Phenotypic Mutation 'Bernie' (pdf version)
AlleleBernie
Mutation Type missense
Chromosome14
Coordinate99,539,666 bp (GRCm39)
Base Change C ⇒ T (forward strand)
Gene Klf5
Gene Name Kruppel-like transcription factor 5
Synonym(s) IKLF, Bteb2, 4930520J07Rik, CKLF
Chromosomal Location 99,536,127-99,550,848 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 Kruppel-like factor subfamily of zinc finger proteins. The encoded protein is a transcriptional activator that binds directly to a specific recognition motif in the promoters of target genes. This protein acts downstream of multiple different signaling pathways and is regulated by post-translational modification. It may participate in both promoting and suppressing cell proliferation. Expression of this gene may be changed in a variety of different cancers and in cardiovascular disease. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2013]
PHENOTYPE: Homozygous null mice die during gestation, while heterozygotes exhibit abnormal cardiovascular remodeling after external stress. Mice homozygous for a floxed allele activated in the prostate exhibit increased cell proliferation and hyperplasia in the prostate without neoplasia. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_009769; MGI:1338056

MappedYes 
Amino Acid Change Arginine changed to Cysteine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000005279] [ENSMUSP00000154786]
AlphaFold Q9Z0Z7
SMART Domains Protein: ENSMUSP00000005279
Gene: ENSMUSG00000005148
AA Change: R360C

DomainStartEndE-ValueType
low complexity region 53 65 N/A INTRINSIC
low complexity region 166 173 N/A INTRINSIC
low complexity region 290 301 N/A INTRINSIC
ZnF_C2H2 362 386 3.83e-2 SMART
ZnF_C2H2 392 416 2.47e-5 SMART
ZnF_C2H2 422 444 1.2e-3 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000005279)
Predicted Effect probably damaging

PolyPhen 2 Score 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000226784)
Meta Mutation Damage Score 0.9055 question?
Is this an essential gene? Essential (E-score: 1.000) question?
Phenotypic Category Autosomal Semidominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(37) : Chemically induced (other)(1) Gene trapped(28) Targeted(8)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01295:Klf5 APN 14 99539157 missense probably benign 0.01
IGL02380:Klf5 APN 14 99538894 missense possibly damaging 0.67
I0000:Klf5 UTSW 14 99540911 missense probably damaging 1.00
R0133:Klf5 UTSW 14 99539318 missense probably benign
R1672:Klf5 UTSW 14 99538986 missense probably damaging 0.98
R1914:Klf5 UTSW 14 99539357 missense probably benign 0.01
R2193:Klf5 UTSW 14 99536406 unclassified probably benign
R3892:Klf5 UTSW 14 99536509 missense probably benign 0.00
R4446:Klf5 UTSW 14 99539666 missense probably damaging 1.00
R5437:Klf5 UTSW 14 99538895 nonsense probably null
R5707:Klf5 UTSW 14 99538944 missense probably benign
R6475:Klf5 UTSW 14 99538817 missense probably benign 0.00
R6552:Klf5 UTSW 14 99539078 missense probably benign
R6982:Klf5 UTSW 14 99550671 missense probably damaging 1.00
R7250:Klf5 UTSW 14 99536455 missense probably benign 0.00
R7643:Klf5 UTSW 14 99550614 missense possibly damaging 0.88
R7938:Klf5 UTSW 14 99536444 missense probably damaging 0.98
R8272:Klf5 UTSW 14 99539540 missense possibly damaging 0.67
R8396:Klf5 UTSW 14 99539670 missense possibly damaging 0.95
R8898:Klf5 UTSW 14 99538922 missense probably damaging 0.99
R9015:Klf5 UTSW 14 99540919 makesense probably null
R9251:Klf5 UTSW 14 99538824 missense possibly damaging 0.95
R9560:Klf5 UTSW 14 99539034 missense probably benign 0.06
R9717:Klf5 UTSW 14 99539189 missense probably damaging 1.00
Mode of Inheritance Autosomal Semidominant
Local Stock
Repository
Last Updated 2018-08-08 8:46 AM by Anne Murray
Record Created 2016-08-23 3:17 PM
Record Posted 2018-08-08
Phenotypic Description

Figure 1. Bernie mice exhibited weight loss 7 days after DSS treatment. 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. Bernie mice exhibited weight loss 10 days after DSS treatment. 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 Bernie phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4446, some of which showed susceptibility to dextran sodium sulfate (DSS)-induced colitis at 7 (Figure 1) and 10 days (Figure 2) after DSS exposure; weight loss is used to measure DSS susceptibility.

Nature of Mutation

Figure 3. Linkage mapping of the DSS susceptibility phenotype using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 45 mutations (X-axis) identified in the G1 male of pedigree R4446. 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 45 mutations. The DSS sensitivity phenotype was linked by continuous variable mapping to a mutation in Klf5:  a C to T transition at base pair 99,302,230 (v38) on chromosome 14, or base pair 3,540 in the GenBank genomic region NC_000080 encoding Klf5. The strongest association was found with an additive model of inheritance to the DSS susceptibility phenotype at day 10, wherein five variant homozygotes departed phenotypically from 24 homozygous reference mice and 29 heterozygous mice with a P value of 3.635 x 10-7 (Figure 3).  

The mutation corresponds to residue 1,382 in the mRNA sequence NM_009769 within exon 2 of 4 total exons.

1367 GATCTGGAGAAGCGACGTATCCACTTCTGCGAT

355  -D--L--E--K--R--R--I--H--F--C--D-

The mutated nucleotide is indicated in red.  The mutation results in an arginine (R) to cysteine (C) substitution at position 360 (R360C) in the KLF5 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 4. Domain organization of KLF5. The location of the Bernie mutation is indicated. Phosphorylation (P), sumoylation (Su), acetylation (Ac), and ubiquitination (Ub) sites are noted. Domain information is from SMART and UniProt.

Krüppel-like factor 5 (KLF5; alternatively, basic transcription element binding protein 2 [BTEB2] or intestinal Krüppel-like factor [IKLF]) is a member of the KLF subfamily of zinc finger proteins.

KLF5 has a proline-rich transactivation domain (TAD) at the N-terminus, a short basic region, and three consecutive Cys2–His2 zinc fingers near the C-terminus (1;2) (Figure 4). The basic region and zinc fingers regulate DNA binding; the KLF proteins bind to GC boxes within the promoters of target genes (3).

KLF5 interacts with several proteins, including post-translational modifiers (e.g., PKC, HDAC1/2, SET, and p300), transcriptional co-regulators (e.g., CBP, CEBPα/β/δ, NK-κB, PPARδ, SREBP1, FOXO3, SMAD2/3/4, NCoR1/2, ERβ, MYC, JUN, and p53), and basal transcriptional components (e.g., TFIIβ, TFIIEβ, TFIIFβ, and TBP) [reviewed in (4;5)].

KLF5 undergoes several posttranslational modifications. KLF5 phosphorylation increased the transactivation ability of KLF5. For example, PKC-mediated phosphorylation of Ser153 enhances the KLF—CBP interaction (1). KLF5 is phosphorylated on Ser406 downstream of the MEK/ERK and p38 signaling pathways. MEK/ERK/p38-associated phosphorylation of KLF5 enhances the interaction of KLF5 with c-JUN (6). Ser406 phosphorylation also increases the interaction with unliganded RARα(7;8). KLF5 can also be phosphorylated on Ser303, Thr234, and Thr323. KLF5 is acetylated at Lys369 by p300 in response to PMA stimulation in vascular smooth muscle cells; p300-mediated acetylation of KLF5 increases the expression of target genes (e.g., Pdgfa) (9-12). In epithelial keratinocytes, TGFβ stimulates acetyl-KLF5/p300 to recruit SMADS2/3/4 and FOXO3 to form a complex on the Cdkn2b promoter, inhibiting cell proliferation (9;10). The oncogenic regulator SET and the histone deacetylase HDAC1 can bind KLF5 to prevent p300-associated acetylation and activation (11;12). A PY2 motif within the transactivation domain mediates association of KLF5 and the E3 ubiquitin ligase WWP1 (13); WWP1 promotes the polyubiquitination and subsequent degradation of KLF5 via the ubiquitin proteasome (14-16). KLF5 is SUMOylated on Lys152 and Lys209. SUMOylation of KLF5 inactivates the KLF5 nuclear export signal, facilitating KLF5 nuclear localization (17;18).

The Bernie mutation results in an arginine (R) to cysteine (C) substitution at position 360 (R360C) in the KLF5 protein; amino acid 360 immediate precedes the first zinc finger motif.

Expression/Localization

KLF5 is highly expressed in intestinal epithelial cells as well as in vascular smooth muscle cells, adipocytes, neural cells, and leukocytes (19-23). KLF5 is expressed at lower levels in the reproductive organs, prostate, pancreas, skeletal muscle, and lung (5). KLF5 expression is induced after physical injury, exposure to bacterial pathogens, or irradiation. Several signaling pathways (e.g., Ras/MAPK, PKC, and TGFβ) promote KLF5 expression, including growth factor-stimulated Ras/MAPK signaling pathways as well as the Wnt- and angiotensin II-stimulated PKC signaling pathways [reviewed in (5)]. KLF5 primarily localizes to the nucleus.

Background
Figure 5. Transactivation function of KLF5.  KLF5 can function as both a transcriptional activator and a repressor. Stimuli and signaling pathways which regulate (A) phosphorylation (P), (B) sumoylation (Su), and (C) acetylation (Ac) of the KLF5 protein. Arrows downstream of KLF5  represent how modified KLF5 subsequently activates or represses target genes through interaction with regulatory co‐factors. Select target genes are listed in Table 1.

KLF5 can function as both a transcriptional activator and a repressor, and as either an oncogene or a tumor suppressor in a cell type-specific manner (5) (Figure 5). KLF5 activates at least 88 targets and represses at least 300 targets [reviewed in (5)]. Select targets of KLF5 are listed in Table 1.

Table 1. Select KLF5 target genes. Table was adapted from (5).

Target genes

Downstream functions

References

Tcl1, Nr0b1, Bmp4, Tgfβ2, Otx2, Pitx2, Gdnf, Nanog, and Oct3/4

Promotes embryonic stem cell stemness and self-renewal

(24-27)

Cyclin D1, cyclin B, Cdc2, p15, p27

Accelerates the G1/S and G2/M cell cycle progression

(28)

Pdgfα, Vegfα

Regulates angiogenesis

(29-31)

Survivin, Pim1

Promotes cell survival (Survivin); promotes apoptosis (Pim1)

(32;33)

NF-κB, Mcp-1

Regulates inflammation

(34;35)

Ilk

Promotes epithelial cell migration

(36)

Mmp9

Promotes cartilage degradation

(37)

SM22a, PAI-1, Egr-1, Pparγ, iNOS

Promotes epithelial cell, smooth muscle cell, and adipocyte differentiation

(19;20;38;39)

Cpt1b, Ucp2, Ucp3, FASN, PPARδ

Regulates fatty acid metabolism

(18;40)

KLF5 functions in embryonic development (24-26;29), apoptosis (32;33;41), cell survival through the inhibition of apoptotic pathways (32;33), cell proliferation by accelerating G1/S and G2/M cell cycle transition (42-46), epithelial cell migration in the gut and skin (36), adipocyte differentiation (19), angiogenesis (29-31), muscle differentiation (47), skeletal development (37), intestinal development (43;48-50), cardiovascular remodeling (29;41;48;51;52), inflammatory stress responses (34;35), and energy metabolism (18;40). KLF5 also functions in reprogramming of somatic cells toward pluripotent stem cells (53) and in the maintenance of embryonic stem cell self-renewal (24). KLF5 maintains embryonic stem cells in an undifferentiated state by regulating the transcription of transcription factors Nanog and Oct3/4 (Pou5f1) transcription (24), which are known factors in embryonic stem cell self-renewal (54;55). KLF5 can also inhibit the proliferation of intestine (56), prostate (57), and esophageal (58) cancer cell lines.

KLF5 is aberrantly expressed in several human cancer cell lines [reviewed in (5)]. KLF5 expression is upregulated in cancer cell lines of the bladder (46), esophageal (58), and salivary gland (59) as well as in gastric carcinomas (60), some leukemia cell lines (33), and prostate cancers (57;61). In contrast, KLF5 expression is reduced in colon/intestine (45;56), breast (62;63), and nasopharyngeal (64) cancers as well as in melanomas (65). KLF5 regulates the expression of SM22α, Egf-1, and PDGF. KLF5-mediated regulation of these genes putatively linking KLF5 to the development of vascular smooth muscle cells-related diseases, such as atherosclerosis, restenosis after angioplasty, cardiac hypertrophy, and hypertension [reviewed in (5)]. Also, KLF5 regulates factors in inflammation (34), obesity (18), schizophrenia (21), and ulcerative colitis (66).

Klf5-deficient (Klf5-/-) mice exhibited embryonic lethality between embryonic day (E) 6.5 and E8.5 putatively due to defects in signaling that promotes the specification of trophoblast versus inner cell mass fate during the transition from morula to blastocyst stages (27;29;67). Klf5+/- mice exhibited reduced response to injury, skeletal growth retardation, and defects in adipocyte differentiation (19;29;37). Klf5+/- mice exhibited shorter intestinal crypts and villi and decreased thickening of arterial walls (48). Klf5+/- mice showed resistance to high fat-induced obesity, hypercholesterolemia, and glucose intolerance putatively due to increased energy expenditure (18). Klf5+/- mice also exhibited reduced angiogenesis, cardiac hypertrophy, and interstitial fibrosis after low-intensity transverse aortic constriction compared to wild-type mice (29;48).

Homozygous mice with conditional knockout of KLF5 in respiratory epithelial cells in the fetal lung died of respiratory distress immediately after birth, showing aberrant lung maturation and morphogenesis (68). Homozygous mice with hematopoietic-specific knockout of KLF5 showed increased numbers of short-term hematopoietic stem cells and multipotent progenitors in the spleen, reduction in the fraction of neutrophils in peripheral blood and bone marrow, and increased frequency of eosinophils in the peripheral blood, bone marrow, and lung (69). Homozygous mice with ocular surface-specific knockout of KLF5 showed abnormal eyelids with malformed meibomian glands and a conjunctiva devoid of mucin-producing goblet cells (70). Mice overexpressing KLF5 in the basal layer of the epidermis showed disrupted epithelial-mesenchymal interactions, exencephaly, craniofacial defects, ectodermal dysplasia, hypolastic epidermis, and abdominal herniation (71).

Putative Mechanism
Figure 6. KLF5 functions in crypt/epithelial proliferation and migration in the colon. Signaling pathways within the intestinal crypt and the relative location of cells of different lineages are shown. Stem cells give rise to terminally differentiated enterocytes, Goblet and Paneth cells. Stem cell differentiation depends on Wnt concentration as well as Notch expression. Interaction between Ephrin B ligands and EphB receptors direct cellular localization and migratory behavior within the crypt.

The intestinal epithelium is a single layer of cells that are renewed every four to five days throughout the life of a mouse (Figure 6). Stem cells at the base of the crypts divide, differentiate, and migrate up the villi to replace the shedding epithelium. The NOTCH, WNT, BMP, EphB1, EphB2, and EphB3 pathways control the stem cell self-renewal, differentiation, amplification, and migration. KLF5 functions in crypt/epithelial proliferation and migration in the colon. Loss of KLF5 expression in mice with DSS-induced colitis resulted in increased disease severity due to loss of cell proliferation and migration in the colon (49;72).

Primers PCR Primer
Bernie_pcr_F: GTCCCGATAGACAAGCTGAGATG
Bernie_pcr_R: TGTAGCCCAGGTTAGTTTGAACG

Sequencing Primer
Bernie_seq_F: ATGCTGCAGAATCTCACCCC
Bernie_seq_R: ACGTGTAATCTTCCTGTCTC
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 425 nucleotides is amplified (chromosome 14, + strand):


1   gtcccgatag acaagctgag atgctgcaga atctcacccc acctccgtcc tatgccgcta
61  caattgcttc caaactggcg attcacaacc caaatttacc tgccactctg ccagttaatt
121 cgccaactct cccacctgtc agatacaaca gaaggagtaa cccggatctg gagaagcgac
181 gtatccactt ctgcgattat aatggtatgt ggtctgtgtg ctcagggcat caggagggtt
241 tcatgttcca gagtgccaat aggtagggta tacctgacag ttaaagaaat cacctattcg
301 taatatttct gtgttttatg ctgatgtata atggcataaa acaccagcca tggcaattca
361 tgcctttaat cttagcaatg aggccgagac aggaagatta cacgttcaaa ctaacctggg
421 ctaca


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

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsEmre Turer, Kuan-Wen Wang, William McAlpine, and Bruce Beutler