Phenotypic Mutation 'kaburo'

• Allele: kaburo
• Chromosome: 14
• Gene: Hr
• Gene Name: lysine demethylase and nuclear receptor corepressor
• Synonym(s): rh-bmh, rh, N, bldy, ba
• Chromosomal Location: 70,789,652-70,810,988 bp (+) (GRCm39)
• MGI Phenotype: FUNCTION: This gene encodes a protein that is involved in hair growth. This protein functions as a transcriptional corepressor of multiple nuclear receptors, including thyroid hormone receptor, the retinoic acid receptor-related orphan receptors and the vitamin D receptors, and it interacts with histone deacetylases. The translation of this protein is modulated by a regulatory ORF that exists upstream of the primary ORF. Mutations in this upstream ORF, U2HR, cause Marie Unna hereditary hypotrichosis (MUHH), an autosomal dominant form of genetic hair loss in human. [provided by RefSeq, Oct 2014] ; PHENOTYPE: Mutant homozygotes exhibit hair loss, usually wrinkled skin with epidermal cysts. Females do not nurse their pups well. [provided by MGI curators]
• Mapped: Yes
• Institutional Source: Beutler Lab
• Ref Sequences:
  Ensembl: ENSMUSP00000022691
  Ensembl: ENSMUSP00000124042
• Gene Model: not available
• AlphaFold: Q61645
• Meta Mutation Damage Score: Not available
• Is this an essential gene?: Non Essential (E-score: 0.000)
• Phenotypic Category: Autosomal Semidominant
• Mode of Inheritance: Autosomal Semidominant
• Local Stock: Sperm, gDNA
• MMRRC Submission: 036518-MU
• Last Updated: 2018-03-02 4:49 PM by Diantha La Vine
• Record Created: 2011-02-22 10:04 PM by Wataru Tomisato
• Record Posted: 2012-04-10
• Science Writers: Anne Murray
• Illustrators: Victoria Webster
• Authors: Wataru Tomisato, Bruce Beutler

Accession Number

Ncbi Refseq: NM_021877.2; MGI:96223

Alleles Listed at MGI

All alleles(26) : Targeted(3) Transgenic(1) Spontaneous(16) Chemically induced(6)

Phenotypic Description

Figure 1. Representative Kaburo animals at 4.5 weeks. (A) A wild-type male mouse. (B) A Kaburo heterozygote displays thinning hair compared to the age-matched wild-type animal. (C) The Kaburo homozygote displays an almost complete loss of hair.

Figure 2. Comparison of the ventral side of a wild-type (A) and Kaburo heterozygote (B). The age-matched Kaburo heterozygote has body-wide thinning of the hair that is especially visible on the ventral side of the animal.

The Kaburo mutant pheotype was observed in a N-ethyl-N-nitrosourea (ENU)-induced G3 mutant mouse.  The mice displayed an alopecia phenotype and by 4.5 weeks of age, Kaburo homozygous animals have an almost complete loss of fur (Figure 1C).  The heterozygous Kaburo mutants displayed thinning of their fur (Figure 1B) that is especially noticable when comparing the wild-type (Figure 2A) to the heterozygote (Figure 2B) on the ventral side.

Nature of Mutation

Sequencing of the hr locus detected an A to G transition (A12173G) in the Hr gene chromosome 14 (position 12173 in Genbank genomic region NC_000080.5 for linear genomic DNA sequence of hr). 

In ENSMUST00000163060, the mutation location corresponds to intron 9 (36 bp from exon 9, 81 bp from exon 10):

<--exon 9  <--intron 9-->     exon 10-->  2394  ...CCGGATTCAC....TCTGCCCAGT  ....CCTGAGGAC....  GATGACCGC.... 2448 799   ...--R--I--H-....--L--P--S-                     -D--D--R-....  814

The mutated A is in red lettering.

In ENSMUST00000022691, the mutation location corresponds to intron 11 (36 bp from exon 11, 81 bp from exon 12):

<--exon 11  <--intron 11-->    exon 12-->  3001  ...CCGGATTCAC....TCTGCCCAGT  ....CCTGAGGAC....  GATGACCGC.... 3060 769   ...--R--I--H-....--L--P--S-                     -D--D--R-....  788

The mutated A is in red lettering.

Protein Prediction

Figure 3. HR functional domains. The N-terminal domain varies between the two isoforms of HR; only the longer isoform is shown. The position of the Kaburo mutation is indiated in red and results in a A to G transition in intron 9 of isoform encoded by the ENSMUST00000163060 cDNA transcript, or A to G transition in intron 11 of the isoform encoded  by the ENSMUST00000022691 cDNA transcript (not shown). NMTS, nuclear matrix targeting signal; RD, repression domain; NLS, nuclear localization signal; ID, interacting domain; ZF, zinc finger domain; ROR, retinoic acid receptor related orphan receptor; TR, thyroid hormone receptor. Click on the image to view other mutations found in HR. Click on each mutation for more specific information.

The Kaburo mutation may result in changes in the intron (albeit not to a donor splice site, acceptor splice site, or branch site) that result in the skipping of exon 11, 12, and/or others (Figure 3).  Exon 12 encodes repressive domain (RD) 2 (aa 725-839).  If any or all of exon 12 is affected by the Kaburo mutation, portions of the RD2 domain that interact with the thyroid hormone receptor (TR), the retinoic acid receptor related orphan receptor α (RORα) and the vitamin D receptor (VDR) could also be affected (see prune for more details) (1-3).  Domains that have been shown to interact with TR are located at amino acids 792-805 (TR-ID1 for TR interacting domain 1) and amino acids 1001-1013 (TR-ID2) (1).  RORα-interacting domains (ROR-ID) are located at amino acids 561-565 and 753-757 (2). Expression analysis of Kaburo, indicates that there is no alterations in splicing within the fragment that was assayed (primers were designed to amplify exons 10-13) (Figure 4A).  The band size, as determined by RT-PCR, was maintained, although the amount of product appears reduced.  qRT-PCR analysis confirmed that there was a drastic reduction in the amount of Hr cDNA expression in Kaburo (Figure 4B).  

Figure 4. Expression analysis of Kaburo . (A) RT-PCR analysis of Kaburo . The expected band size is 590 bp. The Kaburo mutants generate the correct band size. The upper bands are predicted to result from genomic DNA contamination. (B) qRT-PCR analysis of Kaburo . The homozygous Kaburo mouse ( Hr Kaburo /Hr Kaburo ) expresses less than 1/20 of Hr to the wild-type ( Hr + /Hr + ) mouse.

Please see prune and mister clean for more information about Hr.

Putative Mechanism

Mice with the classical hairless (hr) and rhino (hr^rh) mutations develop a normal first coat, but do not reinitiate subsequent hair cycles, resulting in alopecia (4-6). The Kaburo mutation results in reduction in the expression of the mutant cDNA, similar to some of the rhino alleles (4).  The Kaburo phenotype does not appear as severe as the prune, mister clean, or hr knockout phenotypes suggesting that some retention of HR function occurs, at least initially, in these animals.

Primers

Primers cannot be located by automatic search.

Genotyping

Kaburo genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.  

Primers for PCR amplification

Kaburo(F): 5’- AGGGTCTCTCTGAACCTGAAGCTG-3’

Kaburo(R): 5’- TCAATGTCTTCTGGATGCCTGACAC-3’

PCR program

1) 94°C             2:00

2) 94°C             0:30

3) 56°C             0:30

4) 72°C             1:00

5) repeat steps (2-4) 29X

6) 72°C             7:00

7) 4°C               ∞

Primers for sequencing

Kaburo _seq(F): 5’- ATGACCGGATTCACATGGC-3'

Kaburo _seq(R): 5’- TGACACTAACACGGGCTG-3’

The following sequence of 1203 nucleotides (from Genbank genomic region NC_000080.5 for linear DNA sequence of hr) is amplified:

11521                                       agggt ctctctgaac ctgaagctgt

11581 attagtggcc agcaaacctc agccagcagt gtccctagct ccacgccacc tcttccccaa

11641 cccaagagct gaggtggcaa gcaaaacaat aacattttat ttgtttgttg tttgggtttt

11701 ttttgttttt gttttttttg tgagtgttgg ggatttgaac ttagatcctg acatttctta

11761 cttacacaga aattctgtgt gcttgcctgc tgagctgtct cacagttcaa gactcccatt

11821 tggagccact cttctcagag acgctgcaac ttcctttagg aaagcagaga tggggcgaca

11881 cccagacata gtacagtaaa gttgaggtgc tattgaagta aagactaaag gccaagcaga

11941 aagccactca ctcactattt tcttcttcaa cagagacccc agactccact gagagcccag

12001 cagaggatgg tgctggccgg tcaccccttc cttgtccctc tctctgtgag ctgctagcct

12061 ctactgctgt caaactctgc ctggggcatg accggattca catggccttt gctccggtca

12121 ccccagctct gcccagtgtg agcaagggct cagagggagg gccaagaacc tgaggaccag

12181 aggctgggtg gccatcctta attggctgaa ttggtcctga cctttcttgc tcctccatcc

12241 ctgcctcccc acaggatgac cgcattacca acatcctgga cagcattatt gcgcaggtag

12301 tagaacggaa gatccaagag aaagccctgg ggccaggcct gcgagcaggg tcaggcttac

12361 gcaagggcct gagccttcca ttgtcaccag tgcgaacccg gctgtctcct cctggagctt

12421 tgctgtggct gcaggagccc aggcctaagc atggcttcca tctcttccag gaacactggc

12481 ggcagggcca ggtaagctgg cctacctctc ccttcccaac actatggctt cccctgcttc

12541 acagggtcct agtccccaag cttagagggt cctcctggtg ttcagaggga gcaagcaaag

12601 gctggtcttt cctcaatccc ttccccccac atgttctgga gatgttacaa catgccccag

12661 cctggccact atgggctccc caacaattct tttctcttct tttctttttc ttccttctct

12721 gcagcccgtg ttagtgtcag gcatccagaa gacattga  

PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray (overlapping sequences between the PCR primer and sequencing primer are italicized); the mutated A is shown in red text.

References

  1. Potter, G. B., Beaudoin, G. M.,3rd, DeRenzo, C. L., Zarach, J. M., Chen, S. H., and Thompson, C. C. (2001) The Hairless Gene Mutated in Congenital Hair Loss Disorders Encodes a Novel Nuclear Receptor Corepressor. Genes Dev.. 15, 2687-2701.

  2. Moraitis, A. N., Giguere, V., and Thompson, C. C. (2002) Novel Mechanism of Nuclear Receptor Corepressor Interaction Dictated by Activation Function 2 Helix Determinants. Mol. Cell. Biol.. 22, 6831-6841.

  3. Hsieh, J. C., Sisk, J. M., Jurutka, P. W., Haussler, C. A., Slater, S. A., Haussler, M. R., and Thompson, C. C. (2003) Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling. J. Biol. Chem.. 278, 38665-38674.

  4. Cachon-Gonzalez, M. B., Fenner, S., Coffin, J. M., Moran, C., Best, S., and Stoye, J. P. (1994) Structure and Expression of the Hairless Gene of Mice. Proc. Natl. Acad. Sci. U. S. A.. 91, 7717-7721.

  5. Cachon-Gonzalez, M. B., San-Jose, I., Cano, A., Vega, J. A., Garcia, N., Freeman, T., Schimmang, T., and Stoye, J. P. (1999) The Hairless Gene of the Mouse: Relationship of Phenotypic Effects with Expression Profile and Genotype. Dev. Dyn.. 216, 113-126.

  6. Panteleyev, A. A., Paus, R., Ahmad, W., Sundberg, J. P., and Christiano, A. M. (1998) Molecular and Functional Aspects of the Hairless (Hr) Gene in Laboratory Rodents and Humans. Exp. Dermatol.. 7, 249-267.