A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease

Article metrics

Abstract

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case–based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: cis-regulatory SNP in NF-κB binding site alters HTT expression.
Figure 2: NF-κB binds to the huntingtin promoter in vitro and in vivo.
Figure 3: cis-regulatory variants in the HTT promoter alter NF-κB binding.
Figure 4: Targeting of NF-κB modulates HTT expression.
Figure 5: rs13102260 (A) variant modulates HD AO.

References

  1. 1

    Strong, T.V. et al. Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 5, 259–265 (1993).

  2. 2

    Li, S.H. et al. Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron 11, 985–993 (1993).

  3. 3

    Sotrel, A. et al. Morphometric analysis of the prefrontal cortex in Huntington's disease. Neurology 41, 1117–1123 (1991).

  4. 4

    Leavitt, B.R. et al. Wild-type huntingtin protects neurons from excitotoxicity. J. Neurochem. 96, 1121–1129 (2006).

  5. 5

    Cattaneo, E. et al. Loss of normal huntingtin function: new developments in Huntington's disease research. Trends Neurosci. 24, 182–188 (2001).

  6. 6

    Ross, C.A. & Tabrizi, S.J. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 10, 83–98 (2011).

  7. 7

    Andrew, S.E. et al. The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease. Nat. Genet. 4, 398–403 (1993).

  8. 8

    Langbehn, D.R., Brinkman, R.R., Falush, D., Paulsen, J.S. & Hayden, M.R. A new model for prediction of the age of onset and penetrance for Huntington's disease based on CAG length. Clin. Genet. 65, 267–277 (2004).

  9. 9

    Brinkman, R.R., Mezei, M.M., Theilmann, J., Almqvist, E. & Hayden, M.R. The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size. Am. J. Hum. Genet. 60, 1202–1210 (1997).

  10. 10

    Albert, F.W. & Kruglyak, L. The role of regulatory variation in complex traits and disease. Nat. Rev. Genet. 16, 197–212 (2015).

  11. 11

    Wexler, N.S. et al. Venezuelan kindreds reveal that genetic and environmental factors modulate Huntington's disease age of onset. Proc. Natl. Acad. Sci. USA 101, 3498–3503 (2004).

  12. 12

    Graham, R.K. et al. Levels of mutant huntingtin influence the phenotypic severity of Huntington disease in YAC128 mouse models. Neurobiol. Dis. 21, 444–455 (2006).

  13. 13

    Leavitt, B.R. et al. Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo. Am. J. Hum. Genet. 68, 313–324 (2001).

  14. 14

    Van Raamsdonk, J.M. et al. Loss of wild-type huntingtin influences motor dysfunction and survival in the YAC128 mouse model of Huntington disease. Hum. Mol. Genet. 14, 1379–1392 (2005).

  15. 15

    Coles, R., Caswell, R. & Rubinsztein, D.C. Functional analysis of the Huntington's disease (HD) gene promoter. Hum. Mol. Genet. 7, 791–800 (1998).

  16. 16

    Holzmann, C. et al. Isolation and characterization of the rat huntingtin promoter. Biochem. J. 336, 227–234 (1998).

  17. 17

    Feng, Z. et al. p53 tumor suppressor protein regulates the levels of huntingtin gene expression. Oncogene 25, 1–7 (2006).

  18. 18

    Wang, R. et al. Sp1 regulates human huntingtin gene expression. J. Mol. Neurosci. 47, 311–321 (2012).

  19. 19

    Emond, M.J. et al. Exome sequencing of extreme phenotypes identifies DCTN4 as a modifier of chronic Pseudomonas aeruginosa infection in cystic fibrosis. Nat. Genet. 44, 886–889 (2012).

  20. 20

    Ott, J., Kamatani, Y. & Lathrop, M. Family-based designs for genome-wide association studies. Nat. Rev. Genet. 12, 465–474 (2011).

  21. 21

    Barnett, I.J., Lee, S. & Lin, X. Detecting rare variant effects using extreme phenotype sampling in sequencing association studies. Genet. Epidemiol. 37, 142–151 (2013).

  22. 22

    Hayles, B., Yellaboina, S. & Wang, D. Comparing transcription rate and mRNA abundance as parameters for biochemical pathway and network analysis. PLoS ONE 5, e9908 (2010).

  23. 23

    Hodgson, J.G. et al. Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype. Hum. Mol. Genet. 5, 1875–1885 (1996).

  24. 24

    Ehrlich, M.E. et al. ST14A cells have properties of a medium-size spiny neuron. Exp. Neurol. 167, 215–226 (2001).

  25. 25

    Chen, F.E., Huang, D.B., Chen, Y.Q. & Ghosh, G. Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature 391, 410–413 (1998).

  26. 26

    Escalante, C.R., Shen, L., Thanos, D. & Aggarwal, A.K. Structure of NF-kappaB p50/p65 heterodimer bound to the PRDII DNA element from the interferon-beta promoter. Structure 10, 383–391 (2002).

  27. 27

    Natarajan, K., Singh, S., Burke, T.R. Jr., Grunberger, D. & Aggarwal, B.B. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc. Natl. Acad. Sci. USA 93, 9090–9095 (1996).

  28. 28

    Nørremolle, A. et al. 4p16.3 haplotype modifying age at onset of Huntington disease. Clin. Genet. 75, 244–250 (2009).

  29. 29

    Andrew, S.E., Goldberg, Y.P., Theilmann, J., Zeisler, J. & Hayden, M.R.A. CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing. Hum. Mol. Genet. 3, 65–67 (1994).

  30. 30

    Pêcheux, C. et al. Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes. J. Med. Genet. 32, 399–400 (1995).

  31. 31

    Coles, R., Leggo, J. & Rubinsztein, D.C. Analysis of the 5′ upstream sequence of the Huntington's disease (HD) gene shows six new rare alleles which are unrelated to the age at onset of HD. J. Med. Genet. 34, 371–374 (1997).

  32. 32

    Nadeau, J.H. Modifier genes in mice and humans. Nat. Rev. Genet. 2, 165–174 (2001).

  33. 33

    Génin, E., Feingold, J. & Clerget-Darpoux, F. Identifying modifier genes of monogenic disease: strategies and difficulties. Hum. Genet. 124, 357–368 (2008).

  34. 34

    Rubinsztein, D.C. et al. Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease. Proc. Natl. Acad. Sci. USA 94, 3872–3876 (1997).

  35. 35

    Nazé, P., Vuillaume, I., Destee, A., Pasquier, F. & Sablonniere, B. Mutation analysis and association studies of the ubiquitin carboxy-terminal hydrolase L1 gene in Huntington's disease. Neurosci. Lett. 328, 1–4 (2002).

  36. 36

    Metzger, S. et al. Huntingtin-associated protein-1 is a modifier of the age-at-onset of Huntington's disease. Hum. Mol. Genet. 17, 1137–1146 (2008).

  37. 37

    Weydt, P. et al. The gene coding for PGC-1alpha modifies age at onset in Huntington's Disease. Mol. Neurodegener. 4, 3 (2009).

  38. 38

    Metzger, S. et al. Age at onset in Huntington's disease is modified by the autophagy pathway: implication of the V471A polymorphism in Atg7. Hum. Genet. 128, 453–459 (2010).

  39. 39

    Kasowski, M. et al. Variation in transcription factor binding among humans. Science 328, 232–235 (2010).

  40. 40

    Meffert, M.K. & Baltimore, D. Physiological functions for brain NF-kappaB. Trends Neurosci. 28, 37–43 (2005).

  41. 41

    Ge, B. et al. Global patterns of cis variation in human cells revealed by high-density allelic expression analysis. Nat. Genet. 41, 1216–1222 (2009).

  42. 42

    Liu, W. et al. Increased steady-state mutant Huntingtin mRNA in Huntington's disease brain. J. Huntingtons Dis. 2, 491–500 (2013).

  43. 43

    Van Raamsdonk, J.M., Pearson, J., Murphy, Z., Hayden, M.R. & Leavitt, B.R. Wild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in the YAC128 mouse model of Huntington disease. BMC Neurosci. 7, 80 (2006).

  44. 44

    Sah, D.W. & Aronin, N. Oligonucleotide therapeutic approaches for Huntington disease. J. Clin. Invest. 121, 500–507 (2011).

  45. 45

    Andersen, M.C. et al. In silico detection of sequence variations modifying transcriptional regulation. PLoS Comput. Biol. 4, e5 (2008).

  46. 46

    Sandelin, A., Alkema, W., Engstrom, P., Wasserman, W.W. & Lenhard, B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94 (2004).

  47. 47

    Nørremolle, A. et al. Mosaicism of the CAG repeat sequence in the Huntington disease gene in a pair of monozygotic twins. Am. J. Med. Genet. A. 130A, 154–159 (2004).

Download references

Acknowledgements

The authors wish to thank M. Davey and N. Schintu for technical assistance, R.H. Newbury for helpful discussions of the manuscript, J. Tedroff for critical assessment of clinical data, R.A. Harris for critical reading of the manuscript, and L.V. Olsen for excellent technical assistance. This work was supported by the Swedish Research Council (K.B., O.H.), Huntington Society of Canada (K.B.), the Canadian Institutes of Health Research (B.R.L., M.R.H. and W.W.W.) and the CHDI foundation (M.R.H. and B.R.L.).

Author information

K.B. conceived, designed and performed the experiments, analyzed and interpreted the data, conceived and designed the study, and wrote the manuscript. T.L., S.J.N., S.M., G.G., C.D. and J.B. performed experiments. A.N. performed SNP genotyping and haplotype identification of Danish cohort, and contributed with fibroblast lines. C.N.K., J.A.C. and S.C.W. conducted haplotype identification experiments of the UBC cohort. D.A. and E.P.-C. performed in silico analysis. N.L. and S.J.T. contributed with DNA and conducted SNP genotyping of EHDN Registry samples. C.C. and R.A.G.D. contributed with cell culture work. O.H. contributed to ChIP and siRNA experiments. M.R.H. provided the UBC HD biobank samples. D.R.L. performed statistical analysis of cohort data. W.W.W. conceived the experiments. B.R.L. conceived the study. All authors reviewed the manuscript.

Correspondence to Kristina Bečanović or Blair R Leavitt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Department of Neurology, University Hospital of Ulm, Ulm, Germany.

Integrated supplementary information

Supplementary Figure 1 NF-ĸB TFBS in mouse, rat and human HTT promoters.

Supplementary Figure 2 Binding of CFI/USP and AML-1 to the HTT promoter assessed by EMSA.

Binding of the CFI/USP and AML-1 to the allele 4 and 5 target sequences was assessed following incubation with ST14A nuclear extract. (a) Incubation of the oligonucleotides spanning the CF1/USP site with nuclear extract produced a series of shifted bands with decreased binding to the allele 4 compared to allele 5 oligonucleotide. The strongest shifted band was eliminated upon addition of unlabeled competitor oligonucleotide indicating that this interaction is specific. (b) Incubation of the oligonucleotides spanning the AML-1 site with the ST14A nuclear extract also produced multiple shifted bands, of which the strongest was abolished in the presence of unlabeled competitor oligonucleotide. Lanes 1–2, no nuclear extract; lanes 3, 5, 7, allele 4 oligonucleotide and lanes 4, 6, 8, allele 5 oligonucleotide incubated with 2 µg, 4 µg and 6 µg ST14A nuclear extract, respectively. Lanes 9–10, unlabeled competitor oligonucleotide added to labeled oligonucleotide + protein extract.

Supplementary Figure 3 Effects of NF-κB knockdown on p53 expression and CAPE on basal HTT transcriptional activity.

(a) HEK293 cells were treated with control or NF-κB targeted siRNA. p53 was down-regulated in the siRNA treated group compared to the control (t(12) = 3.46, P = 0.0047, two-tailed unpaired t-test) (n = 7 per group). (b) ST14A cells transfected with construct 4 or 5 were stimulated with CAPE. The untreated construct 5 displayed a significant reduction in transcriptional activity compared to construct 4 (F(3,38) = 29.9, P < 0.0001, one-way ANOVA with Dunnett’s multiple comparison test). Constructs 4 and 5 displayed similar levels in transcriptional activity when the ST14A cells were treated with CAPE in the absence of a NF-κB pathway activating treatment. The data from four independent reporter assay experiments is shown for each panel (n = 12 per group). Data is presented as mean ± s.e.m.

Supplementary Figure 4 Interaction between rs13102260 SNP and expected age of onset.

Evidence for interaction between the rs13102260 (G/A) genotype and CAG-based expected age of onset (F = 5.80, df = 73.9, P = 0.0185).

Supplementary Figure 5 Genotype distribution of rs13102260 (G/A) and age of onset percentiles for HD patients representing a random population.

Analysis included HD patients with CAG repeat length 41–55 obtained from the EHDN Registry (n = 450). Legend indicates ratio AO percentile; number of patients; percentage of total patients. WT = wild-type HTT allele. HD = HD disease allele.

Supplementary Figure 6 The rs13102260 (A) variant correlates with two distinct haplotype patterns.

The 5′ upstream region and the polymorphic CCG region in exon 1 of the HD gene were analyzed using direct sequencing of 26 unrelated HD patients (UBC Biobank) carrying the rs13102260 (A) sequence variant. 55% (12/22) of HD subjects with the (A) sequence variant carried two repeat alleles at the 6bp VNTR loci in conjunction with the (CCG)7(CCT)3 polymorphism (referred to as CCG8), while 45% (10/22) carried the one-repeat allele at the 6bp VNTR (referred to as CCG7). (a) The 5’ upstream region including the 6bp VNTR and the rs13102260 (G/A) is indicated. Numbering of the sequence start is in relation to the transcriptional start site. (b) The CCG polymorphisms identified are indicated.

Supplementary Figure 7 Genotype at rs13102260 drives allele-specific expression of HTT.

The effect of the rs13102260 (A) sequence variant on wild-type and mutant HTT (mHTT) protein expression levels was measured with western blot analysis in HD patient fibroblast lines. Wild-type HTT protein was reduced in the samples with the (A) variant phased to the wild-type allele compared to the (G) variant (G[WT] vs A[WT]) (t(6) = 3.27, P = 0.0169, two-tailed unpaired t-test). mHTT protein was reduced in the samples with the (A) variant phased on the HD disease allele compared to the (G) variant (G[HD] vs A[HD]) (t(6) = 2.46, P = 0.0495). We observed a general reduction of mHTT protein compared to the wild-type protein regardless of genotype. The mHTT protein in the A[WT]G[HD] fibroblast lines was expressed at lower levels compared to the G[WT]G[HD] (t(6) = 3.36, P = 0.0152). The data represents two different fibroblast lines per genotype group including two pellets per cell line (mean ± s.e.m, n = 4). Alleles carrying the rs13102260 (A) sequence variant are labelled in red; (G) variant carriers are indicated in white. For each genotype group, the left bar indicates wild-type HTT levels, and the right bar indicates mHTT levels.

Supplementary Figure 8 Model for rs13102260 (G/A) as a bidirectional genetic modifier of HD age of onset.

rs13102260 (G/A) is located in the identified NF-κB TFBS in the HTT promoter immediately proximal to the 5’UTR of the HTT gene. The rs13102260 (A) sequence variant impaired NF-κB binding, and reduced transcriptional activity of the HTT gene and HTT protein levels. Presence of the rs13102260 (A) variant on the wild-type allele associated with reduced wild-type HTT protein levels and earlier AO, while the (A) variant on the HD disease allele associated with lower mutant HTT (mHTT) protein levels and delayed AO in HD patients.

Supplementary Figure 9 Full-length uncropped western blot presented in figure 4d.

Western blot shows TNF-alpha and CAPE stimulated ST14A cells. This blot includes stimulation of ST14A cells with lipopolysaccharide (LPS), which is not presented in the main manuscript. Molecular-weight marker indicates protein size (kDa).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1–5 (PDF 775 kb)

Supplementary Methods Checklist (PDF 531 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bečanović, K., Nørremølle, A., Neal, S. et al. A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease. Nat Neurosci 18, 807–816 (2015) doi:10.1038/nn.4014

Download citation

Further reading