Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise
© Whitelaw et al.; licensee BioMed Central Ltd. 2010
Received: 30 June 2010
Accepted: 19 November 2010
Published: 19 November 2010
Inbred individuals reared in controlled environments display considerable variance in many complex traits but the underlying cause of this intangible variation has been an enigma. Here we show that two modifiers of epigenetic gene silencing play a critical role in the process.
Inbred mice heterozygous for a null mutation in DNA methyltransferase 3a (Dnmt3a) or tripartite motif protein 28 (Trim28) show greater coefficients of variance in body weight than their wild-type littermates. Trim28 mutants additionally develop metabolic syndrome and abnormal behavior with incomplete penetrance. Genome-wide gene expression analyses identified 284 significantly dysregulated genes in Trim28 heterozygote mutants compared to wild-type mice, with Mas1, which encodes a G-protein coupled receptor implicated in lipid metabolism, showing the greatest average change in expression (7.8-fold higher in mutants). This gene also showed highly variable expression between mutant individuals.
These studies provide a molecular explanation of developmental noise in whole organisms and suggest that faithful epigenetic control of transcription is central to suppressing deleterious levels of phenotypic variation. These findings have broad implications for understanding the mechanisms underlying sporadic and complex disease in humans.
Experiments designed to analyze the significance of genes and environment on quantitative traits using laboratory rats and mice have found that 70 to 80% of all variation is of unknown origin . Gartner  carried out experiments over a period of 20 years to analyze the significance of different components of random variability in quantitative traits. Reduction of genetic variability, by using inbred strains, and reduction of environmental variability, by standardized husbandry, did not significantly reduce the range of random phenotypic variability. Similarly, moving the animals into the wild to increase environmental variability did not increase random phenotypic variability, hence the term 'intangible variance' . For example, only 20 to 30% of the range of the body weights of inbred mice was estimated to be the result of postnatal environment, with the remaining 70 to 80%, which Gartner termed 'the third component', being of unknown origin. These and other studies suggested that this phenotypic variation, also known as 'developmental noise' , is determined early in ontogeny [4, 5].
Comparisons of classic quantitative traits, such as body weight and behavior, across mouse strains have been hampered by the difficulty of controlling for maternal effects. In the experiments described here, such effects have been ruled out by comparing mutant with wild-type littermates, raised in the same cage by the same dam. The studies have been carried out using mice heterozygous for known modifiers of epigenetic reprogramming, one of which (Trim28 MommeD9/+ ) emerged from a dominant screen for modifiers of epigenetic reprogramming. In this screen N-ethyl-N-nitrosourea (ENU) mutagenesis was carried out on inbred FVB/NJ mice carrying a variegating GFP transgene expressed in red blood cells . The percentage of cells expressing the transgene is sensitive to the dosage of epigenetic modifiers. The screen has identified both known (Dnmt1, Smarca5, Hdac1, Baz1b) and novel (SmcHD1) genes [7–9] and has provided us with mouse models (MommeDs) to study the role of epigenetic reprogramming in whole organisms and populations.
Mice with reduced levels of DNA methyltransferases  and other modifiers of epigenetic reprogramming (for example, Suv39 h, Hdac1, Smarca5, Mel18) are viable, reproduce and are superficially phenotypically normal [11–13]. We were keen to discover subtle phenotypic abnormalities in MommeD mice and found that cohorts heterozygous for some modifiers of epigenetic gene silencing display greater phenotypic noise.
We were keen to discover whether similar effects would be seen with other proteins involved in epigenetic reprogramming. We have previously reported that MommeD2 mice carry a mutation in the Dnmt1 gene that destabilizes the protein and heterozygotes are haploinsufficient for Dnmt1 . This mouse strain was produced and maintained on the FVB/NJ background. In Dnmt1 MommeD2/+ mice there was no difference in the mean, the range, or the coefficient of variance of body weight at weaning (Figure 1). Similarly, we have published previously that haploinsufficiency for Snf2 h (the protein disrupted in Smarca5 MommeD4/+ mice) resulted in smaller mean body weight but with no obvious increase in the coefficient of variance  and that haploinsufficiency for Baz1b (the protein disrupted in Baz1b MommeD10 mice) resulted in no change to the mean body weight, nor the coefficient of variance .
Transcriptional noise at the cellular level has been documented in single cell organisms [23, 24]. Gordon and colleagues  have shown, using single cell observation of the bistable lac operon in Escherichia coli, that reduction in the levels of proteins regulating transcription can result in heritable aberrant behavior in genetically identical cells. Intrinsic variability in expression state at a number of genes in yeast has been shown to be associated with changes in the epigenetic state of their promoters [26–28]. The manifestation of this transcriptional noise at the level of multicellular organisms or populations is rarely considered. Interestingly, Raj and colleagues  have recently shown that increased transcriptional noise can lead to intestinal cell fate changes in Caenorhabditis elegans and that chromatin proteins may be involved. Our data are consistent with this finding. Here we have shown that reduced levels of two proteins involved in transcriptional gene silencing, Dnmt3a and Trim28, cause increased phenotypic variance in inbred littermates.
While developmental flexibility with respect to cell fate is necessary for complex organisms to produce multiple cell types, unfettered transcriptional noise appears to be detrimental. Not all inbred colonies haploinsufficient for epigenetic modifiers display changes in body weight (for example, Baz1b , Dnmt1) but more extensive phenotypic analysis using a broader range of measurements may reveal other traits with increased variation. Perhaps transcriptional noise at critical stages in early development results in increased variance in cell fate decisions among mutant offspring leading to changes in the proportions of different tissue types in the adult. While it is theoretically possible that reduced levels of epigenetic modifier proteins lead to increased genetic changes, we see no evidence of this using comparative genomic hybridization arrays (data not shown). Our data suggest that disrupting the epigenome can change gene regulatory networks and that this results in increased phenotypic variation.
The capacity of an organism to ensure the production of a standard phenotype in spite of environmental disturbances is called canalization . Our studies show that modifiers of epigenetic gene silencing are fundamental to this process and suggest that their levels have been fine-tuned by evolutionary pressures to allow cells to acquire different patterns of gene expression during differentiation, but at the same time to lock-in the transcriptional profile of differentiated cell types. Numerous studies in vertebrates and invertebrates using isogenic individuals raised in controlled environments show considerable variance for many phenotypic traits, for example, body weight and bristle number. This is the first report of any mechanism that can change the degree of variance at the level of the whole organism in mammals. Our findings have broad implications for the mechanisms underlying phenotype and disease in all multicellular organisms.
Materials and methods
Mouse strains and genotyping
Wild-type inbred C57BL/6J mice were purchased from ARC Perth (Perth, WA, Australia). Procedures were approved by the Animal Ethics Committee of the Queensland Institute of Medical Research. The ENU screen was carried out in an FVB/NJ inbred line that carry a GFP transgene, as described previously . Dnmt1 MommeD2 mice and Trim28 MommeD9 mice were maintained in this background unless stated otherwise. Dnmt1 MommeD2 mice and Trim28 MommeD9 mice were classed as heterozygous or wild-type by fluorescence-activated cell sorting (FACS) analysis of GFP expression as described previously [7, 9]. The Dmnt3a - knockout allele was maintained on a C57BL/6 background and detected by PCR primers specific for the neo cassette, as described at the Jackson Laboratory website .
FVB/NJ MommeD9 heterozygotes, homozygous for the GFP transgene, were backcrossed twice to C57BL/6 and phenotyped for GFP expression by flow cytometry, as previously described . DNA from tail tips was used to perform a genome-wide linkage scan, which identified the linked interval on chromosome 7 . We have reduced the linked interval from that reported by using additional SNP markers. Fine mapping using microsatellite and SNP markers polymorphic between FVB/NJ and C57BL/6 was carried out on 127 wild types and 103 heterozygotes to define the linked interval. Estimating the probability of ENU-induced coding mutations was performed using formulas accessible on the 'enuMutRat on zeon' website .
RNA and cDNA analysis
Poly(A)+ RNA was purified from the livers of 4-week-old male Trim28 MommeD9/+ mice and Trim28 +/+ littermates. RNA was separated on a 1% denaturing agarose gel, transferred and hybridized with a fragment encompassing Trim28 exons 11 and 12 using PCR primers (Additional file 2). cDNA was prepared from total RNA from the livers of 4-week-old Trim28 MommeD9/+ mice and Trim28 +/+ littermates using random priming and the Superscript®III system (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR reactions were prepared using SYBR® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). PCRs were run on standard programs using a Rotor-Gene 3000 (Corbett/Qiagen, Valencia, CA, USA). Mas1 mRNA was amplified with primers: 5′-AAGCCTCTAGCCCTCTGTCC-3′ (forward) and 5′-GGTCCATGAGGAGTTCTTGA-3′ (reverse).
Nuclear extracts were prepared from the spleens of 4-week-old MommeD9 mice. Approximately 5 μg of proteins were separated by SDS-PAGE on a 4 to 12% Bis-tris polyacrylamide gel (Invitrogen) and were analyzed with a monoclonal antibody to Trim28 (MAB3662, Millipore, Billerica, MA, USA).
For Illumina BeadArray analysis, total liver RNA from Trim28 MommeD9/+ mice (n = 4) and Trim28 +/+ mice (n = 4) was assessed for integrity using the Agilent Bioanalyzer 2100, and RNA integrity (RIN) scores above 8 were present in all samples. RNA was amplified using the Illumina TotalPrep RNA Amplification kit (Ambion, Carlsbad, CA, USA). Amplified cRNA was assessed for quantity and quality also using the Agilent Bioanalyzer 2100. RNA was hybridized to MouseRef-8 v2.0 Expression BeadChip (Illumina, Carlsbad, CA, USA) according to the manufacturer's instructions. Technical replicates were performed for all samples. BeadChip arrays were scanned with Illumina BeadStation Scanner and data values with detection scores were compiled using BeadStudio (Illumina). The gene expression data were analyzed by the GenomeStudio Gene Expression Module (Illumina). Genes with significantly different expression (difference score > 16) were analyzed using Ingenuity Pathway Analysis. The expression data have been deposited in NCBI's Gene Expression Omnibus (GEO), and is accessible through GEO Series accession number [GEO:GSE23512] .
Trim28 MommeD9/+ mice and Trim28 +/+ littermates between the ages of 5 and 11 months were placed into a 40 cm × 40 cm box with a grid dividing it into 16 squares (10 × 10 cm). Mice were placed in the open field and scored for the number of squares entered, and number of times the mouse reared up on its hind legs over a 2-minute period. Data were collected by two independent investigators, one of whom was blind to genotype. The data were the average of the two scores and scores were 90% concordant.
Gene Expression Omnibus
green fluorescent protein
haematoxylin and eosin
single nucleotide polymorphism.
We would like to thank Paul Fahey (QIMR/RBWH Statistics Unit) for his assistance with statistical analysis. This study was supported by NHMRC Project Grants to EW. NCW, DKM, TJB and AA were supported by Australian Postgraduate Awards. EW is supported by a NHMRC Australia Fellowship.
- Falconer DS: The genetics of litter size in mice. J Cell Comp Physiol. 1960, 56 (Suppl 1): 153-167. 10.1002/jcp.1030560414.PubMedView ArticleGoogle Scholar
- Gartner K: A third component causing random variability beside environment and genotype. A reason for the limited success of a 30 year long effort to standardize laboratory animals?. Lab Anim. 1990, 24: 71-77. 10.1258/002367790780890347.PubMedView ArticleGoogle Scholar
- Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM: Introduction to Genetic Analysis. 1996, Freeman: New York, 6Google Scholar
- Biggers JD, Mc LA, Michie D: Variance control in the animal house. Nature. 1958, 182: 77-80. 10.1038/182077a0.PubMedView ArticleGoogle Scholar
- Durrant A, Mather K: Heritable variation in a long inbred line of Drosophila. Genetica. 1954, 27: 97-119. 10.1007/BF01664156.PubMedView ArticleGoogle Scholar
- Blewitt ME, Vickaryous NK, Hemley SJ, Ashe A, Bruxner TJ, Preis JI, Arkell R, Whitelaw E: An N-ethyl-N-nitrosourea screen for genes involved in variegation in the mouse. Proc Natl Acad Sci USA. 2005, 102: 7629-7634. 10.1073/pnas.0409375102.PubMedPubMed CentralView ArticleGoogle Scholar
- Chong S, Vickaryous N, Ashe A, Zamudio N, Youngson N, Hemley S, Stopka T, Skoultchi A, Matthews J, Scott HS, de Kretser D, O'Bryan M, Blewitt M, Whitelaw E: Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat Genet. 2007, 39: 614-622. 10.1038/ng2031.PubMedPubMed CentralView ArticleGoogle Scholar
- Blewitt ME, Gendrel AV, Pang Z, Sparrow DB, Whitelaw N, Craig JM, Apedaile A, Hilton DJ, Dunwoodie SL, Brockdorff N, Kay GF, Whitelaw E: SmcHD1, containing a structural-maintenance-of-chromosomes hinge domain, has a critical role in × inactivation. Nat Genet. 2008, 40: 663-669. 10.1038/ng.142.PubMedView ArticleGoogle Scholar
- Ashe A, Morgan DK, Whitelaw NC, Bruxner TJ, Vickaryous NK, Cox LL, Butterfield NC, Wicking C, Blewitt ME, Wilkins SJ, Anderson GJ, Cox TC, Whitelaw E: A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development. Genome Biol. 2008, 9: R182-10.1186/gb-2008-9-12-r182.PubMedPubMed CentralView ArticleGoogle Scholar
- Li E, Bestor TH, Jaenisch R: Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992, 69: 915-926. 10.1016/0092-8674(92)90611-F.PubMedView ArticleGoogle Scholar
- Stopka T, Skoultchi AI: The ISWI ATPase Snf2 h is required for early mouse development. Proc Natl Acad Sci USA. 2003, 100: 14097-14102. 10.1073/pnas.2336105100.PubMedPubMed CentralView ArticleGoogle Scholar
- Peters AH, O'Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A, Opravil S, Doyle M, Sibilia M, Jenuwein T: Loss of the Suv39 h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell. 2001, 107: 323-337. 10.1016/S0092-8674(01)00542-6.PubMedView ArticleGoogle Scholar
- Lagger G, O'Carroll D, Rembold M, Khier H, Tischler J, Weitzer G, Schuettengruber B, Hauser C, Brunmeir R, Jenuwein T, Seiser C: Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J. 2002, 21: 2672-2681. 10.1093/emboj/21.11.2672.PubMedPubMed CentralView ArticleGoogle Scholar
- Okano M, Bell DW, Haber DA, Li E: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999, 99: 247-257. 10.1016/S0092-8674(00)81656-6.PubMedView ArticleGoogle Scholar
- Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ: SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 2002, 16: 919-932. 10.1101/gad.973302.PubMedPubMed CentralView ArticleGoogle Scholar
- Schultz DC, Friedman JR, Rauscher FJ: Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD. Genes Dev. 2001, 15: 428-443. 10.1101/gad.869501.PubMedPubMed CentralView ArticleGoogle Scholar
- Keays DA, Clark TG, Campbell TG, Broxholme J, Valdar W: Estimating the number of coding mutations in genotypic and phenotypic driven N-ethyl-N-nitrosourea (ENU) screens: revisited. Mamm Genome. 2007, 18: 123-124. 10.1007/s00335-006-0065-z.PubMedView ArticleGoogle Scholar
- Cammas F, Mark M, Dolle P, Dierich A, Chambon P, Losson R: Mice lacking the transcriptional corepressor TIF1beta are defective in early postimplantation development. Development. 2000, 127: 2955-2963.PubMedGoogle Scholar
- Barness LA, Opitz JM, Gilbert-Barness E: Obesity: genetic, molecular, and environmental aspects. Am J Med Genet A. 2007, 143A: 3016-3034. 10.1002/ajmg.a.32035.PubMedView ArticleGoogle Scholar
- Santos SH, Fernandes LR, Mario EG, Ferreira AV, Porto LC, Alvarez-Leite JI, Botion LM, Bader M, Alenina N, Santos RA: Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism. Diabetes. 2008, 57: 340-347. 10.2337/db07-0953.PubMedView ArticleGoogle Scholar
- Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O'Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007, 448: 553-560. 10.1038/nature06008.PubMedPubMed CentralView ArticleGoogle Scholar
- Jakobsson J, Cordero MI, Bisaz R, Groner AC, Busskamp V, Bensadoun JC, Cammas F, Losson R, Mansuy IM, Sandi C, Trono D: KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. Neuron. 2008, 60: 818-831. 10.1016/j.neuron.2008.09.036.PubMedView ArticleGoogle Scholar
- Blake WJ, Balazsi G, Kohanski MA, Isaacs FJ, Murphy KF, Kuang Y, Cantor CR, Walt DR, Collins JJ: Phenotypic consequences of promoter-mediated transcriptional noise. Mol Cell. 2006, 24: 853-865. 10.1016/j.molcel.2006.11.003.PubMedView ArticleGoogle Scholar
- Blake WJ, M KA, Cantor CR, Collins JJ: Noise in eukaryotic gene expression. Nature. 2003, 422: 633-637. 10.1038/nature01546.PubMedView ArticleGoogle Scholar
- Gordon AJ, Halliday JA, Blankschien MD, Burns PA, Yatagai F, Herman C: Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network. PLoS Biol. 2009, 7: e44-10.1371/journal.pbio.1000044.PubMedView ArticleGoogle Scholar
- Choi JK, Kim YJ: Intrinsic variability of gene expression encoded in nucleosome positioning sequences. Nat Genet. 2009, 41: 498-503. 10.1038/ng.319.PubMedView ArticleGoogle Scholar
- Lam FH, Steger DJ, O'Shea EK: Chromatin decouples promoter threshold from dynamic range. Nature. 2008, 453: 246-250. 10.1038/nature06867.PubMedPubMed CentralView ArticleGoogle Scholar
- Raser JM, O'Shea EK: Control of stochasticity in eukaryotic gene expression. Science. 2004, 304: 1811-1814. 10.1126/science.1098641.PubMedPubMed CentralView ArticleGoogle Scholar
- Raj A, Rifkin SA, Andersen E, van Oudenaarden A: Variability in gene expression underlies incomplete penetrance. Nature. 2010, 463: 913-918. 10.1038/nature08781.PubMedPubMed CentralView ArticleGoogle Scholar
- Waddington CH: Canalization of development and the inheritance of acquired characters. Nature. 1942, 150: 563-565. 10.1038/150563a0.View ArticleGoogle Scholar
- The Jackson Laboratory. [http://www.jax.org/]
- enuMutRat on zeon: Estimating the probability of ENU-induced coding mutations. [http://zeon.well.ox.ac.uk/git-bin/enuMutRat]
- NCBI Gene Expression Omnibus. [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23512]
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.