Showing papers by "Richard A. Jorgensen published in 2000"

Silencing Morpheus awakens transgenes.

Abstract: Forty years ago, R. Alexander Brink1 first proposed that in addition to their genetic function, chromosomes possess a paragenetic function. By paragenetics, he was referring to chromosomally based mechanisms in epigenetics—the catchall term we now use for heritable changes in gene expression associated with mechanisms superimposed upon an unchanged primary DNA sequence. One example of epigenetic regulation with significant impact on biotechnology is transgene silencing. In many animal and plant systems cytosine methylation is a reversible covalent modification often associated with transgene silencing2. However, epigenetic gene regulation occurs in organisms without DNA methylation3, indicating that methylationindependent mechanisms exist for heritably perpetuating altered transcriptional states. A recent article in Nature describes a very interesting gene, MOM (Morpheus molecule, for the Greek god of dreams), which is required to maintain transcriptional silencing of a heavily methylated transgene array in Arabidopsis4. When this gene is disrupted, the transgene array becomes transcriptionally active, yet the extensive cytosine methylation associated with the array remains. This work, together with previous studies on another mutant in Arabidopsis (antisense MET1), suggests that extensive DNA methylation is neither required nor sufficient for transgene silencing (summarized in Fig. 1). Transgenes or endogenous genes, organized in direct or inverted repeats, often become transcriptionally silenced2. In organisms with DNA methylation mechanisms, the inactivation often correlates with extensive DNA methylation relative to the transcriptionally active state. In addition to cis-inactivation, some sequences can transcriptionally inactivate homologous sequences in allelic or nonallelic positions, and this inactivation is heritable in the absence of the inducing transgene. These trans-inactivation events are very similar to cases of endogenous gene silencing referred to as paramutation5. Mutations in MOM could provide an important tool for genetic engineering, as silent transgenes are activated in the absence of pleiotropic developmental abnormalities. MOM efficiently reactivates two different transcriptionally silent transgenes, suggesting that the MOM protein maintains transcriptional silencing in Arabidopsis4. If mutations in the MOM ortholog in crop species behave similarly, introducing transgenes into mutant lines could mitigate the problem of transgene silencing. Efficient resilencing of the transgene in heterozygous MOM/mom plants4 will require that mom be homozygous in marketable crop varieties. The MOM gene is the first known molecular component essential for transcriptional silencing of transgenes that does not affect DNA methylation levels. All other mutants that relieve transcriptional silencing also have reduced levels of DNA methylation within the transgene locus6,7. The putative protein sequences of the two silencing mutants that have been cloned provide a link with chromatin remodeling. The predicted MOM gene product is a 2001-amino acid novel nuclear protein that has a region of similarity to the C-terminal portion of the putative helicase region of SWI2/SNF2 DNAdependent ATPases. Another gene in Arabidopsis that is required for transgene silencing7, DDM1, is also a member of the SWI2/SNF2 family of DNA-dependent ATPases8. DDM1 is most similar to the subset of putative helicases involved in chromatin remodeling8, and it shares sequence similarity throughout a larger region of SWI2/SNF2 relative to MOM4,8. DDM1 was originally identified by a screen for mutants that exhibited reduced methylation in the highly repeated centromeric and ribosomal sequences9. Multiple alleles of ddm1 were subsequently isolated as mutants that activate a transcriptionally silent transgene7. In ddm1 mutants, transgene activation correlated with decreased methylation7. Whereas MOM plants have normal developmental phenotypes after nine generations of inbreeding4, plants homozygous for ddm1 for several generations frequently show developmental abnormalities that when outcrossed segregate as new mutations independent of ddm110. As many of these mutations are semidominant and unstable, they are likely to represent epimutations that result from the inappropriate regulation of particular genes. Similarly, reduced expression of the major DNA methyltransferase in Arabidopsis, MET1, by means of antisense, also produces plants with abnormal development11. When antisense MET1 plants were tested for relief of transgene silencing, DNA methylation of the transgene was significantly reduced, but the transgene remained silent (see Fig. 1)7. The observation that DNA methylation is neither sufficient (mom) nor required (antisense MET1) for the maintenance of transgene silencing raises the issue of what aspects of chromatin are mediating silencing. These mutants are an excellent resource for addressing this issue. Silencing Morpheus awakens transgenes

Directed cell-to-cell movement of functional proteins: do transcription factors double as signal molecules in plants?

Abstract: Cell fate in plants is predominantly determined by position during development. Jorgensen discusses a new mechanism by which cells can regulate gene expression in neighboring cells. Several lines of evidence suggest that transcription factors expressed in one cell can traffic through plasmodesmata to regulate gene expression in adjacent cells that are not expressing the transcription factor themselves. In plants, some transcription factors appear to be intercellular signaling molecules, as well as intracellular regulators of gene expression.