRL-CMV (Promega), and either of 20 ng of pEF-hARNT-IRES-Neo or 50 ng of pEF-hARNT2-IRESNeo, using FuGENE® HD according to the manufacturer’s instructions (Promega). At 7 h after transfection, media was replaced with full media supplemented with 1 μg/ml doxycyline. After 16 h of doxycycline treatment, cells were harvested and assayed for luciferase activity. For HIF (hypoxia-inducible factor) reporter assays, cells were transfected with 200 ng of pGL34HRE luciferase reporter construct and 0.5 ng of pRL-CMV and media was replaced 7 h after transfection with full media containing 1 μg/ml doxycycline with or without 1 mM DMOG (dimethyloxalylglycine). Cells were harvested and assayed after 16 h of treatment. For SIM2s truncation construct reporter assays, HEK-293T cells were transfected with 200 ng of pML-6CME luciferase reporter construct, 50 ng of pEF-hARNT-IRES-Neo, 0.5 ng of pRL-CMV and 20 ng of pEF-hSIM2s-2Myc-IRESPuro, empty vector or truncated SIM2s expression plasmid. Cells were harvested and assayed 24 h after transfection. For all reporter c The Authors Journal compilation c 2014 Biochemical Society
Immunoblotting
Whole-cell extracts of cell lines were taken as previously described [13]. Samples were separated using SDS/PAGE, transferred on to nitrocellulose, and immunoblotted using antiMyc antibodies (4A6, Millipore; or ab9106, Abcam), anti-FLAG antibody (M2, Sigma), anti-ARNT antibody (ab2, Abcam), antiARNT2 antibody (M-165, Santa Cruz Biotechnology) and antiα-tubulin (MCA78G, AbD Serotec). Immunoprecipitation
293 Flp-In TRex cells were co-treated with 1 μg/ml doxycycline (6 h) and 10 μM MG132 (6.5 h) before being harvested for whole-cell extracts. HEK-293T cells were harvested 24 h after transient transfection. Whole-cell extracts were incubated with anti-Myc antibody (4A6, Millipore) for 3 h and then rec-Protein G–Sepharose 4B conjugate (Invitrogen) for a further 1 h, or with anti-FLAG M2 affinity gel (Sigma) for 4 h. Samples were separated by SDS/PAGE and analysed by immunoblotting. Immunocytochemistry
HEK-293T cells or 293 Flp-In TRex cells were seeded on to glucose-coated glass coverslips in a 24-well tray. HEK-293T cells were transfected with 200 ng of pcDNA5-FRT/TO-hSIM2s3FLAG WT (wild-type) or variant constructs using FuGENE® 6 (Promega) according to the manufacturer’s instructions. 293 FlpIn TRex cells were induced with 1 μg/ml doxycycline for 16 h. At 24 h after transfection or 16 h after doxycycline treatment, cells were fixed with 4 % paraformaldehyde/PBS and immunostained with anti-FLAG antibody (M2, Sigma) or anti-Myc antibody (4A6, Millipore) and Alexa Fluor® 594-conjugated anti-mouse secondary antibody. Coverslips were mounted on to slides using ProLong Gold antifade reagent with DAPI (Invitrogen). Homology modelling
The ICM-Pro program suite was used to perform all homology modelling and carried out using the homology add-on [14]. Individual models for each SIM1 variant were created (without a dimerization partner first) using residues Asn230 –Leu332 (Uniprot number P81133) and the HIF-2α subunit of the previously reported HIF2α–ARNT crystal structure (PDB code 3F1P [15]) as the starting model. After creation of the individual models, they were subjected to regularization and model refinement within ICM-Pro (to optimize model geometry, carry out energy minimization and alleviate clashing side chains). These structures were then docked to ARNT using the HIF2α–ARNT crystal structure as a guide (PDB code 3F1P). Further regularization and model refinement was carried out to ensure the integrity of the dimer interface. Figure 7 was created using UCSF Chimera [16]. Statistics
To assess the significance of the activity of each variant relative to WT in reporter assays, univariate ANOVA was performed on the
Dimerization defects in SIM1 and SIM2 protein variants
Figure 1 Position of SIM1 and SIM2 amino acid changes due to nonsynonymous SNVs (A) Activity-impairing mutations caused by SNVs previously identified in juvenile-onset obesity patients (black text) and SNVs identified in human genomic databases (grey text) are located in the bHLH and two PAS repeats (PASA and PASB) that form the interface of dimerization with a partner factor. In particular, a cluster of low-activity mutations is found within PASB. Domain boundaries of SIM1 and SIM2 have been estimated based on sequence alignments with other bHLH PAS proteins and structure prediction by PsiPred software. The NLS in SIM1 and SIM2 has been previously defined [22]. (B) Human SIM1 and SIM2 proteins have 62 % primary sequence identity overall, but 89 % identity to the end of PASB. All mutated residues are fully conserved (highlighted). The positions of the basic region, helices and PAS folds (grey) are estimated using alignments and structure prediction by PsiPred. Alignments were performed by Clustal W2.
log values of relative luciferase units using IBM SPSS version 20. For comparisons between SIM1 and SIM2 variants as a percentage of WT activity in reporter assays, unpaired Student’s t test was used. RESULTS Transcriptional activities of reciprocal SIM1/SIM2 variants
Obesity-related SIM1 variants previously reported to harbour T A>G A>G A>G A>T C>A A>C G>A
SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM2 SIM2
R171H T46R H323Y T292A R296G S309G V290E V326F N316T E334K
3 7 2 – – – 1 1 – 11
Obesity Obesity Obesity – – – Unknown Unknown – Unknown
[9] [8] [8] I.S. Farooqi, unpublished work P. Froguel, unpublished work P. Froguel, unpublished work rs202065103 rs41285857 – rs143650216
Figure 2
Variants produce near-identical decreased activity in SIM1 and SIM2
(A) Expression of empty vector (Empty), WT or mutant proteins in stable 293 Flp-In TRex cell lines induced with doxycycline (1 μg/ml, 16 h) show some variation in protein levels, but in a similar manner between equivalent mutations in SIM1–2Myc and SIM2s–3FLAG. (B) 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct (6×CME-Luc) and plasmids expressing ARNT (left-hand histogram) or ARNT2 (right-hand histogram), and treated with doxycycline (1 μg/ml, 16 h). Equivalent mutant SIM1 and SIM2 proteins partnered with ectopic ARNT or ARNT2 show near-identical decreases in function compared with WT proteins, with the exceptions of T46R (with ARNT2) and R296G (with ARNT or ARNT2) which showed statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) (all other SIM1/SIM2 comparisons were not significant). Histograms show the mean fold change in luciferase units + − S.E.M. relative to WT for n = 6 (Empty) or n = 3 (all others) independent experiments. WB, Western blot.
PASB (T292A, R296G, S309G and H323Y) and caused decreased activities in both SIM1 and SIM2 proteins, we looked for other non-synonymous SNVs within or about this region in the 1000 Genomes and dbSNP human genomic databases. We identified two non-synonymous SNVs within the SIM1-coding region that were close to the cluster region: V290E (rs202065103) and V326F (rs41285857) (Figure 1A). We also identified two nonsynonymous SNVs within the SIM2-coding region that were likely to disrupt SIM2 protein function and lay close to or within the PASB cluster: N316T and E334K (rs143650216). At the time of analysis, these were the best candidate SNVs within our area of interest. Unfortunately, N316T was removed in a later build of the database as a false-positive variant. These SNVs were cloned into SIM2s–3FLAG or SIM1–2Myc expression plasmids and 293 Flp-In TRex stable cell lines were generated. Inducible expression c The Authors Journal compilation c 2014 Biochemical Society
of each variant protein was tested by Western blot analysis and protein levels were similar to WT in all cases (Figure 3). The variants were assessed for function when partnered with ARNT or ARNT2 using the 6×CME–Luciferase reporter gene assay. V290E SIM1–2Myc showed only 25 % WT activity with ARNT (P < 0.001) but retained 65 % WT activity with ARNT2 (P = 0.032). V326F SIM1–2Myc also demonstrated decreased activity compared with WT SIM1 with either of ARNT (P = 0.003) and ARNT2 (P = 0.010) (Figure 3A). Although no phenotypic information regarding individuals with these variants is available, this verifies that previously unidentified SIM1 lowactivity variants exist in the population. N316T SIM2s–3FLAG showed decreased activity compared with WT when partnered with either ARNT (P = 0.013) or ARNT2 (P < 0.001), but E334K SIM2–3FLAG showed no
Dimerization defects in SIM1 and SIM2 protein variants
Figure 3
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Select SIM1 and SIM2 variants reported in human genomic databases also show impaired function
Expression of empty vector (Empty), WT or mutant proteins in stable 293 Flp-In TRex cell lines induced with doxycycline (1 μg/ml, 16 h) show that mutations in SIM1–2Myc (A) and SIM2s–3FLAG (B) do not alter protein stability compared with WT. 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct (6×CME-Luc) and plasmids expressing ARNT (black) or ARNT2 (grey), and treated with doxycycline (1 μg/ml, 16 h). Significantly decreased activity compared with WT was observed for V290E SIM1–2Myc, V326F SIM1–2Myc (A) and N316T SIM2s–3FLAG (B) with both ARNT and ARNT2 (*P < 0.05, **P < 0.01, ***P < 0.001, ns is not significant). Histograms show the mean fold change in luciferase units + − S.E.M. relative to WT for n = 3 independent experiments. WB, Western blot.
significant change in activity compared with WT (Figure 3B). In total, three out of the four variants tested within or about the observed ‘mutational hot-spot’ region showed decreased function on a reporter gene.
Dimerization of SIM1/SIM2 variants with partner proteins
As the weakest SIM variants described thus far lie within the bHLH or PAS repeat domains, which constitute the dimerization interfaces of bHLH PAS proteins, co-immunoprecipitations were performed for each variant to assess whether dimerization with ARNT or ARNT2 was impaired. SIM1 T46R, T292A, R296G, S309G, H323Y and V290E showed decreased binding to ARNT2. SIM1 R171H and V326F, as well as some other previously tested SIM1 variants identified in individuals with early-onset obesity {S71R, L238R [9] and K51N (I.S. Farooqi, unpublished work)} showed no decrease in binding to ARNT2 compared with SIM1 WT (Figure 4A). All SIM2 variants with reduced activity, except R171H and N316T, showed decreased binding to ARNT (Figure 4B) and ARNT2 (results not shown) relative to SIM2 WT. This is consistent with their positions within domains critical for association with partner proteins, and provides evidence that impaired heterodimerization is a common cause of severely depleted activities in SIM1 and SIM2 variants.
Nuclear localization of SIM1/SIM2 variants
To determine that the observed loss of dimerization with nuclear proteins ARNT/ARNT2 was not due to mis-localization of SIM1 or SIM2 mutants to the nucleus, immunocytochemistry was performed. All variants localized predominantly to the nucleus, although some cytoplasmic staining was also observed for SIM1 V290E (Figure 5B), and T292A and R296G (results not shown). Variants T46R, T292A, R296G, S309G and H323Y showed similar results when tested in SIM1–2Myc (results not shown) and SIM2–3FLAG (Figure 5A). Although this mis-localization may contribute to the decrease in binding with ARNT and ARNT2, it is unlikely to be the singular cause as distribution still appears to be primarily nuclear. These variants are not located within the previously defined NLS (nuclear localization sequence) for SIM1 (amino acids 368–388) and SIM2 (amino acids 367–389) [22], and so increased protein in the cytoplasm may be an indication of increased nuclear export rather than decreased import.
Repression of HIF activity by SIM2 variants
SIM1 and SIM2 compete with other Class I bHLH PAS transcription factors, such as HIF-1α, for binding to common partner proteins ARNT and ARNT2 [23]. HIF-1α protein, which is expressed ubiquitously, is degraded under normal cellular conditions but becomes stabilized when oxygen is scarce c The Authors Journal compilation c 2014 Biochemical Society
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SIM1 and SIM2 variants show impaired dimerization with partner proteins
Whole-cell extracts from 293 TRex lines co-treated with doxycycline (1 μg/ml, 6 h) and proteasome inhibitor (10 μM MG132, 6.5 h) were used in co-immunoprecipitations of (A) SIM1–2Myc variants with endogenous ARNT2 and (B) SIM2s–3FLAG variants with endogenous ARNT. T46R, T292A, R296G, S309G and H323Y variants in both SIM1 and SIM2s caused a decrease in dimerization compared with WT proteins, whereas no change was seen in R171H, N316T and V326F variants. Some other SIM1 activity-deficient variants identified in individuals with early-onset obesity (K51N, S71R and L238R) were also tested but showed no loss of dimerization with ARNT2. IP, immunoprecipitation; WB, Western blot.
(hypoxia). HIF-1α heterodimerizes with ARNT, which is also known as HIF-1β, and binds HREs (hypoxic-response elements) to up-regulate target genes involved in hypoxic survival and cell development. SIM1 and SIM2 are also capable of binding HREs, and SIM1 has been shown to activate expression of HREbased reporter constructs [23]. Conversely, HRE-bound SIM2 is known to inhibit expression of both reporter genes and the endogenous HIF-1 target gene BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3) [24]. To confirm the impaired activity of a representative selection of SIM2s variants using an alternative system, 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct containing four HRE repeats (pGL3-4HRE) and treated with the hypoxia mimetic DMOG, which stabilizes HIF-1α protein (Figure 6). In the absence of SIM2s (Empty), DMOG treatment induces reporter activity greater than 20-fold. SIM2s–3FLAG WT protein does not alter expression of luciferase in the absence of DMOG, but represses induction to background levels by competing with HIF-1α for partner factors and binding to HREs. SIM2s R171H, which does not show impaired dimerization, represses DMOG induction in an identical manner with SIM2s WT. Conversely SIM2s T46R, T292A and R296G variants, which are dimerization-deficient, fail to fully inhibit induction of the reporter gene presumably due to reduced competition with HIF c The Authors Journal compilation c 2014 Biochemical Society
1α for ARNT and reduced binding of HRE sequences by SIM2s– ARNT heterodimers. Despite being similarly deficient in dimerization when analysed by co-immunoprecipitation (Figure 4B), SIM2s T46R was more effective than SIM2s T292A and R296G variants at inhibiting reporter gene induction. This is potentially due to the observed exclusive nuclear localization of SIM2s T46R, whereas T292A and R296G variants were partially localized to the cytoplasm (Figure 5A). Competition between SIM2s and HIF-1α for other unknown cofactors may also contribute to observed changes in reporter gene activity. In general, these results are consistent with previous observations of SIM2s variant activity.
Homology modelling of SIM1 PASB
A total of seven weak-activity variants in SIM1 and SIM2 cluster to a ‘hot-spot’ region in PASB (V290E, T292A, R296G, S309G, N316T, H323Y and V326F), and five also impair dimerization with a partner factor (V290E, T292A, R296G, S309G and H323Y). To further investigate how these variants cluster on a tertiary PAS fold, homology modelling of SIM1 PASB was performed (Figure 7A, blue chain) based on the most closely related protein structure available: HIF-2α PASB (42 % identity
Dimerization defects in SIM1 and SIM2 protein variants
Figure 5
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Activity-deficient variants show predominantly nuclear localization
(A) HEK-293T cells were transiently transfected with plasmids expressing SIM2s–3FLAG WT or mutant proteins, then fixed and stained with and anti-FLAG antibody (red) and DAPI nuclear stain (blue). Nuclear localization was observed for all SIM2s proteins, although some cytoplasmic staining was also seen for T292A and R296G variants (×600 magnification). (B) 293 Flp-In TRex cell lines were treated with doxycycline (1 μg/ml, 16 h), then fixed and stained with an anti-Myc antibody (red) and DAPI nuclear stain (blue). V326F SIM1–2Myc showed exclusively nuclear localization, but V290E SIM1–2Myc also showed some cytoplasmic localization (×600 magnification).
with SIM1 PASB), which was co-crystallized with ARNT PASB (Figure 7B, right brown chain) (PDB code 3F1P). Of the amino acids that cause dimerization defects when mutated (red), four out of five (Val290 , Thr292 , Arg296 and Ser309 ) roughly cluster in a region on the β-sheet face of the PASB fold. The remaining residue, His323 , is located at the edge of a loop region which may be highly mobile or differentially structured. Other low-activity variant residues (Leu238 , Asn316 and Val326 ) not associated with impaired dimerization (green) were not located in a common region. The homology-modelled SIM1 PASB was then superimposed on the position of HIF-2α PASB in the HIF-2α–ARNT cocrystal structure and subjected to more rounds of minimization and regularization, in order to model the association between SIM1 PASB and ARNT PASB. However, the locations of the variants are not entirely consistent with this dimerization interface (Figure 7B). In particular, R296G variants demonstrated the lowest dimerization ability, but Arg296 is located at a distal point from the modelled dimerization interface. This indicates that the homology model is insufficient to fully characterize interactions between the PASB folds of SIM1 and ARNT, but also that the SIM1–ARNT dimerization interface is possibly structurally unique from those of previously reported structures of PAS domain proteins. DISCUSSION
Weak-activity variants in SIM1 have previously been identified in juvenile-onset obesity patients [8,9], but the mechanisms
of impairment were only speculated. In the present study we determined that two (T46R and H323Y) SIM1 variants found in human obesity patients, as well as a further three low-activity variants (T292A, R296G and S309G), experienced reduced function due to impaired binding of partner factor ARNT2. This is consistent with the location of the variants within domains that mediate dimerization. Only one SIM1 variant, R171H, showed no deficiency in dimerization. It is possible that this particular variant may impair PASA interactions with other unidentified proteins that are required for target gene activation. We also demonstrated that activity-deficient SIM1 variants (V290E and V326F) are present in the general population. Unfortunately no phenotypic data was available, but we predict that the individuals harbouring these SNVs may have an above-average BMI (body mass index). In total, we have confirmed deficiencies for nine variants in either or both SIM1 and SIM2, eight of which are located in the PAS repeat domain. This is consistent with the importance of the PAS region in the function of bHLH PAS transcription factors. The PAS repeat domain is considered the primary means of selective dimerization between specific bHLH PAS family members, to the exclusion of other subfamily bHLH PAS proteins and species of the broader bHLH superfamily [25]. PASB is particularly critical to the function of SIM2 protein such that when this repeat is lost through truncation, the SIM2 protein loses all ability to dimerize with ARNT and becomes completely transcriptionally inactive (Supplementary Figure S1 at http://www.biochemj.org/bj/461/bj4610403add.htm and [26]). This importance of PASB to the function of full-length SIM c The Authors Journal compilation c 2014 Biochemical Society
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Figure 6 SIM2s variants with impaired dimerization show reduced repression of HIF activity Treatment with the hypoxia mimetic DMOG (1 mM, 16 h) was used to stabilize HIF-1α protein and induce luciferase expression from a transiently transfected HIF-responsive reporter construct (4×HRE-Luc) in 293 Flp-In TRex cell lines. Expression of empty vector (Empty), WT or mutant proteins was simultaneously induced with doxycycline (1 μg/ml, 16 h). SIM2s WT protein has no effect on reporter activity alone (WT no DMOG), but reduces HIF-mediated induction to non-induced levels. SIM2s T46R, T292A and R296G variants, which have impaired dimerization with ARNT, fail to completely repress induction of the reporter gene. SIM2s R171H, which has no loss of dimerization, shows similar repression to SIM2s WT (***P < 0.001, ns is not significant). Histogram shows the mean fold change in luciferase units + − S.E.M. relative to Empty (no DMOG) for n = 4 independent experiments.
proteins is also evident in the observed critical loss of activity caused by missense SNVs within our defined mutational hot-spot region in PASB.
Figure 7
There is also evidence that PAS domains contribute to the specific binding of DNA sequences and target gene recognition [25,27]. The importance of PAS domains in the unique function of individual bHLH PAS proteins was demonstrated in a key experiment whereby the PAS domain of the Drosophila melanogaster orthologue of HIF-1α, Trh (Trachealess), was replaced with the PAS domain of the SIM1/SIM2 orthologue, Sim (Single-minded). The Trh–Sim PAS chimaeric protein lost the ability to regulate Trh target genes and instead induced target genes and phenotypes specific to Sim [28]. Exactly how the PAS domain dictates this unique activity is not well understood. In a previous study, a random mutagenesis screen was performed for key residues in the PASA repeat of ARNT that mediate heterodimerization with the PASA repeat of AhR (aryl hydrocarbon receptor). A total of 22 individual mutations were found to inhibit or ablate AhR–ARNT dimerization but, interestingly, four of these variants did not affect activity of ARNT with other partners such as SIM1, SIM2 or NPAS4 (neuronal PAS domain protein 4) [13]. This indicates that although the general interface of ARNT PASA with its partner may be conserved, there are key residues which are critical to partner-specific interactions. This conclusion is consistent with the observation that the R296G variant, located in PASB, produces significantly different changes in activity when present in SIM1 or SIM2s and when partnered with either ARNT or ARNT2. R296G SIM1–2Myc retains 42 % of WT activity when partnered with ARNT2, but appears to be completely inactive when partnered with ARNT, indicating that different residues may be critical in ARNT- and ARNT2-specific interactions with SIM1. That R296G SIM2s– 3FLAG shows below-basal activity in all cases indicates that, despite sharing high sequence identity, there are some partner interactions that are specific to SIM1 or SIM2. These specific PAS-mediated interactions may also be a factor that differentiates the biological activities of SIM1 and SIM2 in vivo. The T46R variant, which is located in the second helix of the bHLH domain, also produced significantly different effects on activity in SIM1 compared with SIM2s when partnered with ARNT but not ARNT2. This may indicate that some SIM1- and
Mapping of variants on the SIM1 PASB homology model
(A) Homology modelling of SIM1 PASB (blue chain) based on HIF-2α PASB (PDB code 3F1P) shows the position of residues that, when mutated, either impaired (red; Val290 , Thr292 , Arg296 , Ser309 or His323 ) or did not alter (green; Leu238 , Asn316 or Val326 ) dimerization with ARNT2. With the exception of His323 , red residues roughly cluster together at the β-sheet face. Green residues do not seem to cluster with red residues or each other. (B) ARNT PASB (brown chain, right), which was co-crystallized with HIF-2α PASB, does not seem to interact closely with the red cluster. c The Authors Journal compilation c 2014 Biochemical Society
Dimerization defects in SIM1 and SIM2 protein variants
SIM2-specific interactions are also mediated by the bHLH region, although in all cases T46R variant activity was approximate to basal levels and so the true relevance of this difference in activity is uncertain. Specificity of partner selection is likely to also be influenced by the structural orientation and interfacing of PASB. Although structural data of bHLH PAS proteins is limited, the PASB repeats of family members CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) have been revealed to associate in an approximately parallel manner [29], whereas isolated PASB folds of HIF-2α and ARNT show an antiparallel orientation and β-sheet interface [15]. This may explain the preference of CLOCK for BMAL1 rather than ARNT, and raises the question of how the PASB dimerization interface varies among other family members. Homology modelling of SIM1 PASB on HIF-2α PASB revealed that hot-spot activity-impaired variants that impair dimerization cluster roughly to a region on the β-sheet face of the fold, indicating that the dimerization interface of SIM1 is more similar to that of HIF-2α than that of CLOCK. However, the exact location of the cluster relative to ARNT PASB suggests that this model is incomplete, and implies that the PASB dimerization interface may vary between SIM1 and HIF-2α. It is also possible that the isolated HIF-2α PASB and ARNT PASB folds, which lack the structural context of the larger protein, do not accurately depict the natural dimerization interface. The structure of SIM1 will need to be solved to ultimately resolve these questions, but data regarding the influence of point mutations on protein activity is still important when assessing and interpreting solved structures; for example, data from the aforementioned AhR/ARNT PASA mutational study [13] was used in analysis of the recently generated AhR PASA crystal structure [30]. Therefore the present study will be useful to future SIM or bHLH PAS structural determination projects. In conclusion, we have identified a hot-spot region in SIM1 and SIM2 spanning amino acids 290–326 which is critical for protein function, and defined a subset of human variants within this hotspot which inhibit dimerization with partner proteins ARNT and ARNT2. These data may be used in the future for assessment of deleterious human SNVs in SIM1 and SIM2, and in understanding the mechanistic deficiencies in protein variants that currently exist in the population. Severely deficient SIM1 variants that were identified in obese patients are possibly monogenic causes of obesity, but less-deficient variants could also contribute to weight gain in combination with other factors. In the future, identifying any genetic factors contributing to development of obesity may be important in order to manage the patient condition effectively. Thus screening patients for SIM1 variants and testing activity with molecular assays is likely to be an important diagnostic tool. AUTHOR CONTRIBUTION Adrienne Sullivan, Anne Raimondo, Daniel Peet and Murray Whitelaw conceived the project and designed the experiments. Adrienne Sullivan, Anne Raimondo and Tanja Schwab conducted experiments. John Bruning generated the SIM1 homology model. Philippe Froguel and I. Sadaf Farooq provided reagents and information about SIM1 variants. Adrienne Sullivan and Murray Whitelaw wrote the paper.
ACKNOWLEDGEMENT We thank the third-year Biochemistry students of the University of Adelaide for their work in cloning variants.
FUNDING This work was supported by the Australian Research Council.
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Acids Res. 39, 3695–3709 CrossRef 14 Cardozo, T., Totrov, M. and Abagyan, R. (1995) Homology modeling by the ICM method. Proteins 23, 403–414 CrossRef PubMed 15 Scheuermann, T. H., Tomchick, D. R., Machius, M., Guo, Y., Bruick, R. K. and Gardner, K. H. (2009) Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor. Proc. Natl. Acad. Sci. U.S.A. 106, 450–455 CrossRef PubMed 16 Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004) UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 CrossRef PubMed 17 Goshu, E., Jin, H., Fasnacht, R., Sepenski, M., Michaud, J. L. and Fan, C. M. (2002) Sim2 mutants have developmental defects not overlapping with those of Sim1 mutants. Mol. Cell. Biol. 22, 4147–4157 CrossRef PubMed 18 DeYoung, M. P., Tress, M. and Narayanan, R. (2003) Identification of Down’s syndrome critical locus gene SIM2-s as a drug therapy target for solid tumors. Proc. Natl. Acad. Sci. U.S.A. 100, 4760–4765 CrossRef PubMed 19 Muenke, M., Bone, L. J., Mitchell, H. F., Hart, I., Walton, K., Hall-Johnson, K., Ippel, E. F., Dietz-Band, J., Kvaloy, K., Fan, C. M. et al. (1995) Physical mapping of the holoprosencephaly critical region in 21q22.3, exclusion of SIM2 as a candidate gene for holoprosencephaly, and mapping of SIM2 to a region of chromosome 21 important for Down syndrome. Am. J. Hum. Genet. 57, 1074–1079 PubMed 20 Ema, M., Ikegami, S., Hosoya, T., Mimura, J., Ohtani, H., Nakao, K., Inokuchi, K., Katsuki, M. and Fujii-Kuriyama, Y. (1999) Mild impairment of learning and memory in mice overexpressing the mSim2 gene located on chromosome 16: an animal model of Down’s syndrome. Hum. Mol. Genet. 8, 1409–1415 CrossRef PubMed 21 Metz, R. P., Kwak, H. I., Gustafson, T., Laffin, B. and Porter, W. W. (2006) Differential transcriptional regulation by mouse single-minded 2s. J. Biol. Chem. 281, 10839–10848 CrossRef PubMed 22 Yamaki, A., Kudoh, J., Shimizu, N. and Shimizu, Y. (2004) A novel nuclear localization signal in the human single-minded proteins SIM1 and SIM2. Biochem. Biophys. Res. Commun. 313, 482–488 CrossRef PubMed c The Authors Journal compilation c 2014 Biochemical Society
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23 Woods, S. L. and Whitelaw, M. L. (2002) Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop-helix/per-Arnt-Sim homology transcription factors. J. Biol. Chem. 277, 10236–10243 CrossRef PubMed 24 Farrall, A. L. and Whitelaw, M. L. (2009) The HIF1α-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element. Oncogene 28, 3671–3680 CrossRef PubMed 25 Pongratz, I., Antonsson, C., Whitelaw, M. L. and Poellinger, L. (1998) Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor. Mol. Cell. Biol. 18, 4079–4088 PubMed 26 Moffett, P., Reece, M. and Pelletier, J. (1997) The murine Sim-2 gene product inhibits transcription by active repression and functional interference. Mol. Cell. Biol. 17, 4933–4947 PubMed Received 9 December 2013/15 April 2014; accepted 12 May 2014 Published as BJ Immediate Publication 12 May 2014, doi:10.1042/BJ20131618
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27 Chapman-Smith, A., Lutwyche, J. K. and Whitelaw, M. L. (2004) Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators. J. Biol. Chem. 279, 5353–5362 CrossRef PubMed 28 Zelzer, E., Wappner, P. and Shilo, B. Z. (1997) The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins. Genes Dev. 11, 2079–2089 CrossRef PubMed 29 Huang, N., Chelliah, Y., Shan, Y., Taylor, C. A., Yoo, S. H., Partch, C., Green, C. B., Zhang, H. and Takahashi, J. S. (2012) Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex. Science 337, 189–194 CrossRef PubMed 30 Wu, D., Potluri, N., Kim, Y. and Rastinejad, F. (2013) Structure and dimerization properties of the aryl hydrocarbon receptor PAS-A domain. Mol. Cell. Biol. 33, 4346–4356 CrossRef PubMed
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SUPPLEMENTARY ONLINE DATA
Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2 Adrienne E. SULLIVAN*, Anne RAIMONDO*1 , Tanja A. SCHWAB*, John B. BRUNING*, Philippe FROGUEL†‡§, I. Sadaf FAROOQI, Daniel J. PEET* and Murray L. WHITELAW*2 *School of Molecular and Biomedical Science (Biochemistry) and Centre for Molecular Pathology, University of Adelaide, Adelaide 5005, South Australia, Australia †CNRS-UMR8199, Lille Pasteur Institute, 59010 Lille, France ‡Lille Nord de France University, 59044 Lille, France §Department of Genomics of Common Disease, School of Public Health, Imperial College London, Hammersmith Hospital, London W12 ONN, U.K. University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, U.K.
Figure S1
SIM2 PASB is essential for transcriptional activation and heterodimerization
Expression of SIM2s truncations lacking the C-terminal transrepression domain (3) or both PASB and the transrepression domain (2) (A) was confirmed by transient expression in HEK-293T cells and Western blot analysis (B). Activity of the truncated proteins was assessed by transiently co-transfecting with plasmids expressing ARNT and luciferase reporter construct (6 × CME-Luc) in HEK-293T cells (C). Loss of the transrepression domain does not inhibit SIM2s activity, but loss of PASB ablates all reporter gene induction. Co-immunoprecipitation experiments using co-transfected FLAG-tagged ARNT demonstrate that this loss of activity corresponds with a loss of dimerization with ARNT (D). Histograms show the mean fold change in luciferase units + − S.E.M. relative to ARNT only for n = 4 independent experiments (*P
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*School of Molecular and Biomedical Science (Biochemistry) and Centre for Molecular Pathology, University of Adelaide, Adelaide 5005, South Australia, Australia †CNRS-UMR8199, Lille Pasteur Institute, 59010 Lille, France ‡Lille Nord de France University, 59044 Lille, France §Department of Genomics of Common Disease, School of Public Health, Imperial College London, Hammersmith Hospital, London W12 ONN, U.K. University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, U.K.
The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Lowactivity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization
defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hotspot’ in SIM1 that is critical for protein function.
INTRODUCTION
within the SIM1 coding region in individuals with severe earlyonset obesity. Many of these variants cause a decrease in SIM1 protein activity [8,9]. However, the molecular mechanisms by which SIM1 variants impair function have not been explored to date. SIM1 forms a functional transcription factor by dimerizing with a partner bHLH PAS protein, specifically one of either ARNT (aryl hydrocarbon nuclear translocator) or ARNT2, via the bHLH and PAS repeat domains. Although SIM1–ARNT and SIM1–ARNT2 dimers behave similarly in cell-based assays [10], ARNT2 is likely to be the obligate partner of SIM1 in vivo as ARNT2 expression is mostly restricted to neurons and Arnt2-null mice appear to phenocopy Sim1-null mice [11]. In the present study we characterize the molecular basis of impaired function in selected human SIM1 variants, focusing on poor dimerization with ARNT2 and anomalous intracellular localization. To determine whether the effects of mutation are unique to the function and dimerization interface of SIM1, comparison is made with a series of equivalent variants in the coding region of the highly conserved paralogue SIM2 (singleminded 2). These data are then used to predict that specific SNVs listed in the 1000 Genomes and dbSNP human genomic databases will also be deficient in activity. Finally, the cumulative data is used to make observations about the specific interfacing of SIM1 and ARNT2 PAS domains, and define a ‘hot-spot’ in SIM1 and SIM2 which spans protein residues 290–326 and is
SIM1 (single-minded 1) is a transcription factor of the bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) family, a set of mammalian proteins which typically function in early development and stress-response pathways [1]. SIM1 is expressed most prominently in neurons of distinct regions in the hypothalamus, including the SON (supraoptic nucleus) and the PVN (paraventricular nucleus). Sim1 − / − mice die perinatally, presumably due to hypodevelopment of these nuclei as a result of impaired neuronal migration and differentiation [2]. SIM1 haploinsufficient mice (Sim1 − / + ) demonstrate hyperphagia and develop diet-related obesity without a change in energy expenditure, which is consistent with the role of the PVN in the regulation of energy homoeostasis and the feeding response [3,4]. Obesity is also observed in mice with a postnatally conditional knockout allele of Sim1 [5], whereas Agouti yellow (Ay ) mice, which are an established mouse obesity model, show a decrease in food intake when overexpressing ectopic SIM1 [6]. This indicates that SIM1 is also critical for post-developmental function of the PVN. SIM1 is also strongly associated with obesity in humans: a chromosomal translocation affecting the SIM1 gene was observed in a patient with juvenile-onset obesity and hyperphagia [7]. In addition, two recent studies have identified multiple cases of rare non-synonymous SNVs (single nucleotide variants) occurring
Key words: aryl hydrocarbon nuclear translocator 2 (ARNT2), obesity, Per/Arnt/Sim (PAS), single-minded (SIM), single nucleotide variant (SNV).
Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon nuclear translocator; bHLH, basic helix–loop–helix; BMAL1, brain and muscle ARNT-like 1; CLOCK, circadian locomotor output cycles kaput; CME, central midline element; DMOG, dimethyloxalylglycine; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; HRE, hypoxic-response element; NLS, nuclear localization sequence; PAS, Per/Arnt/Sim; PVN, paraventricular nucleus; SIM, single-minded; SNV, single nucleotide variant; Trh, Trachealess; WT, wild-type. 1 Present address: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LE, U.K. 2 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2014 Biochemical Society
Biochemical Journal
Adrienne E. SULLIVAN*, Anne RAIMONDO*1 , Tanja A. SCHWAB*, John B. BRUNING*, Philippe FROGUEL†‡§, I. Sadaf FAROOQI, Daniel J. PEET* and Murray L. WHITELAW*2
www.biochemj.org
Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2
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critical for function. The work described in the present study is of particular interest when considering the contributions of SIM1 and SIM2 activity to the development of obesity and select cancers respectively in human patients.
assays, each sample had triplicate wells assayed using the DualLuciferase Assay System (Promega), in at least three independent experiments. Firefly luciferase units were normalized to Renilla luciferase units for each well to give relative luciferase units, and the mean was calculated for each triplicate.
MATERIALS AND METHODS Plasmid construction
pcDNA5-FRT/TO-hSIM1-2Myc has been described previously [9]. V290E and V326F mutations were introduced via Gibson Assembly using Phusion High-Fidelity DNA Polymerase to pEFhSIM1-2Myc-IRES-Puro, and then subcloned into pcDNA5FRT/TO-hSIM1-2Myc. The SIM2s-2myc cassette from pEFhSIM2s-2Myc-IRES-Puro (described previously [12]) was removed with NotI/PmeI, and ligated into pENTR1A (Invitrogen) cut with DraI/NotI. The 2Myc sequence was removed and replaced with a 3FLAG tag sequence. Mutations and diagnostic silent restriction enzyme digestion sites were introduced to pENTR1A-hSIM2s-3FLAG using overlap extension PCR. The SIM2s-3FLAG cassette was then subcloned into pcDNA5FRT/TO-Gateway using LR recombination (Invitrogen). SIM2s truncation constructs were generated by PCR amplification and subcloning into pEF-2Myc-IRES-Puro cut with NheI/EcoRV. The details of all primers used are available in Supplementary Table S1 (at http://www.biochemj.org/bj/461/bj4610403add.htm). Plasmids pEF-hARNT-IRES-Neo and pEF-hARNT2-IRES-Neo [9] have been described previously. pML-6CME was a gift from Dr J. Pelletier (Department of Biochemistry, McGill University, Montreal, Canada). Generation and maintenance of cell lines
Doxycycline-inducible stable cell lines were generated using pcDNA5-FRT/TO-based plasmids and the 293 Flp-In TRex system (Invitrogen) according to the manufacturer’s instructions. All 293 Flp-In TRex and HEK (human embryonic kidney)293T cell lines were maintained in DMEM (Dulbecco’s modified Eagle’s medium; Gibco) supplemented with 10 % FBS (Gibco), 2 mM GlutaMAX (Gibco), 10 000 units/ml penicillin and 10 mg/ml streptomycin (Invitrogen) at 37 ◦ C with 5 % CO2 . Dual-luciferase assays
Cells were seeded in a 24-well tray the day before and transfected with 400 ng of pML-6CME luciferase reporter construct, 0.5 ng of pRL-CMV (Promega), and either of 20 ng of pEF-hARNT-IRES-Neo or 50 ng of pEF-hARNT2-IRESNeo, using FuGENE® HD according to the manufacturer’s instructions (Promega). At 7 h after transfection, media was replaced with full media supplemented with 1 μg/ml doxycyline. After 16 h of doxycycline treatment, cells were harvested and assayed for luciferase activity. For HIF (hypoxia-inducible factor) reporter assays, cells were transfected with 200 ng of pGL34HRE luciferase reporter construct and 0.5 ng of pRL-CMV and media was replaced 7 h after transfection with full media containing 1 μg/ml doxycycline with or without 1 mM DMOG (dimethyloxalylglycine). Cells were harvested and assayed after 16 h of treatment. For SIM2s truncation construct reporter assays, HEK-293T cells were transfected with 200 ng of pML-6CME luciferase reporter construct, 50 ng of pEF-hARNT-IRES-Neo, 0.5 ng of pRL-CMV and 20 ng of pEF-hSIM2s-2Myc-IRESPuro, empty vector or truncated SIM2s expression plasmid. Cells were harvested and assayed 24 h after transfection. For all reporter c The Authors Journal compilation c 2014 Biochemical Society
Immunoblotting
Whole-cell extracts of cell lines were taken as previously described [13]. Samples were separated using SDS/PAGE, transferred on to nitrocellulose, and immunoblotted using antiMyc antibodies (4A6, Millipore; or ab9106, Abcam), anti-FLAG antibody (M2, Sigma), anti-ARNT antibody (ab2, Abcam), antiARNT2 antibody (M-165, Santa Cruz Biotechnology) and antiα-tubulin (MCA78G, AbD Serotec). Immunoprecipitation
293 Flp-In TRex cells were co-treated with 1 μg/ml doxycycline (6 h) and 10 μM MG132 (6.5 h) before being harvested for whole-cell extracts. HEK-293T cells were harvested 24 h after transient transfection. Whole-cell extracts were incubated with anti-Myc antibody (4A6, Millipore) for 3 h and then rec-Protein G–Sepharose 4B conjugate (Invitrogen) for a further 1 h, or with anti-FLAG M2 affinity gel (Sigma) for 4 h. Samples were separated by SDS/PAGE and analysed by immunoblotting. Immunocytochemistry
HEK-293T cells or 293 Flp-In TRex cells were seeded on to glucose-coated glass coverslips in a 24-well tray. HEK-293T cells were transfected with 200 ng of pcDNA5-FRT/TO-hSIM2s3FLAG WT (wild-type) or variant constructs using FuGENE® 6 (Promega) according to the manufacturer’s instructions. 293 FlpIn TRex cells were induced with 1 μg/ml doxycycline for 16 h. At 24 h after transfection or 16 h after doxycycline treatment, cells were fixed with 4 % paraformaldehyde/PBS and immunostained with anti-FLAG antibody (M2, Sigma) or anti-Myc antibody (4A6, Millipore) and Alexa Fluor® 594-conjugated anti-mouse secondary antibody. Coverslips were mounted on to slides using ProLong Gold antifade reagent with DAPI (Invitrogen). Homology modelling
The ICM-Pro program suite was used to perform all homology modelling and carried out using the homology add-on [14]. Individual models for each SIM1 variant were created (without a dimerization partner first) using residues Asn230 –Leu332 (Uniprot number P81133) and the HIF-2α subunit of the previously reported HIF2α–ARNT crystal structure (PDB code 3F1P [15]) as the starting model. After creation of the individual models, they were subjected to regularization and model refinement within ICM-Pro (to optimize model geometry, carry out energy minimization and alleviate clashing side chains). These structures were then docked to ARNT using the HIF2α–ARNT crystal structure as a guide (PDB code 3F1P). Further regularization and model refinement was carried out to ensure the integrity of the dimer interface. Figure 7 was created using UCSF Chimera [16]. Statistics
To assess the significance of the activity of each variant relative to WT in reporter assays, univariate ANOVA was performed on the
Dimerization defects in SIM1 and SIM2 protein variants
Figure 1 Position of SIM1 and SIM2 amino acid changes due to nonsynonymous SNVs (A) Activity-impairing mutations caused by SNVs previously identified in juvenile-onset obesity patients (black text) and SNVs identified in human genomic databases (grey text) are located in the bHLH and two PAS repeats (PASA and PASB) that form the interface of dimerization with a partner factor. In particular, a cluster of low-activity mutations is found within PASB. Domain boundaries of SIM1 and SIM2 have been estimated based on sequence alignments with other bHLH PAS proteins and structure prediction by PsiPred software. The NLS in SIM1 and SIM2 has been previously defined [22]. (B) Human SIM1 and SIM2 proteins have 62 % primary sequence identity overall, but 89 % identity to the end of PASB. All mutated residues are fully conserved (highlighted). The positions of the basic region, helices and PAS folds (grey) are estimated using alignments and structure prediction by PsiPred. Alignments were performed by Clustal W2.
log values of relative luciferase units using IBM SPSS version 20. For comparisons between SIM1 and SIM2 variants as a percentage of WT activity in reporter assays, unpaired Student’s t test was used. RESULTS Transcriptional activities of reciprocal SIM1/SIM2 variants
Obesity-related SIM1 variants previously reported to harbour T A>G A>G A>G A>T C>A A>C G>A
SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM1 SIM2 SIM2
R171H T46R H323Y T292A R296G S309G V290E V326F N316T E334K
3 7 2 – – – 1 1 – 11
Obesity Obesity Obesity – – – Unknown Unknown – Unknown
[9] [8] [8] I.S. Farooqi, unpublished work P. Froguel, unpublished work P. Froguel, unpublished work rs202065103 rs41285857 – rs143650216
Figure 2
Variants produce near-identical decreased activity in SIM1 and SIM2
(A) Expression of empty vector (Empty), WT or mutant proteins in stable 293 Flp-In TRex cell lines induced with doxycycline (1 μg/ml, 16 h) show some variation in protein levels, but in a similar manner between equivalent mutations in SIM1–2Myc and SIM2s–3FLAG. (B) 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct (6×CME-Luc) and plasmids expressing ARNT (left-hand histogram) or ARNT2 (right-hand histogram), and treated with doxycycline (1 μg/ml, 16 h). Equivalent mutant SIM1 and SIM2 proteins partnered with ectopic ARNT or ARNT2 show near-identical decreases in function compared with WT proteins, with the exceptions of T46R (with ARNT2) and R296G (with ARNT or ARNT2) which showed statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) (all other SIM1/SIM2 comparisons were not significant). Histograms show the mean fold change in luciferase units + − S.E.M. relative to WT for n = 6 (Empty) or n = 3 (all others) independent experiments. WB, Western blot.
PASB (T292A, R296G, S309G and H323Y) and caused decreased activities in both SIM1 and SIM2 proteins, we looked for other non-synonymous SNVs within or about this region in the 1000 Genomes and dbSNP human genomic databases. We identified two non-synonymous SNVs within the SIM1-coding region that were close to the cluster region: V290E (rs202065103) and V326F (rs41285857) (Figure 1A). We also identified two nonsynonymous SNVs within the SIM2-coding region that were likely to disrupt SIM2 protein function and lay close to or within the PASB cluster: N316T and E334K (rs143650216). At the time of analysis, these were the best candidate SNVs within our area of interest. Unfortunately, N316T was removed in a later build of the database as a false-positive variant. These SNVs were cloned into SIM2s–3FLAG or SIM1–2Myc expression plasmids and 293 Flp-In TRex stable cell lines were generated. Inducible expression c The Authors Journal compilation c 2014 Biochemical Society
of each variant protein was tested by Western blot analysis and protein levels were similar to WT in all cases (Figure 3). The variants were assessed for function when partnered with ARNT or ARNT2 using the 6×CME–Luciferase reporter gene assay. V290E SIM1–2Myc showed only 25 % WT activity with ARNT (P < 0.001) but retained 65 % WT activity with ARNT2 (P = 0.032). V326F SIM1–2Myc also demonstrated decreased activity compared with WT SIM1 with either of ARNT (P = 0.003) and ARNT2 (P = 0.010) (Figure 3A). Although no phenotypic information regarding individuals with these variants is available, this verifies that previously unidentified SIM1 lowactivity variants exist in the population. N316T SIM2s–3FLAG showed decreased activity compared with WT when partnered with either ARNT (P = 0.013) or ARNT2 (P < 0.001), but E334K SIM2–3FLAG showed no
Dimerization defects in SIM1 and SIM2 protein variants
Figure 3
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Select SIM1 and SIM2 variants reported in human genomic databases also show impaired function
Expression of empty vector (Empty), WT or mutant proteins in stable 293 Flp-In TRex cell lines induced with doxycycline (1 μg/ml, 16 h) show that mutations in SIM1–2Myc (A) and SIM2s–3FLAG (B) do not alter protein stability compared with WT. 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct (6×CME-Luc) and plasmids expressing ARNT (black) or ARNT2 (grey), and treated with doxycycline (1 μg/ml, 16 h). Significantly decreased activity compared with WT was observed for V290E SIM1–2Myc, V326F SIM1–2Myc (A) and N316T SIM2s–3FLAG (B) with both ARNT and ARNT2 (*P < 0.05, **P < 0.01, ***P < 0.001, ns is not significant). Histograms show the mean fold change in luciferase units + − S.E.M. relative to WT for n = 3 independent experiments. WB, Western blot.
significant change in activity compared with WT (Figure 3B). In total, three out of the four variants tested within or about the observed ‘mutational hot-spot’ region showed decreased function on a reporter gene.
Dimerization of SIM1/SIM2 variants with partner proteins
As the weakest SIM variants described thus far lie within the bHLH or PAS repeat domains, which constitute the dimerization interfaces of bHLH PAS proteins, co-immunoprecipitations were performed for each variant to assess whether dimerization with ARNT or ARNT2 was impaired. SIM1 T46R, T292A, R296G, S309G, H323Y and V290E showed decreased binding to ARNT2. SIM1 R171H and V326F, as well as some other previously tested SIM1 variants identified in individuals with early-onset obesity {S71R, L238R [9] and K51N (I.S. Farooqi, unpublished work)} showed no decrease in binding to ARNT2 compared with SIM1 WT (Figure 4A). All SIM2 variants with reduced activity, except R171H and N316T, showed decreased binding to ARNT (Figure 4B) and ARNT2 (results not shown) relative to SIM2 WT. This is consistent with their positions within domains critical for association with partner proteins, and provides evidence that impaired heterodimerization is a common cause of severely depleted activities in SIM1 and SIM2 variants.
Nuclear localization of SIM1/SIM2 variants
To determine that the observed loss of dimerization with nuclear proteins ARNT/ARNT2 was not due to mis-localization of SIM1 or SIM2 mutants to the nucleus, immunocytochemistry was performed. All variants localized predominantly to the nucleus, although some cytoplasmic staining was also observed for SIM1 V290E (Figure 5B), and T292A and R296G (results not shown). Variants T46R, T292A, R296G, S309G and H323Y showed similar results when tested in SIM1–2Myc (results not shown) and SIM2–3FLAG (Figure 5A). Although this mis-localization may contribute to the decrease in binding with ARNT and ARNT2, it is unlikely to be the singular cause as distribution still appears to be primarily nuclear. These variants are not located within the previously defined NLS (nuclear localization sequence) for SIM1 (amino acids 368–388) and SIM2 (amino acids 367–389) [22], and so increased protein in the cytoplasm may be an indication of increased nuclear export rather than decreased import.
Repression of HIF activity by SIM2 variants
SIM1 and SIM2 compete with other Class I bHLH PAS transcription factors, such as HIF-1α, for binding to common partner proteins ARNT and ARNT2 [23]. HIF-1α protein, which is expressed ubiquitously, is degraded under normal cellular conditions but becomes stabilized when oxygen is scarce c The Authors Journal compilation c 2014 Biochemical Society
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SIM1 and SIM2 variants show impaired dimerization with partner proteins
Whole-cell extracts from 293 TRex lines co-treated with doxycycline (1 μg/ml, 6 h) and proteasome inhibitor (10 μM MG132, 6.5 h) were used in co-immunoprecipitations of (A) SIM1–2Myc variants with endogenous ARNT2 and (B) SIM2s–3FLAG variants with endogenous ARNT. T46R, T292A, R296G, S309G and H323Y variants in both SIM1 and SIM2s caused a decrease in dimerization compared with WT proteins, whereas no change was seen in R171H, N316T and V326F variants. Some other SIM1 activity-deficient variants identified in individuals with early-onset obesity (K51N, S71R and L238R) were also tested but showed no loss of dimerization with ARNT2. IP, immunoprecipitation; WB, Western blot.
(hypoxia). HIF-1α heterodimerizes with ARNT, which is also known as HIF-1β, and binds HREs (hypoxic-response elements) to up-regulate target genes involved in hypoxic survival and cell development. SIM1 and SIM2 are also capable of binding HREs, and SIM1 has been shown to activate expression of HREbased reporter constructs [23]. Conversely, HRE-bound SIM2 is known to inhibit expression of both reporter genes and the endogenous HIF-1 target gene BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3) [24]. To confirm the impaired activity of a representative selection of SIM2s variants using an alternative system, 293 Flp-In TRex cell lines were transfected with a luciferase reporter construct containing four HRE repeats (pGL3-4HRE) and treated with the hypoxia mimetic DMOG, which stabilizes HIF-1α protein (Figure 6). In the absence of SIM2s (Empty), DMOG treatment induces reporter activity greater than 20-fold. SIM2s–3FLAG WT protein does not alter expression of luciferase in the absence of DMOG, but represses induction to background levels by competing with HIF-1α for partner factors and binding to HREs. SIM2s R171H, which does not show impaired dimerization, represses DMOG induction in an identical manner with SIM2s WT. Conversely SIM2s T46R, T292A and R296G variants, which are dimerization-deficient, fail to fully inhibit induction of the reporter gene presumably due to reduced competition with HIF c The Authors Journal compilation c 2014 Biochemical Society
1α for ARNT and reduced binding of HRE sequences by SIM2s– ARNT heterodimers. Despite being similarly deficient in dimerization when analysed by co-immunoprecipitation (Figure 4B), SIM2s T46R was more effective than SIM2s T292A and R296G variants at inhibiting reporter gene induction. This is potentially due to the observed exclusive nuclear localization of SIM2s T46R, whereas T292A and R296G variants were partially localized to the cytoplasm (Figure 5A). Competition between SIM2s and HIF-1α for other unknown cofactors may also contribute to observed changes in reporter gene activity. In general, these results are consistent with previous observations of SIM2s variant activity.
Homology modelling of SIM1 PASB
A total of seven weak-activity variants in SIM1 and SIM2 cluster to a ‘hot-spot’ region in PASB (V290E, T292A, R296G, S309G, N316T, H323Y and V326F), and five also impair dimerization with a partner factor (V290E, T292A, R296G, S309G and H323Y). To further investigate how these variants cluster on a tertiary PAS fold, homology modelling of SIM1 PASB was performed (Figure 7A, blue chain) based on the most closely related protein structure available: HIF-2α PASB (42 % identity
Dimerization defects in SIM1 and SIM2 protein variants
Figure 5
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Activity-deficient variants show predominantly nuclear localization
(A) HEK-293T cells were transiently transfected with plasmids expressing SIM2s–3FLAG WT or mutant proteins, then fixed and stained with and anti-FLAG antibody (red) and DAPI nuclear stain (blue). Nuclear localization was observed for all SIM2s proteins, although some cytoplasmic staining was also seen for T292A and R296G variants (×600 magnification). (B) 293 Flp-In TRex cell lines were treated with doxycycline (1 μg/ml, 16 h), then fixed and stained with an anti-Myc antibody (red) and DAPI nuclear stain (blue). V326F SIM1–2Myc showed exclusively nuclear localization, but V290E SIM1–2Myc also showed some cytoplasmic localization (×600 magnification).
with SIM1 PASB), which was co-crystallized with ARNT PASB (Figure 7B, right brown chain) (PDB code 3F1P). Of the amino acids that cause dimerization defects when mutated (red), four out of five (Val290 , Thr292 , Arg296 and Ser309 ) roughly cluster in a region on the β-sheet face of the PASB fold. The remaining residue, His323 , is located at the edge of a loop region which may be highly mobile or differentially structured. Other low-activity variant residues (Leu238 , Asn316 and Val326 ) not associated with impaired dimerization (green) were not located in a common region. The homology-modelled SIM1 PASB was then superimposed on the position of HIF-2α PASB in the HIF-2α–ARNT cocrystal structure and subjected to more rounds of minimization and regularization, in order to model the association between SIM1 PASB and ARNT PASB. However, the locations of the variants are not entirely consistent with this dimerization interface (Figure 7B). In particular, R296G variants demonstrated the lowest dimerization ability, but Arg296 is located at a distal point from the modelled dimerization interface. This indicates that the homology model is insufficient to fully characterize interactions between the PASB folds of SIM1 and ARNT, but also that the SIM1–ARNT dimerization interface is possibly structurally unique from those of previously reported structures of PAS domain proteins. DISCUSSION
Weak-activity variants in SIM1 have previously been identified in juvenile-onset obesity patients [8,9], but the mechanisms
of impairment were only speculated. In the present study we determined that two (T46R and H323Y) SIM1 variants found in human obesity patients, as well as a further three low-activity variants (T292A, R296G and S309G), experienced reduced function due to impaired binding of partner factor ARNT2. This is consistent with the location of the variants within domains that mediate dimerization. Only one SIM1 variant, R171H, showed no deficiency in dimerization. It is possible that this particular variant may impair PASA interactions with other unidentified proteins that are required for target gene activation. We also demonstrated that activity-deficient SIM1 variants (V290E and V326F) are present in the general population. Unfortunately no phenotypic data was available, but we predict that the individuals harbouring these SNVs may have an above-average BMI (body mass index). In total, we have confirmed deficiencies for nine variants in either or both SIM1 and SIM2, eight of which are located in the PAS repeat domain. This is consistent with the importance of the PAS region in the function of bHLH PAS transcription factors. The PAS repeat domain is considered the primary means of selective dimerization between specific bHLH PAS family members, to the exclusion of other subfamily bHLH PAS proteins and species of the broader bHLH superfamily [25]. PASB is particularly critical to the function of SIM2 protein such that when this repeat is lost through truncation, the SIM2 protein loses all ability to dimerize with ARNT and becomes completely transcriptionally inactive (Supplementary Figure S1 at http://www.biochemj.org/bj/461/bj4610403add.htm and [26]). This importance of PASB to the function of full-length SIM c The Authors Journal compilation c 2014 Biochemical Society
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Figure 6 SIM2s variants with impaired dimerization show reduced repression of HIF activity Treatment with the hypoxia mimetic DMOG (1 mM, 16 h) was used to stabilize HIF-1α protein and induce luciferase expression from a transiently transfected HIF-responsive reporter construct (4×HRE-Luc) in 293 Flp-In TRex cell lines. Expression of empty vector (Empty), WT or mutant proteins was simultaneously induced with doxycycline (1 μg/ml, 16 h). SIM2s WT protein has no effect on reporter activity alone (WT no DMOG), but reduces HIF-mediated induction to non-induced levels. SIM2s T46R, T292A and R296G variants, which have impaired dimerization with ARNT, fail to completely repress induction of the reporter gene. SIM2s R171H, which has no loss of dimerization, shows similar repression to SIM2s WT (***P < 0.001, ns is not significant). Histogram shows the mean fold change in luciferase units + − S.E.M. relative to Empty (no DMOG) for n = 4 independent experiments.
proteins is also evident in the observed critical loss of activity caused by missense SNVs within our defined mutational hot-spot region in PASB.
Figure 7
There is also evidence that PAS domains contribute to the specific binding of DNA sequences and target gene recognition [25,27]. The importance of PAS domains in the unique function of individual bHLH PAS proteins was demonstrated in a key experiment whereby the PAS domain of the Drosophila melanogaster orthologue of HIF-1α, Trh (Trachealess), was replaced with the PAS domain of the SIM1/SIM2 orthologue, Sim (Single-minded). The Trh–Sim PAS chimaeric protein lost the ability to regulate Trh target genes and instead induced target genes and phenotypes specific to Sim [28]. Exactly how the PAS domain dictates this unique activity is not well understood. In a previous study, a random mutagenesis screen was performed for key residues in the PASA repeat of ARNT that mediate heterodimerization with the PASA repeat of AhR (aryl hydrocarbon receptor). A total of 22 individual mutations were found to inhibit or ablate AhR–ARNT dimerization but, interestingly, four of these variants did not affect activity of ARNT with other partners such as SIM1, SIM2 or NPAS4 (neuronal PAS domain protein 4) [13]. This indicates that although the general interface of ARNT PASA with its partner may be conserved, there are key residues which are critical to partner-specific interactions. This conclusion is consistent with the observation that the R296G variant, located in PASB, produces significantly different changes in activity when present in SIM1 or SIM2s and when partnered with either ARNT or ARNT2. R296G SIM1–2Myc retains 42 % of WT activity when partnered with ARNT2, but appears to be completely inactive when partnered with ARNT, indicating that different residues may be critical in ARNT- and ARNT2-specific interactions with SIM1. That R296G SIM2s– 3FLAG shows below-basal activity in all cases indicates that, despite sharing high sequence identity, there are some partner interactions that are specific to SIM1 or SIM2. These specific PAS-mediated interactions may also be a factor that differentiates the biological activities of SIM1 and SIM2 in vivo. The T46R variant, which is located in the second helix of the bHLH domain, also produced significantly different effects on activity in SIM1 compared with SIM2s when partnered with ARNT but not ARNT2. This may indicate that some SIM1- and
Mapping of variants on the SIM1 PASB homology model
(A) Homology modelling of SIM1 PASB (blue chain) based on HIF-2α PASB (PDB code 3F1P) shows the position of residues that, when mutated, either impaired (red; Val290 , Thr292 , Arg296 , Ser309 or His323 ) or did not alter (green; Leu238 , Asn316 or Val326 ) dimerization with ARNT2. With the exception of His323 , red residues roughly cluster together at the β-sheet face. Green residues do not seem to cluster with red residues or each other. (B) ARNT PASB (brown chain, right), which was co-crystallized with HIF-2α PASB, does not seem to interact closely with the red cluster. c The Authors Journal compilation c 2014 Biochemical Society
Dimerization defects in SIM1 and SIM2 protein variants
SIM2-specific interactions are also mediated by the bHLH region, although in all cases T46R variant activity was approximate to basal levels and so the true relevance of this difference in activity is uncertain. Specificity of partner selection is likely to also be influenced by the structural orientation and interfacing of PASB. Although structural data of bHLH PAS proteins is limited, the PASB repeats of family members CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) have been revealed to associate in an approximately parallel manner [29], whereas isolated PASB folds of HIF-2α and ARNT show an antiparallel orientation and β-sheet interface [15]. This may explain the preference of CLOCK for BMAL1 rather than ARNT, and raises the question of how the PASB dimerization interface varies among other family members. Homology modelling of SIM1 PASB on HIF-2α PASB revealed that hot-spot activity-impaired variants that impair dimerization cluster roughly to a region on the β-sheet face of the fold, indicating that the dimerization interface of SIM1 is more similar to that of HIF-2α than that of CLOCK. However, the exact location of the cluster relative to ARNT PASB suggests that this model is incomplete, and implies that the PASB dimerization interface may vary between SIM1 and HIF-2α. It is also possible that the isolated HIF-2α PASB and ARNT PASB folds, which lack the structural context of the larger protein, do not accurately depict the natural dimerization interface. The structure of SIM1 will need to be solved to ultimately resolve these questions, but data regarding the influence of point mutations on protein activity is still important when assessing and interpreting solved structures; for example, data from the aforementioned AhR/ARNT PASA mutational study [13] was used in analysis of the recently generated AhR PASA crystal structure [30]. Therefore the present study will be useful to future SIM or bHLH PAS structural determination projects. In conclusion, we have identified a hot-spot region in SIM1 and SIM2 spanning amino acids 290–326 which is critical for protein function, and defined a subset of human variants within this hotspot which inhibit dimerization with partner proteins ARNT and ARNT2. These data may be used in the future for assessment of deleterious human SNVs in SIM1 and SIM2, and in understanding the mechanistic deficiencies in protein variants that currently exist in the population. Severely deficient SIM1 variants that were identified in obese patients are possibly monogenic causes of obesity, but less-deficient variants could also contribute to weight gain in combination with other factors. In the future, identifying any genetic factors contributing to development of obesity may be important in order to manage the patient condition effectively. Thus screening patients for SIM1 variants and testing activity with molecular assays is likely to be an important diagnostic tool. AUTHOR CONTRIBUTION Adrienne Sullivan, Anne Raimondo, Daniel Peet and Murray Whitelaw conceived the project and designed the experiments. Adrienne Sullivan, Anne Raimondo and Tanja Schwab conducted experiments. John Bruning generated the SIM1 homology model. Philippe Froguel and I. Sadaf Farooq provided reagents and information about SIM1 variants. Adrienne Sullivan and Murray Whitelaw wrote the paper.
ACKNOWLEDGEMENT We thank the third-year Biochemistry students of the University of Adelaide for their work in cloning variants.
FUNDING This work was supported by the Australian Research Council.
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Biochem. J. (2014) 461, 403–412 (Printed in Great Britain)
doi:10.1042/BJ20131618
SUPPLEMENTARY ONLINE DATA
Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2 Adrienne E. SULLIVAN*, Anne RAIMONDO*1 , Tanja A. SCHWAB*, John B. BRUNING*, Philippe FROGUEL†‡§, I. Sadaf FAROOQI, Daniel J. PEET* and Murray L. WHITELAW*2 *School of Molecular and Biomedical Science (Biochemistry) and Centre for Molecular Pathology, University of Adelaide, Adelaide 5005, South Australia, Australia †CNRS-UMR8199, Lille Pasteur Institute, 59010 Lille, France ‡Lille Nord de France University, 59044 Lille, France §Department of Genomics of Common Disease, School of Public Health, Imperial College London, Hammersmith Hospital, London W12 ONN, U.K. University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, U.K.
Figure S1
SIM2 PASB is essential for transcriptional activation and heterodimerization
Expression of SIM2s truncations lacking the C-terminal transrepression domain (3) or both PASB and the transrepression domain (2) (A) was confirmed by transient expression in HEK-293T cells and Western blot analysis (B). Activity of the truncated proteins was assessed by transiently co-transfecting with plasmids expressing ARNT and luciferase reporter construct (6 × CME-Luc) in HEK-293T cells (C). Loss of the transrepression domain does not inhibit SIM2s activity, but loss of PASB ablates all reporter gene induction. Co-immunoprecipitation experiments using co-transfected FLAG-tagged ARNT demonstrate that this loss of activity corresponds with a loss of dimerization with ARNT (D). Histograms show the mean fold change in luciferase units + − S.E.M. relative to ARNT only for n = 4 independent experiments (*P
