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EPIGENETICS
Cold Spring Harbor Symposium 69 Live:  
Dispatches

 

Sunday, June 6/Afternoon
EPIGENETIC TWISTS
Catherine Kidner

David Haig described epigenetics as 'anything that doesn't look like conventional genetics'.  This meeting has shown that recent work in epigenetics has been so fruitful that now we are close to a ‘conventional epigenetics’.  This afternoon's session, chaired by Bruce Stillman, dealt with phenomena both very old (paramutation) and very new (the hothead mutation) that are still definitely unconvential epigenetics.

A context for the variety of epigenetic effects discussed was provided by the first talk. David Haig of the department of Organismic and Evolutionary Biology at Harvard discussed how the meanings of 'epigenetic' have evolved. The earliest modern use of the term was by Conrad Waddington in 1942, he defined it as the causal mechanisms of development. The term was taken up by developmental biologists and used to describe both the unravelling of information and genetic interactions in the growing organism and the influences such as other organisms, physical surroundings, temperature and even gravity that cause variation.  

After the discovery of DNA the term was revived by Nanney  to refer to self perpetuating states of the cytoplasm as opposed to a totally DNA-based source for the phenotype. At a number of meetings in the late fifties the term and the phenomena to which it referred were debated.  It was eventually conceded that the nucleus was the main source of heritable information although 'not everything inherited is genetic', and there was discussion of a role for non-sequence based aspects of inheritance due to changes associated with modifications of DNA.  Our current use of the term stems from Robin Holliday who first used it in 1979 to refer to non-sequence changes in DNA and in 1987 published 'The inheritance of epigenetic defects' in Science.  

The definition of epigenetics as anything that appears non-mendelian is well suited to the subject of the second talk of the afternoon. Michel George of the University of Leige described the strange behavior of the callipyge (CLPG) mutation in sheep.  This mutation arose in the ram Solid Gold in Oklahoma in the 1980’s.  It was noticed that his offspring had particulary well developed buttocks, hence the name.  This is a very desirable trait in sheep and the lineage attracted the interest of animal breeders.  The callipyge phenotype showed an interesting polar over dominance: the phenotype was only apparent on inheritance of the mutant allele from the father, in the absence of a maternally inherited mutation.  George presented evidence that this effect may derive from a combination of imprinting and miRNA regulation.

By positional cloning, the mutation was localized to the DLK1-GTL2 domain, an area of about 1MB that includes 4 protein encoding genes and genes for 4 large noncoding RNAs.  This region is rich in pri-miRNA genes. The mutation itself is an A to G substitution in a conserved 12bp sequence that affects levels of expression of the protein coding genes - ectopic expression of DLK1 is responsible for the hypertrophic muscle phenotype.  Imprinting of the paternal chromosome blocks the expression of the protein coding genes but allows the expression of the noncoding RNA genes. The loss of the effect in homozygote mutants suggests that the noncoding RNAs are imprinted in the maternal allele and downregulate the protein coding transcripts. Computational analysis has suggested that one of the pri miRNAs may encode a regulator of DLK1 transcript. 

callipyge is a very recent phenomenon compared to paramatution of the B locus in maize described by Vicky Chandler of the University of Arizona, Tuscon.   Study of this effect dates back to the early days of the term 'epigenetics'.  Plants carrying B-I  produce copius dark anthocyanin pigments, while those carrying B' produce little anthocyanin.  B-I reverts to B' at a frequency of 0.1 to 10%.  Paramutaion refers to the ability of B' to convert B-I alleles to B'. This effect is notable for its stability and penetrance.  Sequencing of the locus revealed that B' and B-I alleles both had seven tandem repeats of a 853bp sequence 100kb upstream of the CDS for the B gene.  In alleles not susceptible to the paramutation (most B alleles) there was only a single 853bp sequence.  The extra repeats were required both for the high expression levels in B-I and the paramutation effect of B'. Methylation occurs in paramutaion but is a response to the silencing rather than a cause of it. Most excitingly Chandler described experiments that randomly inserted the repeats into the genome of a neutral line and showed that this could confer susceptibility even though the transgenes were unlinked to the B locus.  This supports an RNA mediated effect or long distance direct pairing.

The two following talks dealt with prions, which might be seen to fit the 'cytoplasmic inheritance' definition of epigenetics current in the 1950's.  Reed Wickner of NIH, Bethesda, described the characteristics of these proteins and their behavior:  The effect is non-chromosomal, 'cures' are reversible, overproduction of the protein leads to more prions and the prion phenotype requires both the chromosomal gene and the phenotypic prion.  He also described four different forms of prion, two bad, two good: those which form toxic amyloids, such as BSE; those that form inactive amyloids such as [URE3] which he described in detail; those that from active amyloids (Het-s); and a new form - the self activation enzyme such as b, 'crippled growth' in Podospora anseria and PaASK1, a MAP kinase kinase kinase.

The N terminus of Ure2P controls prion [URE3] formation.  The crystal structure of the amyloid showed that the N terminal domain formed b sheet filaments surrounded by the ‘blobs’ of the C terminal domain. Wickner also described experiments by Eric Ross  in his lab which showed that it is the amino acid composition rather than sequence that is important for prion formation.

A role for prions in normal development was suggested by Kausik Si, of Columbia University.  He has studied the role of the neuronal isoform of Cytoplasmic Polyadenylation Element Binding protein (CPEB) in activating transcription of genes required for synaptic facillitation. Like many prions, the N' terminus of the protein is rich in glutamine and aspargine. Assays in yeast using the b gal system showed that the protein exists in two distinct metastable forms, the active form being dominant. Using GFP fusions he showed that the active form is produced by overexpression, together with seratonin signaling, and exists as self perpetuating aggregates.

The most intriguing talk of the afternoon was Bob Pruitt of Purdue University's presentation of hothead (hth), an Arabidopsis mutation that causes organ fusion and changes in DNA sequence.  Homozygotes revert to wild type at a frequency from 1 to 10%.  This reversion is due not to 'classical epigenetic' effects, such as methylation of DNA or modifications in histone proteins, but to reversion of the DNA sequence to the sequence of the plant's wild type progenitors. Analysis of the effect of hth on a genome wide polymorphic background produced by a cross between two different ecotypes showed that the reversion was not limited to the HTH locus. SNPs were converted to parental at the same frequency as the hth mutation itself. This effect did, however, depend upon the hth/hth genotype. Conversions have been detected only in reproductive tissue and appear to occur preferentially in the male. The mechanism of this effect remains wholely unknown.  Pruitt suggested that sequence information might be retained by RNA templates cytoplasmically inherited from the wild type progenitors and amplified within the hth/hth plant. The discussion of this exciting effect was unfortunately cut short by the need to clear the hall for the next lecture but hopefully discussion over the evening will have suggested several possible explanations for this most un-mendelian of epigenetic effects.

Other Dispatches   
Symposium 69 Live   
Symposia Past (a bit of history and photographs from previous Symposia)
Online Symposium Volumes (searchable database of past Symposia volumes and currently received manuscripts)


Catherine Kidner
(Martienssen lab)

 

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