Phenotypic Mutation 'delargo' (pdf version)
Alleledelargo
Mutation Type missense
Chromosome14
Coordinate103,326,854 bp (GRCm39)
Base Change G ⇒ A (forward strand)
Gene Fbxl3
Gene Name F-box and leucine-rich repeat protein 3
Synonym(s) Fbxl3a, Play68, Ovtm, Fbl3a
Chromosomal Location 103,317,675-103,337,002 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a member of the F-box protein family which is characterized by an approximately 40 amino acid motif, the F-box. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which function in phosphorylation-dependent ubiquitination. The F-box proteins are divided into 3 classes: Fbws containing WD-40 domains, Fbls containing leucine-rich repeats, and Fbxs containing either different protein-protein interaction modules or no recognizable motifs. The protein encoded by this gene belongs to the Fbls class and, in addition to an F-box, contains several tandem leucine-rich repeats and is localized in the nucleus. [provided by RefSeq, Jul 2008]
PHENOTYPE: Both heterozygous and homozygous mutant mice display a longer free running period than that of wild-type mice. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_015822 (variant 1), NM_001347600 (variant 2), NM_001347601 (variant 3); MGI:1354702

MappedYes 
Amino Acid Change Serine changed to Leucine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000022720] [ENSMUSP00000117701] [ENSMUSP00000115843] [ENSMUSP00000120691] [ENSMUSP00000116044]
AlphaFold Q8C4V4
SMART Domains Protein: ENSMUSP00000022720
Gene: ENSMUSG00000022124
AA Change: S176L

DomainStartEndE-ValueType
FBOX 39 79 5.92e-7 SMART
low complexity region 235 247 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.143 (Sensitivity: 0.92; Specificity: 0.86)
(Using ENSMUST00000022720)
SMART Domains Protein: ENSMUSP00000117701
Gene: ENSMUSG00000022124

DomainStartEndE-ValueType
FBOX 39 79 2.46e-4 SMART
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000115843
Gene: ENSMUSG00000022124
AA Change: S128L

DomainStartEndE-ValueType
FBOX 39 79 2.46e-4 SMART
low complexity region 187 199 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.127 (Sensitivity: 0.93; Specificity: 0.86)
(Using ENSMUST00000132004)
SMART Domains Protein: ENSMUSP00000120691
Gene: ENSMUSG00000022124
AA Change: S176L

DomainStartEndE-ValueType
FBOX 39 79 5.92e-7 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000144141)
SMART Domains Protein: ENSMUSP00000116044
Gene: ENSMUSG00000022124
AA Change: S176L

DomainStartEndE-ValueType
FBOX 39 79 5.92e-7 SMART
low complexity region 235 247 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.143 (Sensitivity: 0.92; Specificity: 0.86)
(Using ENSMUST00000145693)
Meta Mutation Damage Score 0.1582 question?
Is this an essential gene? Probably nonessential (E-score: 0.240) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(33) : Chemically induced (ENU)(3) Chemically induced (other)(1) Gene trapped(27) Targeted(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00492:Fbxl3 APN 14 103332730 missense probably damaging 1.00
IGL01981:Fbxl3 APN 14 103332900 missense possibly damaging 0.79
IGL03208:Fbxl3 APN 14 103320376 nonsense probably null
PIT4403001:Fbxl3 UTSW 14 103332900 missense possibly damaging 0.79
R0282:Fbxl3 UTSW 14 103332661 missense probably damaging 1.00
R0462:Fbxl3 UTSW 14 103320322 missense probably damaging 1.00
R0710:Fbxl3 UTSW 14 103326751 missense probably damaging 1.00
R1075:Fbxl3 UTSW 14 103332839 missense probably benign 0.00
R2263:Fbxl3 UTSW 14 103320648 nonsense probably null
R4239:Fbxl3 UTSW 14 103326854 missense probably damaging 1.00
R4362:Fbxl3 UTSW 14 103329749 missense probably damaging 1.00
R4497:Fbxl3 UTSW 14 103320313 missense probably damaging 1.00
R4585:Fbxl3 UTSW 14 103320526 missense probably damaging 0.99
R4586:Fbxl3 UTSW 14 103320526 missense probably damaging 0.99
R5347:Fbxl3 UTSW 14 103320730 missense probably damaging 0.97
R5349:Fbxl3 UTSW 14 103333012 intron probably benign
R5885:Fbxl3 UTSW 14 103320667 missense probably benign 0.06
R6744:Fbxl3 UTSW 14 103320730 missense probably damaging 0.99
R8314:Fbxl3 UTSW 14 103326876 missense probably benign 0.04
R9015:Fbxl3 UTSW 14 103329790 missense possibly damaging 0.78
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:42 PM by Anne Murray
Record Created 2016-08-15 1:45 PM
Record Posted 2017-03-31
Phenotypic Description
Figure 1. Delargo mice exhibit reduced circadian active phase duration. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 2. Delargo mice exhibit a decrease in circadian tau activity onset. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3. Delargo mice exhibit an increase in the circadian tau DD by eye. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 4. Delargo mice exhibit an increase in the tau chi squared periodogram. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 5. Delargo mice exhibit fasting hyperglycemia. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The delargo phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4239, some of which showed decrease in circadian active phase duration (Figure 1) and a decrease in circadian tau activity onset (Figure 2) with a concomitant increase in circadian tau DD by eye (Figure 3) and an increase in the tau chi squared periodogram (Figure 4). Some mice also showed fasting hyperglycemia (Figure 5).

Nature of Mutation

Figure 6. Linkage mapping of the reduced circadian tau activity onset using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 55 mutations (X-axis) identified in the G1 male of pedigree R4239. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 55 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Fbxl3:  a C to T transition at base pair 103,089,418 (v38) on chromosome 14, or base pair 10,122 in the GenBank genomic region NC_000080 encoding Fbxl3. The strongest association was found with a recessive model of linkage to the normalized diminished circadian tau activity onset phenotype, wherein six variant homozygotes departed phenotypically from 23 homozygous reference mice and 27 heterozygous mice with a P value of 2.334 x 10-21 (Figure 6).  A substantial semidominant effect was observed in several of the assays but the mutation is preponderantly recessive. 

The mutation corresponds to residue 899 in the mRNA sequence NM_015822 within exon 4 of 5 total exons.

 
884 TCCAAGTCCCTGTCCTCACTTAAGATAGACGAC
171 -S--K--S--L--S--S--L--K--I--D--D-
 

The mutated nucleotide is indicated in red.  The mutation results in a serine (S) to leucine (L) substitution at position 176 (S176L) in the FBXL3 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 7. Domain structure of Fbxl3. The location of the F-box is indicated. UniProt has the locations of seven LRR domains; however crystal structures indicate that Fbxl3 has 12 LRRs. The delargo mutation results in a serine (S) to leucine (L) substitution at position 176 (S176L).

Fbxl3 encodes Fbxl3, an orphan F-box SCF (Skp1/cullin/F-box protein) phosphorylation-dependent E3 ubiquitin ligase. There are over 70 SCF ubiquitin ligases in mammals. Each is composed of four subunits: Skp1, a cullin protein (Cul1 in metazoans), Roc1 (alternatively, Rbx1), and an F-box protein. Each SCF ligase differs among the F-box protein subunit (1;2). The F-box proteins differ within the substrate-associated region. The Fbxw family have WD40 repeats, the Fbxl proteins have leucine-rich repeats (LRRs), and the Fbxo proteins have other domains. SCF ligases bring Ubc or Ubc4 ubiquitin conjugating enzymes to substrates. Cul1 functions as a scaffold in which the SKP1 and Rbx1 proteins assemble. The Cul1-Rbx1 complex recruits an E2 ubiquitin ligase. The F-box proteins recruit substrates.

The crystal structure of Fbxl3 in complex with Skp1 and CRY2 has been solved [PDB:4I6J; (3)]. Mammalian CRY2 binds FAD with an open cofactor pocket. Fbxl3 binds CRY2 by occupying the FAD-binding pocket with its C-terminal tail while concomitantly burying its PER-binding interface. The CRY2 interaction can be disrupted by FAD and Per proteins (Per1/2). The CRY2-Fbxl3-Skp1 complex has the globular CRY2 sitting on top of the Fbxl3-Skp1 complex. The F-box domain of Fbxl3 is composed of three helices, which interact with Skp1. The LRR domain folds into a curved and sickle-shaped solenoid structure. The concave surface of the LRR wraps around the α-helical domain and the CCS region of CRY2 opposite to the α/β domain. The Fbxl3-CRY2 complex assembles spontaneously in a phosphorylation-independent manner. The Fbxl3 crystal structure revealed 12 LRRs, which pack in tandem and form a semicircular arch. The concave surface of the arch is formed by the parallel β-strands of the repeats and the convex side is formed by the α-helices. LRR7 has a much longer β-strand, which causes a significant offset at the apical ridge. The longer β-strand feature continues in LRR8, but is not observed in LRR9 through LRR12. LRR7 through LRR12 are in close contact with the α-helical domain of CRY2. Most of LRR1 through LRR6, except its four apical loops, are separated from CRY2 by a gap. The C-terminal tail of Fbxl3 sticks out from the flat concave surface comprised of LRR7 through LRR12 and penetrating into CRY2.

The association between the LRR domain of Fbxl3 and substrates affects the interaction of Fbxl3 with Skp1 and Cul1 through its F-box domain. The F-box domain is an approximate 40-amino acid domain that is essential for SCFFbxl3-mediated protein ubiquitination and subsequent proteasomal degradation by binding to the Skp1 component of the SCF complex. The LRRs mediate substrate recognition (4). The LRR domain of Fbxl3 is proposed to prevent the association with Skp1 and Cul1 (5). Formation of the SCFFbxl3 complex is regulated by substrate binding (5). A mutant Fbxl3 that could not bind to CRY1 did not form a SCF complex. Fbxl3 does not form an SCF complex without the Cry1 substrate. Binding of Cry proteins to the Fbxl3 LRR domain induces the formation of the SCFFbxl3 complex putatively through the dissociation of a protein that interferes with the interaction with Skp1-Cul1 (5) .

The delargo mutation results in a serine (S) to leucine (L) substitution at position 176 (S176L). Residue 176 is within an undefined region between LRR1 and LRR2 (via UniProt).

Expression/Localization

Fbxl3 is ubiquitously expressed and is predominantly localized in the nucleus (4;6;7).

Background
Figure 8. Model of mammalian circadian clock. CLOCK-BMAL1 heterodimers bind to E-box DNA to drive the expression of PER and CRY genes in the core feedback loop of the clock.  CLOCK-BMAL1 also drives transcription of Rev-Erbα and RORα/β, which act as negative and positive transcriptional regulators, respectively, of a second feedback loop that controls rhythmic expression of BMAL1. The nuclear translocation and proteasome-mediated degradation of PER proteins are regulated by CK1δ/ε and PP1/5. CRY protein degradation is mediated by the SKP1-CUL1-FBXL3 E3 ubiquitin ligase complex in the nucleus, and by the SKP1-CUL1-FBXL21 E3 ubiquitin ligase complex in the cytoplasm.

Circadian rhythms are intrinsic daily cycles of behavioral and physiological changes driven by an endogenous “clock” or “oscillator” [Figure 8; reviewed in (8-10)]. Changes in sleep/wakefulness, locomotion, feeding, and temperature are all overt signs of the body’s circadian rhythm. These rhythms, which follow a 24 hour cycle or period, are both self-sustained, occurring even in the absence of external inputs, and entrained (synchronized) by environmental cues such as the light-dark cycle. In mammals, the master circadian clock is located in the hypothalamic suprachiasmatic nucleus (SCN) (11), where a network of interconnected and therefore synchronized neurons receives photic input from the retina and provides circadian timing information to the rest of the body (12). This information is conveyed by direct neuronal projections to other brain and peripheral regions, as well as by diffusible factors from the SCN (13;14).  Peripheral oscillators, including the kidney and liver, can run independently of the SCN for a few days, but rely on the central clock for long term synchronization to an internally coherent timing system (15;16).

At the molecular level, the circadian clock consists of several interacting positive and negative transcriptional feedback loops that drive recurrent cycling of RNA and protein levels of clock components (Figure(8-10). The core feedback loop consists of BMAL1 (also called Arntl, aryl hydrocarbon receptor nuclear translocator-like) and CLOCK, components of the positive arm of the loop, and three Period (PER) and two Cryptochrome (CRY) proteins, components of the negative arm of the loop. CLOCK-BMAL1 heterodimers activate the transcription of PerCry, and Rev-Erbα genes through interaction with E-box enhancers (17;18). PER and CRY proteins translocate back into the nucleus where CRY proteins act as negative regulators by directly interacting with CLOCK and BMAL1 and inhibiting transcription (19;20). On the other hand, Rev-Erbα binds to ROREs in the promoter of Bmal1 and acts as a transcriptional repressor, reducing BMAL1 levels (18;21). Remaining CRY and PER proteins entering the nucleus inhibit transcription activated by CLOCK-BMAL1, including that of CryPer, and Rev-Erbα. Rev-Erbα levels decrease, resulting in a de-repression of Bmal1 transcription and beginning the whole cycle again. On top of these loops, regulated protein phosphorylation and turnover provide temporal separation of the signaling events and stretch the period to 24 hours.

Fbxl3 promotes the ubiquitination and subsequent degradation of the CRY proteins (1). As a result, Fbxl3 regulates oscillation of the circadian clock.  In addition to repressing the transcription of their own genes, expression of the Per and Cry transcription factors are regulated by ubiquitin proteasome-mediated proteolysis. β-TrCP (also known as Fbxw1 or Fbxw11) targets Per for degradation, and Fbxl3 targets Cry (1;4;6). AMP-activated protein kinase (AMPK)-mediated phosphorylation of CRY1 promotes SCFFbxl3 binding to CRY1, which promotes its degradation (22). SCFFbxl3-induced ubiquitination is triggered by AMPK-mediated Cry phosphorylation (22) and reversed by the deubiquitinating enzyme USP2a (23). Fbxl3 silencing resulted in loss of the Cry1 expression oscillations and led to a decrease in the expression of Per1 and Per2 (1). Fbxl21, a paralog of Fbxl3, forms a SCF E3 ligase complex that slowly degrades Cry in the cytoplasm, but antagonizes the E3 ligase activity of Fbxl3 in the nucleus (7). Fbxl3 regulates Rev-Erb/retinoic acid receptor-related orphan receptor-binding element (RRE)-mediated transcription by inactivating the Rev-Erbα:histone deacetylase 3 (HDAC3) corepressor complex (24). This Fbxl3 function on Rev-Erbα:HDAC3 is essential for clock function. Deletion of Rev-erbα in Fbxl3-deficient mice rescued its long-circadian period phenotype. Fbxl3 also regulates the amplitudes of E-box–driven gene expression to alter the circadian period and robustness of the clock (24).

In addition to functioning in circadian rhythm, Cry2 and Fbxl3 limit tumor formation by stimulating ubiquitination and subsequent proteasomal-mediated degradation of the oncoprotein c-Myc (25). Cry2 is a proposed scaffold protein that promotes the association of Fbxl3 with Cul1 and the formation of an active SCFFbxl3 complex that ubiquitinates c-Myc.

Loss-of-function Fbxl3 results in period lengthening due to a reduction in the SCFFbxl3-induced ubiquitination-mediated proteasomal degradation of the Cry proteins. The overtime (ovtm) and afterhours (afh) mice maintain a circadian period of approximately 26 and 27 hours in the dark, respectively (4;6;26). The mouse mutants overtime and afterhours both have mutations in the LRRs of Fbxl3, which disrupted the interaction between Fbxl3 and Cry. As a result, the Cry proteins are stabilized, resulting in increased repression of CLOCK-BMAL1 activity (26). The ovtm mutation causes an isoleucine to threonine substitution at amino acid 364 (I364T) (26). Residue 364 is between LRR10 and LRR11. In the ovtm mice, expression of Per1 and Per2 were reduced, but the expression of Cry1 and Cry2 were unchanged. The mRNA expression of Cry1, Cry2, Per1, Per2 and Dbp (all transcriptional targets of the CLOCK/BMAL1 complex) were all reduced in the cerebellum of the ovtm mice. The afh mutation resulted in substitution of Cys358 for a serine (C358S) (4). Similar to that observed in the ovtm mice, the mRNA expression levels of Per, Cry, and Bmal1 were reduced in the afh mice.

Putative Mechanism

The circadian phenotypes in Fbxl3 mutant mice are attributed to a reduced ability of the mutant Fbxl3 protein to bind to and induce the proteolysis of Cry proteins (1).

Primers PCR Primer
delargo_pcr_F: AAGTCTAAGCTCTAGAATACTGCAG
delargo_pcr_R: CAGGGTGCTGCCTTTTAACAG

Sequencing Primer
delargo_seq_F: GGACACTGAAGTTTACTTTGAAGAAG
delargo_seq_R: GCTGCCTTTTAACAGTTTTGCG
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 458 nucleotides is amplified (chromosome 14, - strand):


1   cagggtgctg ccttttaaca gttttgcgat tctttagggt gcagacagac atgcacggta
61  aggaaacctt gtaacatgtc atttctgaag aactgaaact gttttaatct tttgtctttc
121 agtctcactt tatctctgca ctgacagttg tgtttgtaaa ctccaagtcc ctgtcctcac
181 ttaagataga cgacacccca gtcgatgacc catcccttaa agtcctcgtg gccaacaaca
241 gcgacacact caagctgttg aaaatgagca gctgtcctca cgtctctcca gcaggtaggt
301 catgttgtta ccagacattg tttctcaaat tgagaaattt gtttatttaa tagttctgct
361 aatgaacttg taaatgaatt cacaaaacca tttcttcttc aaagtaaact tcagtgtcct
421 aatagcataa tatctgcagt attctagagc ttagactt


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Katherine Timer
AuthorsMarleen de Groot and Joseph Takahashi