Phenotypic Mutation 'Houstonian'

• Allele: Houstonian
• Mutation Type: missense
• Chromosome: 7
• Coordinate: 140,843,935 bp (GRCm39)
• Base Change: T ⇒ C (forward strand)
• Gene: Irf7
• Gene Name: interferon regulatory factor 7
• Chromosomal Location: 140,843,096-140,846,412 bp (-) (GRCm39)
• MGI Phenotype: FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] IRF7 encodes interferon regulatory factor 7, a member of the interferon regulatory transcription factor (IRF) family. IRF7 has been shown to play a role in the transcriptional activation of virus-inducible cellular genes, including interferon beta chain genes. Inducible expression of IRF7 is largely restricted to lymphoid tissue. Multiple IRF7 transcript variants have been identified, although the functional consequences of these have not yet been established. [provided by RefSeq, Jul 2008] ; PHENOTYPE: Homozygous null mice are more vulnerable to viral infection and exhibit decreased serum interferon levels in response to viral infection. [provided by MGI curators]
• Mapped: Yes
• Amino Acid Change: Threonine changed to Alanine
• Institutional Source: Beutler Lab
• Gene Model: predicted gene model for protein(s): [ENSMUSP00000026571] [ENSMUSP00000095565] [ENSMUSP00000101644] [ENSMUSP00000147529 ^†] † probably from a misspliced transcript
• AlphaFold: P70434
• PDB Structure: Crystal structure of IRF-7 DBD apo form [X-RAY DIFFRACTION]
• SMART Domains: Protein: ENSMUSP00000026571; Gene: ENSMUSG00000025498; AA Change: T246A

Domain	Start	End	E-Value	Type
IRF	5	127	1.13e-54	SMART
IRF-3	240	420	1.38e-63	SMART
low complexity region	425	442	N/A	INTRINSIC

• Predicted Effect: probably damaging ; PolyPhen 2 Score 0.992 (Sensitivity: 0.70; Specificity: 0.97)
  (Using ENSMUST00000026571)
• SMART Domains: Protein: ENSMUSP00000095565; Gene: ENSMUSG00000025498; AA Change: T215A

Domain	Start	End	E-Value	Type
IRF	5	127	1.13e-54	SMART
IRF-3	209	389	1.38e-63	SMART
low complexity region	394	411	N/A	INTRINSIC

• Predicted Effect: possibly damaging ; PolyPhen 2 Score 0.928 (Sensitivity: 0.81; Specificity: 0.94)
  (Using ENSMUST00000097952)
• SMART Domains: Protein: ENSMUSP00000101644; Gene: ENSMUSG00000025498; AA Change: T214A

Domain	Start	End	E-Value	Type
IRF	5	127	1.13e-54	SMART
IRF-3	208	388	1.38e-63	SMART
low complexity region	393	410	N/A	INTRINSIC

• Predicted Effect: possibly damaging ; PolyPhen 2 Score 0.949 (Sensitivity: 0.79; Specificity: 0.95)
  (Using ENSMUST00000106023)
• SMART Domains: Protein: ENSMUSP00000121026; Gene: ENSMUSG00000025498; AA Change: T82A

Domain	Start	End	E-Value	Type
IRF	1	69	6.35e-3	SMART
IRF-3	77	251	2.62e-55	SMART

• Predicted Effect: unknown
• Predicted Effect: unknown
• Meta Mutation Damage Score: 0.8884
• Is this an essential gene?: Non Essential (E-score: 0.000)
• Phenotypic Category: Autosomal Semidominant
• Mode of Inheritance: Autosomal Semidominant
• MMRRC Submission: 038163-MU
• Last Updated: 2019-09-04 9:46 PM by Anne Murray
• Record Created: 2015-01-20 2:02 PM by Doan Dao
• Record Posted: 2015-02-11
• Science Writers: Anne Murray
• Illustrators: Peter Jurek
• Authors: Doan Dao, Hexin Shi, Zhao Zhang, Ying Wang, Lei Sun, Jianhui Wang, and Bruce A. Beutler.

Accession Number

NCBI RefSeq: NM_016850; MGI: 1859212 

Alleles Listed at MGI

All mutations/alleles(5) : Chemically induced (ENU)(1) Gene trapped(1) Targeted(3)

Phenotypic Description

Figure 1. Macrophages from Houstonian mice exhibited decreased IFNα secretion in response to dsDNA. IFNα levels were determined by ELISA. 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 Houstonian phenotype was identified among N-Nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R1442, some of which showed reduced type I interferon (IFN) production by macrophages after stimulation with double stranded (ds) DNA (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of reduced IFNα secretion after dsDNA stimulation using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 67 mutations (X-axis) identified in the G1 male of pedigree R1442. Normalized phenotype data are shown for single locus linkage analysis with 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 67 mutations. The reduced type I IFN in response to dsDNA was linked by continuous variable mapping to a mutation in Irf7:  an A to G transition at base pair 141,264,022 (v38) on chromosome 7, or base pair 2,478 in the GenBank genomic region NC_000073.  Linkage was found with a semidominant model of inheritance, wherein 5 variant homozygotes and 23 heterozygotes departed phenotypically from 18 homozygous reference mice with a P value of 1.71 x 10^-4 (Figure 2).  The mutation corresponds to residue 1,179 in the mRNA sequence NM_016850 within exon 8 of 10 total exons.

1164 GGGTTCCTGGATGTGACCATCATGTACAAG 241  -G--F--L--D--V--T--I--M--Y--K-

The mutated nucleotide is indicated in red.  The mutation results in a threonine (T) to alanine (A) substitution at position 246 (T246A) in the IRF7 protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.992).

Protein Prediction

Figure 3. Domain structure of IRF7. Shown are the DNA binding domain (DBD), transactivation domain (TD), autoinhibition domain (AID), and carboxyl-terminal regulation domain (C). There is a nuclear export signal at amino acids 401-415 shown with with diagonal lines. Phosphorylation in the C-terminal is shown with orange circles. The amino acid altered by the Houstonian mutation is shown with a red asterisk.

In mouse and human, the Irf7 gene encodes one of nine members of the interferon regulatory factor (IRF) family of transcription factors, which regulate the transcription of type I interferons (IFN-α/β) and IFN-inducible genes during immune system development, homeostasis and activation by microbes [reviewed by (1;2)]. The N-terminal half of IRF7 (residues 11-122) serves as the DNA binding region (Figure 3). The mouse IRF7 IRF association domain 1 (IAD1) domain occurs at amino acids 240-416 (3), a region that corresponds to an autoinhibitory domain (AID) mapped by deletion constructs (4). The IAD1 domain is mediates interactions with other IRFs, other transcription factors, or homodimerization. These interactions allow the IRFs to modulate their activity and target a variety of genes. A transactivation domain was mapped to amino acids 132-237. Mouse IRF7 also contains a C-terminal regulatory region at residues 423-457 that is phosphorylated upon viral infection and is necessary for dimerization, nuclear localization and transactivation (4;5).  The amino acid mutated by the Houstonian mutation (T246A) lies within the IAD1/AID domain.

Please see the record inept for more information about Irf7.

Putative Mechanism

Type I IFNs are a critical class of cytokines that have antiviral, growth-inhibitory and immunomodulatory functions. Type I IFNs can be divided into two groups: immediate-early response genes that can be induced rapidly in response to immune stimuli without the need for ongoing protein synthesis; and a set of genes that display delayed induction. IFN-β and perhaps IFN-α4 are immediate-early response genes, while other IFN-α subtypes are induced more slowly in response to viral infection and require protein synthesis, a process that is completely dependent on IRF7 transcriptional activity and occurs through a two-step amplification in which IFN-β is initially produced in an IRF3-dependent manner from infected cells (6). Binding of IFN-β to the type I IFN receptor (see the records for macro-1 and macro-2) stimulates the Janus activating kinases (JAK)– signal transducer and activator of transcription (STAT) pathway (see the record for domino), resulting in activation of the transcription factor interferon stimulated gene factor 3 (ISGF3) complex composed of STAT1/STAT2 and IRF9.  The ISGF3 complex binds to upstream regulatory consensus sequences of hundreds of genes including IRF7, providing the type I IFN system with a positive feedback loop that allows amplification of the type I IFN response (5;7). Along with IRF7, IRF8 is also required for the second, amplifying phase of IFN transcription by binding to the promoters of IFN-α/β genes and regulating their transcription (8). IRF7-dependent amplification of the type I IFN response is essential during the immune response as IRF7-deficient mice and cells display a severe reduction of type I IFN in response to immune stimuli including cytosolic DNA (9-11). Although Houstonian mice exhibit diminished IFN production in response to dsDNA, they do not exhibit defective type I IFN production in response to TLR stimulation, indicating that the Houstonian mutation is a hypomorphic allele and that Houstonian mice retain normal type I IFN responses to certain stimuli.

Primers

PCR Primer
Houstonian_pcr_F: AAGCGTCTCTGTGTAGTGCAGC
Houstonian_pcr_R: CAGGAGCAAGACCGTGTTTACGAG

Sequencing Primer
Houstonian_seq_F: GGGCTCTGAAGTTCTTACTGCT
Houstonian_seq_R: TGTTTACGAGGAACCCTATGCAG

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 465 nucleotides is amplified (chromosome 7, - strand):

1   caggagcaag accgtgttta cgaggaaccc tatgcagcat ggcaggtgga agctgtcccc
61  agtcccaggc ctcaacagcc agctctcacc ggtgaggatc tagagcggag gcgctagcac
121 agcttttcct agcctcccgg gcagaattct gccctactgc tccttcacac ctgcatctca
181 gtaggaggtg aatgggtcct tggcaggggg catgggacaa aatgctccac ccaaacctac
241 cactcacagc atggtctata cacagagcgc agccttgggt tcctggatgt gaccatcatg
301 tacaagggcc gcacagtgct acaggcagtg gtggggcacc ccagatgcgt gttcctgtac
361 agccccatgg ccccagcagt aagaacttca gagccccagc cggtgatctt tcccagtcct
421 gctgagctcc cagatcagaa gcagctgcac tacacagaga cgctt

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

References

  1. Tamura, T., Yanai, H., Savitsky, D., and Taniguchi, T. (2008) The IRF Family Transcription Factors in Immunity and Oncogenesis. Annu Rev Immunol. 26, 535-584.

  2. Savitsky, D., Tamura, T., Yanai, H., and Taniguchi, T. (2010) Regulation of Immunity and Oncogenesis by the IRF Transcription Factor Family. Cancer Immunol Immunother. 59, 489-510.

  3. Meraro, D., Hashmueli, S., Koren, B., Azriel, A., Oumard, A., Kirchhoff, S., Hauser, H., Nagulapalli, S., Atchison, M. L., and Levi, B. Z. (1999) Protein-Protein and DNA-Protein Interactions Affect the Activity of Lymphoid-Specific IFN Regulatory Factors. J Immunol. 163, 6468-6478.

  4. Marie, I., Smith, E., Prakash, A., and Levy, D. E. (2000) Phosphorylation-Induced Dimerization of Interferon Regulatory Factor 7 Unmasks DNA Binding and a Bipartite Transactivation Domain. Mol Cell Biol. 20, 8803-8814.

  5. Marie, I., Durbin, J. E., and Levy, D. E. (1998) Differential Viral Induction of Distinct Interferon-Alpha Genes by Positive Feedback through Interferon Regulatory Factor-7. EMBO J. 17, 6660-6669.

  6. Yoneyama, M., Suhara, W., Fukuhara, Y., Fukuda, M., Nishida, E., and Fujita, T. (1998) Direct Triggering of the Type I Interferon System by Virus Infection: Activation of a Transcription Factor Complex Containing IRF-3 and CBP/p300. EMBO J. 17, 1087-1095.

  7. Sato, M., Hata, N., Asagiri, M., Nakaya, T., Taniguchi, T., and Tanaka, N. (1998) Positive Feedback Regulation of Type I IFN Genes by the IFN-Inducible Transcription Factor IRF-7. FEBS Lett. 441, 106-110.

  8. Tailor, P., Tamura, T., Kong, H. J., Kubota, T., Kubota, M., Borghi, P., Gabriele, L., and Ozato, K. (2007) The Feedback Phase of Type I Interferon Induction in Dendritic Cells Requires Interferon Regulatory Factor 8. Immunity. 27, 228-239.

  9. Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., Shimada, N., Ohba, Y., Takaoka, A., Yoshida, N., and Taniguchi, T. (2005) IRF-7 is the Master Regulator of Type-I Interferon-Dependent Immune Responses. Nature. 434, 772-777.

  10. Daffis, S., Samuel, M. A., Suthar, M. S., Keller, B. C., Gale, M.,Jr, and Diamond, M. S. (2008) Interferon Regulatory Factor IRF-7 Induces the Antiviral Alpha Interferon Response and Protects Against Lethal West Nile Virus Infection. J Virol. 82, 8465-8475.

  11. Wang, Z., Choi, M. K., Ban, T., Yanai, H., Negishi, H., Lu, Y., Tamura, T., Takaoka, A., Nishikura, K., and Taniguchi, T. (2008) Regulation of Innate Immune Responses by DAI (DLM-1/ZBP1) and Other DNA-Sensing Molecules. Proc Natl Acad Sci U S A. 105, 5477-5482.