David I. K. Martin

Epigenetic inheritance at the agouti locus in the mouse

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( Avy/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene ( A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A vy dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

Germline epimutation of MLH1 in individuals with multiple cancers

Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.

Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine Avy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the Avy allele. We find that the somatic epigenetic state of Avy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts Avy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the Avy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mothers diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change.

Retrotransposons as epigenetic mediators of phenotypic variation in mammals

Phenotypic variation in mammals is frequently attributed to the action of quantitative trait loci (QTL) or the environment, but may also be epigenetic in origin. Here we consider a mechanism for phenotypic variation based on interference of transcription by somatically active retrotransposons. Transcriptionally competent retrotransposons may number in the tens of thousands in mammalian genomes. We propose that silencing of retrotransposons occurs by cosuppression during early embryogenesis, but that this process is imperfect and produces a mosaic pattern of retrotransposon expression in somatic cells. Transcriptional interference by active retrotransposons perturbs expression of neighboring genes in somatic cells, in a mosaic pattern corresponding to activity of each retrotransposon. The epigenotype of retrotransposon activity is reset in each generation, but incomplete resetting can lead to heritable epigenetic effects. The stochastic nature of retrotransposon activity, and the very large number of genes that may be affected, produce subtle phenotypic variations even between genetically identical individuals, which may affect disease risk and be heritable in a non-mendelian fashion.

Nuclear compartmentalization and gene activity

The regulated expression of genes during development and differentiation is influenced by the availability of regulatory proteins and accessibility of the DNA to the transcriptional apparatus. There is growing evidence that the transcriptional activity of genes is influenced by nuclear organization, which itself changes during differentiation. How do these changes in nuclear organization help to establish specific patterns of gene expression?

A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin

To explore the mechanism by which enhancers maintain gene expression, we have assessed the ability of an enhancer and derivative mutants to influence silencing and nuclear location of a transgene. Using site-specific recombination to place different constructs at the same integration sites, we find that disruption of core enhancer motifs impairs the enhancers ability to suppress silencing. FISH analysis reveals that active transgenes linked to a functional enhancer localize away from centromeres. However, enhancer mutations that result in increased rates of transgene silencing fail to localize the transgene away from centromeric heterochromatin, even when the transgene is in an active state. These mutations thus dissociate transcriptional activity and subnuclear location. Together, our results suggest that the functional enhancer antagonizes gene silencing by preventing localization of a gene near centromeric heterochromatin.

Notch1 and Notch2 Inhibit Myeloid Differentiation in Response to Different Cytokines

ABSTRACT We have compared the ability of two mammalian Notch homologs, mouse Notch1 and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.

The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

Hypomethylation of L1 retrotransposons in colorectal cancer and adjacent normal tissue

Background and aimsMalignant cells often exhibit perturbations in the pattern of cytosine methylation. Hypermethylation of CpG islands has been extensively documented, but genome-wide hypomethylation is also a common feature of malignant cells. The bulk of cytosine methylation in the mammalian genome occurs on repetitive elements. This study analysed the methylation status of L1 retrotransposons in colorectal cancer.Patients and methodsMethylation-sensitive Southern blotting was used to determine L1 promoter methylation in colon tumours, adjacent normal tissue, and normal colonic mucosa from healthy individuals.ResultsHypomethylation of L1 promoter sequences was detected in all tumours but was also detected in the histologically normal colonic mucosa of 6 of 19 cancer patients, even at a considerable distance from the tumour. L1 hypomethylation was not detected in matched normal peripheral blood, lymph node or smooth muscle tissue from cancer patients or in the colonic mucosa of 14 healthy individuals. We also assayed for the total proportion of methylated CpG in normal bowel specimens from normal and colon cancer patients. Normal mucosa from cancer patients exhibited lower levels of genomic methylation than the mucosa from healthy individuals, and levels were significantly lower in those patients exhibiting L1 promoter hypomethylation.ConclusionThese results suggest that genomic hypomethylation is an early event in tumourigenesis. Progressive demethylation of L1 promoter sequences could lead to disturbance of normal gene expression and facilitate the process of neoplastic progression.

To be or not to be active: the stochastic nature of enhancer action.

Transcriptional enhancers are traditionally considered to regulate the rate at which a linked promoter transcribes mRNA, but recent experiments suggest a reevaluation of this model is necessary. Single-cell assays of transgenes reveal that enhancers increase the probability that a reporter gene will be active, but have little or no effect on the transcription rate once a gene has been activated. These results raise the question of how enhancers affect gene expression in their native contexts. A simple interpretation is that enhancers act in a stochastic fashion to increase the probability that a regulated gene will be transcribed; such a model is compatible with programs of cell differentiation in which multiple similar cells subject to similar environmental stimuli do not respond uniformly.