Epigenetic regulation is fundamental to the existence of multicellular organisms, is essential for growth and development of eukaryotic organisms and is also responsible for the maintenance, and reversal of non genetic cellular memory that records developmental and environmental cues (Shilatifard, 2006; Suzuki and Bird, 2008; Bonasio et al., 2010). Epigenetic alterations during development can lead to cancer, neurological disorders, heart disease, and other degenerative conditions (Jiang et al., 2004). Epigenetic genome regulation involves enzyme-mediated chemical modifications of DNA and its associated chromatin proteins (Feng et al., 2010), such modifications include cytosine methylation, post-translational modifications of histone (tails and cores), incorporation of histone variants, the positioning of nucleosomes and the function of noncoding RNAs (Feng et al., 2010; Henderson and Jacobsen, 2007; Law and Jacobsen, 2009). Paramutation
Paramutation is the epigenetic transfer of information from one allele (or homologous sequence) to another that establishes a state of gene expression that is heritable through mitosis and meiosis for generations. Paramutation has been reported between alleles of a gene, between two transgenes, or one transgene and one endogenous gene (Chandler, 2004; Chandler and Stam, 2004; Chandler, 2010). Paramutation was discovered in maize in the 1950s by Alexander Brink (Brink, 1956). The most thoroughly analyzed natural examples of paramutation involve four different loci, r1, b1, pl1 and p1 in maize (Brink, 1956; Chandler et al., 2000; Chandler and Stam, 2004; Coe, 1959; Hollick et al., 1995; Sidorenko and Peterson, 2001), which encode transcription factors that activate the biosynthesis of colored flavonoid pigments (Dooner et al., 1991; Goff et al., 1992). These pigments accumulate in most mature plant tissues and are not essential for plant survival (Dooner et al., 1991). One possible explanation for the discovery of paramutation at these loci is that the amount of pigment observed exquisitely reflects the expression levels of these regulatory genes such that even subtle (i.e. two fold) changes in expression are visually distinguishable (Hollick et al., 2000; Patterson et al., 1993). Paramutation-related processes have also been described in fungi and animals (Chandler, 2007; Chandler and Stam, 2004; Cuzin et al., 2008; Suter and Martin, 2009). The study of paramutation should shed light on fundamental mechanism for gene regulation (Suter and Martin,2009), potential roles of paramutation and consequences include that paramutation provides an adaptive mechanism in the absence of genetic variation through the transfer of favorable expression states to progeny, that paramutation could be a mechanism for establishing functional homozygosity in polyploids, and that it might function in inbreeding depression and hybrid vigor or inheritance associated with complex human diseases (Chandler, 2007; Suter and Martin, 2009). Independent of paramutation’s function or frequency, our understanding of its mechanisms should shed light on potentially novel mechanisms for transmitting epigenetic information across generations (Chandler, 2010). Paramutation at the b1 locus in maize Paramutation at the b1 locus in maize is the most stable and penetrant paramutation natural system described to date (Chandler et al., 2000; Chandler and Stam, 2004; Stam, 2009; Chandler, 2010; Arteaga-Vazquez and Chandler, 2010). The b1 locus encodes a transcription factor required to activate the anthocyanin biosynthetic pathway, resulting in purple pigmentation of most mature plant tissues. The amount of transcription of b1 quantitatively correlates with the pigment levels in plants providing a powerful system for investigating paramutation (Patterson et al., 1993). When an allele’s expression is changed (i.e. sensitive to paramutation), it is referred to as paramutable. The allele that induces the change in expression is referred to as paramutagenic. Following paramutation, previously paramutable (sensitive alleles) are termed paramutant (or paramutated). Paramutated alleles are capable of inducing paramutation; they become paramutagenic. Most alleles do not participate in paramutation (they neither induce a change nor are they changed in the presence of a paramutagenic allele); these types of allele are referred to as neutral (Chandler, 2004). When the highly transcribed, darkly pigmented B-I allele is crossed with B’, which is lightly pigmented and has much lower transcription, paramutation always occurs: B-I is always changed into B’. This new B’ allele (designated as B’*) is as paramutagenic as the parental B’ allele. The key sequences required for b1 paramutation are seven direct tandem repeats of noncoding DNA located ~100 kb upstream of the b1 transcription start site (Stam et al., 2002a; Stam et al., 2002b). Each of the repeat units is 853 bp and both B-I and B’ carry seven tandem repeats, whereas neutral alleles have a single copy of the repeat unit (Stam et al., 2002a; Stam et al., 2002b). Recent results indicate that paramutation involves RNA mediated heritable chromatin changes and a number of genes implicated in RNA-based transcriptional silencing and chromatin modification pathways (Arteaga-Vazquez and Chandler, 2010). Genes required for paramutation
Several genes required for paramutation have been identified through forward genetic screens. The mediator of paramutation (mop) genes (Dorweiler et al., 2000; Sidorenko et al., 2009) and the required to maintain repression (rmr) genes (Erhard et al., 2009; Hollick and Chandler, 2001; Stonaker et al., 2009) have been isolated using the b1 and pl1 systems, respectively. All except one (rmr2) of the cloned genes show homology with proteins that mediate RNA-based transcriptional silencing (Arteaga-Vazquez and Chandler, 2010). In Arabidopsis, this pathway is referred to as RNA-directed DNA methylation (RdDM) and it involves the production of 24-nt short interfering RNAs (siRNAs) that are able to guide de novo cytosine methylation and histone modifications characteristic of heterochromatin, in the homologous genomic regions (Law and Jacobsen, 2010; Matzke et al., 2009). In plants, the vast majority of cellular small RNAs correspond to 24-nt siRNAs derived from transposons that direct the establishment of heterochromatin at these repetitive regions (Matzke et al., 2009; Nobuta et al., 2008). mop1 encodes the maize ortholog of Arabidopsis RNA-dependent RNA polymerase 2 (RDR2) (Alleman et al., 2006; Nobuta et al., 2008), rmr1 encodes a SNF2-like chromatin remodeling factor related to Arabidopsis DRD1 and CLSY1 (Hale et al., 2007), rmr6 is an ortholog of the large subunit of Arabidopsis Pol IV (NRPD1) (Erhard et al., 2009); mop2 and rmr7 are paralogs of NRPD2/NRPE2, the shared second largest subunit of Pol IV and Pol V in Arabidopsis (Sidorenko et al., 2009; Stonaker et al., 2009). Pol-IV and Pol-V are Pol-II related enzyme complexes, limited to the plant kingdom, which are required for RNA-mediated transcriptional silencing of transposons and transgenes (Pikaard et al., 2008; Pikaard and Tucker, 2009). While it is clear that multiple genetic components are in common between paramutation in maize and RdDM in Arabidopsis, there are unique and distinct properties of paramutation that have yet to be demonstrated by RdDM. For example, the silent state established by RdDM is not heritable when separated from the locus driving the production of siRNAs, but most importantly, they are not paramutagenic, they cannot silence active alleles (Chan et al., 2006; Chandler, 2007; Henderson and Jacobsen, 2007; Matzke et al., 2009). Research at the epilab The existence of paramutation challenges traditional paradigms for how genes are regulated and inherited. Although the sequences required for paramutation have been identified for two loci (b1 and p1) and recent results demonstrate a role for RNA and the requirement of components of an RNA-based transcriptional silencing pathway (Arteaga-Vazquez and Chandler, 2010), many questions remain. How do homologous sequences communicate to establish distinct states of expression? Once established, how are the new expression states maintained through subsequent mitotic and meiotic divisions in the absence of DNA sequence changes? What is the heritable molecular mark or signal? (Chandler, 2007). In our lab, we are addressing the following key questions that will shed light on the molecular mechanisms mediating transgenerational epigenetic inheritance and on fundamental aspects of developmental control of gene expression: 1) When and where does the trans-comunication that establishes paramutation occur? 2) What is the developmental role of genes required for paramutation during gamete formation and embryogenesis? 3) What is the widespread influence of paramutation in plants and other multicellular eukaryotes? References Shilatifard, A. (2006). Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annual Review Biochemical 75, 243-269. Suzuki, M.M., and Bird, A. (2008). DNA methylation landscapes: provocative insights from epigenomics. Nature Review Genetics 9, 465-476. Bonasio, R., Tu, S., and Reinberg, D. (2010) Molecular signals of epigenetic states. Science, 330, 612-616. Jiang, Y.H., Bressler, J., and Beaudet, A.L. (2004). Epigenetics and human disease. Annu Rev Genomics Hum Genet 5, 479-510. Feng, S., Jacobsen, S.E., and Reik, W.(2010). Epigenetic reprogramming in plant and animal development. Science 330, 622-627. Henderson, I.R., and Jacobsen, S.E. (2007). Epigenetic inheritance in plants. Nature 447, 418-424. Law, J.A., and Jacobsen, S.E. (2009). Molecular biology. Dynamic DNA methylation. Science 323, 1568-1569. Chandler, V.L. (2010). Paramutation's properties and puzzles. Science 330, 628-629. Chandler, V.L. (2004). Poetry of b1 paramutation: cis- and trans-chromatin communication. Cold Spring Harbor symposia on Quantitative Biology 69, 355-361. Chandler, V.L., and Stam, M. (2004). Chromatin conversations: mechanisms and implications of paramutation. Nature Review Genetics 5, 532-544. Brink, R.A. (1956). A Genetic Change Associated with the R Locus in Maize Which Is Directed and Potentially Reversible. Genetics 41, 872-889. Chandler, V.L., Eggleston, W.B., and Dorweiler, J.E. (2000). Paramutation in maize. Plant Molecular Biology 43, 121-145. Coe, E.H. (1959). A Regular and Continuing Conversion-Type Phenomenon at the B Locus in Maize. Proceedings of the National Academy of Sciences of the United States of America 45, 828-832. Hollick, J.B., Patterson, G.I., Coe, E.H., Jr., Cone, K.C., and Chandler, V.L. (1995). Allelic interactions heritably alter the activity of a metastable maize pl allele. Genetics 141, 709-719. Sidorenko, L.V., and Peterson, T. (2001). Transgene-induced silencing identifies sequences involved in the establishment of paramutation of the maize p1 gene. The Plant Cell 13, 319-335. Dooner, H.K., Robbins, T.P., and Jorgensen, R.A. (1991). Genetic and developmental control of anthocyanin biosynthesis. Annual Review of Genetics 25, 173-199. Goff, S.A., Cone, K.C., and Chandler, V.L. (1992). Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes & Development 6, 864-875. Hollick, J.B., Patterson, G.I., Asmundsson, I.M., and Chandler, V.L. (2000). Paramutation alters regulatory control of the maize pl locus. Genetics 154, 1827-1838. Patterson, G.I., Thorpe, C.J., and Chandler, V.L. (1993). Paramutation, an allelic interaction, is associated with a stable and heritable reduction of transcription of the maize b regulatory gene. Genetics 135, 881-894. Chandler, V.L. (2007). Paramutation: from maize to mice. Cell 128, 641-645. Cuzin, F., Grandjean, V., and Rassoulzadegan, M. (2008). Inherited variation at the epigenetic level: paramutation from the plant to the mouse. Current Opinion in Genetics & Development 18, 193-196. Suter, C.M., and Martin, D.I. (2009). Paramutation: the tip of an epigenetic iceberg?. Trends Genet. 2010 Jan;26(1):9-14. Arteaga-Vazquez, M.A., and Chandler, V.L. (2010). Paramutation in maize: RNA mediated trans-generational gene silencing. Current Opinion in Genetics & Development 20, 156-163. Stam, M., Belele, C., Dorweiler, J.E., and Chandler, V.L. (2002a). Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation. Genes & development 16, 1906-1918. Stam, M., Belele, C., Ramakrishna, W., Dorweiler, J.E., Bennetzen, J.L., and Chandler, V.L. (2002b). The regulatory regions required for B' paramutation and expression are located far upstream of the maize b1 transcribed sequences. Genetics 162, 917-930. Stam, M. (2009). Paramutation: a heritable change in gene expression by allelic interactions in trans. Molecular Plant 2, 578-588. Dorweiler, J.E., Carey, C.C., Kubo, K.M., Hollick, J.B., Kermicle, J.L., and Chandler, V.L. (2000). mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci. The Plant cell 12, 2101-2118. Sidorenko, L., Dorweiler, J.E., Cigan, A.M., Arteaga-Vazquez, M., Vyas, M., Kermicle, J., Jurcin, D., Brzeski, J., Cai, Y., and Chandler, V.L. (2009). A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes. PLoS genetics 5, e1000725. Erhard, K.F., Jr., Stonaker, J.L., Parkinson, S.E., Lim, J.P., Hale, C.J., and Hollick, J.B. (2009). RNA polymerase IV functions in paramutation in Zea mays. Science 323, 1201-1205. Hollick, J.B., and Chandler, V.L. (2001). Genetic factors required to maintain repression of a paramutagenic maize pl1 allele. Genetics 157, 369-378. Stonaker, J.L., Lim, J.P., Erhard, K.F., Jr., and Hollick, J.B. (2009). Diversity of Pol IV function is defined by mutations at the maize rmr7 locus. PLoS Genetics 5, e1000706. Matzke, M., Kanno, T., Daxinger, L., Huettel, B., and Matzke, A.J. (2009). RNAmediated chromatin-based silencing in plants. Current Opinion in Cell Biology 21, 367-376. Nobuta, K., Lu, C., Shrivastava, R., Pillay, M., De Paoli, E., Accerbi, M., Arteaga-Vazquez, M., Sidorenko, L., Jeong, D.H., Yen, Y., et al. (2008). Distinct size distribution of endogeneous siRNAs in maize: Evidence from deep sequencing in the mop1-1 mutant. Proceedings of the National Academy of Sciences of the United States of America 105, 14958-14963. Alleman, M., Sidorenko, L., McGinnis, K., Seshadri, V., Dorweiler, J.E., White, J., Sikkink, K., and Chandler, V.L. (2006). An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 442, 295-298. Hale, C.J., Stonaker, J.L., Gross, S.M., and Hollick, J.B. (2007). A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize. PLoS Biology 5, e275. Pikaard, C.S., Haag, J.R., Ream, T., and Wierzbicki, A.T. (2008). Roles of RNA polymerase IV in gene silencing. Trends in Plant Science 13, 390-397. Pikaard, C.S., and Tucker, S. (2009). RNA-silencing enzymes Pol IV and Pol V in maize: more than one flavor? PLoS genetics 5, e1000736. Chan, S.W., Zhang, X., Bernatavichute, Y.V., and Jacobsen, S.E. (2006). Two-step recruitment of RNA-directed DNA methylation to tandem repeats. PLoS biology 4, e363.