Transgene-induced gene silencing Recommended article: The Plant Journal Volume 16 Issue 6 Page December 1998

Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. Matzke et al. (1989) EMBO J. 8: Doubly-transformed tobacco plants were obtained following sequential transformation steps using two T-DNAs encoding different selection and screening markers: T-DNA-I encoded kanamycin resistance and nopaline synthase; T-DNA-II encoded hygromycin resistance and octopine synthase. A genetic analysis of the inheritance of the selection and screening marker genes in progeny of the doubly- transformed plants revealed that the expression of T-DNA-I genes was often suppressed. This suppression could be correlated with methylation in the promoters of these genes. Surprisingly, both the methylation and inactivation of T- DNA-I genes occurred only in plants containing both T-DNAs: when self- fertilization or backcrossing produced progeny containing only T-DNA-I, expression of the genes on this T-DNA was restored and the corresponding promoters were partially or completely de-methylated. These results indicated that the presence of one T-DNA could affect the state of methylation and expression of genes on a second, unlinked T-DNA in the same genome.

Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. Matzke et al. (1989) EMBO J. 8: NOS promoter NOS transcription terminator 35S promoter and 35S transcription terminator Homologous regions NT= non-transformed ST= single transformed plant with T-DNA I RO-1 to 4 = doubly transformed plants

We attempted to over-express chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale non-clonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germina1 stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the leve1 of the mRNA produced by this gene was reduced 50-fold from wild- type levels. Somatic reversion of plants with white flowers to phenotypically parenta1 violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co- suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation. lntroduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes Ín trans. Carolyn Napoli et al. The Plant Cell, Vol. 2, , April 1990

Transgenic line 1 Transgenic line 2 Transgenic line 3 Transgenic line 4 Phenotypes of Chimeric CHS Transgenotes and Variations among Flowers on Single Plants. A control (parental) flower is shown along with four different CHS transgenotes. Napoli et al. The Plant Cell, Vol. 2, , April 1990 Transgene: 35S pro::CHS::nos3’

Van der Krol et al. The Plant Cell, Vol. 2, , April 1990 To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits. Transgenes: 35S pro::CHS or DFR::nos3’

Van der Krol et al. The Plant Cell, Vol. 2, , April 1990 CHS DFR

Silencing of transgenes Epigenetics: study of mitotically/ meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence. Modes of silencing: Transcriptional silencing (TGS) a. cis TGS b. trans TGS Post-transcriptional silencing (PTGS) a. cis PTGS b. trans PTGS Transgene silencing falls in the field of epigenetics Gene mRNA protein TGS PTGS

TGS: cis inactivation 1. Methylation spread Plant genomic DNA consists of hypo-methylated and hyper-methylated regions. When one or multiple copies of a transgene integrate at a locus located in or next to the hyper-methylated region, the transgene can undergo TGS due to the spread of methylation. 2. Repeat induced silencing (RIGS) Multiple copies integrated into hypo-methylated region can undergo TGS due to the de novo methylation of the repeats. This resembles local heterochromatin formation and silencing in Drosophila. As a result, the repeats possess (in plants and Drosophila) increased resistance to both DNaseI and micrococcal nuclease. Note: Increased resistance of a locus to both DNase I and micrococcal nuclease indicates DNA methylation and chromatin condensation.

The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. F. Prols and P. Meyer. Plant Journal (1992) 2, The regions of integration of a transferred DNA-fragment from three transgenic Petunia hybrida plants were analyzed for their influence on the expression of the foreign DNA. Each of the three transformants, lines 16, 17 and 24, contained a fragment of a plasmid on which two genes were located, an npt II gene which renders the plants resistant to kanamycin and the A1 gene from Zea mays, a visible marker gene that leads to the production of a brick red anthocyanin pigment in the flowers. Inactivation of both genes in line 16 is associated with integration into a region of highly repetitive DNA, while the integration sites of the other two lines were essentially unique. The integration regions of lines 17 and 24, both of which show expression of the foreign genes at characteristically different intensities, showed a distinct methylation pattern that was stably conserved for these regions in both transgenic and wild-type plants. The characteristic methylation pattern of the two integration regions was also imposed on the border region of the integrated fragments and might thus be responsible for the differences in the intensity of gene expression observed among the two lines. A case of methylation spread

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin. RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration FEI YE AND ETHAN R. SIGNER Proc. Natl. Acad. Sci. USA Vol. 93, pp –10886, October 1996 A case of de novo methylation

Silencing was observed when transgene derived from a monocot was inserted into dicot, whereas it was not observed with the corresponding dicot gene, suggesting that strong discrepancy between DNA composition and the genomic integration site can be recognized by the cellular machinery leading to the specific methylation and silencing of the transgene. Elomaa et al. (1995) Mol Gen Genom 248:

Transcriptional trans Inactivation

TGS: trans inactivation (allelic) active silent Unidirectional effect of one locus on another This phenomenon resembles a natural epigenetic phenomenon called paramutation.

Paramutation Paramutator (inactive) alleles inhibit paramutable (active) alleles, which themselves become paramutator. Paramutation indicates that homologous genes can exchange information in somatic cells. Active gene Silenced gene Silenced homologous gene Active homologous gene

Ectopic (non-allelic) trans-inactivation Park et al. (1996) Plant J 9: 183

PTGS: cis-inactivation 1. PTGS cis inactivation has been observed when foreign transgenes were introduced under the control of strong viral promoters such as 35S promoter. 2. PTGS occurred more efficiently in haploid and homozygous plants than in hemizygous plants, indicating a transgene dose effect. 3. PTGS occurred in larger proportion of transgenic plants containing 35S promoter with a double enhancer as compared to original 35S promoter. This suggests that transcription above a threshold level triggered mRNA degradation. But later it was found that the level of transcription in silenced lines was not always higher than the level in expressed lines, thus suggesting that some other parameter is responsible. The presence of repeats at the integration locus was suggested to play the role in PTGS.

Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes Common feature: methylation of coding sequence in the silent gene

Wassenegger et al.(1994) Cell 76:567 RNA-directed de novo methylation of genomic sequences in plants One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation. Southern analysis of the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered. Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants. These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.

PTGS: trans-inactivation (co-suppression) One of the first few cases of transgene silencing described in literature falls in this category. (Napoli et al Plant Cell 2: ). PTGS was originally discovered as the reciprocal and coordinated silencing of transgenes and homologous host genes. eg., petunia plants transformed with 35S: CHS resulted in silencing of chalcone synthase genes. Co-suppression occurs more efficiently in haploids and homozygous plant: suggesting gene dosage effect. Most of the PTGS cases consist of strong promoter indicating role of RNA threshold but weakly transcribed transgenes and promoterless transgenes have also been shown to silence homologous host gene. These cases consisted of transgene repeats Invoking the hypothesis of the role of abrerrant RNA or cRNA in PTGS.

Post-transcriptional virus resistance When the transgene which undergoes PTGS encodes part of the genome of a plant RNA virus, silenced transgenic plants become resistant against virus infection. In some cases virus resistance can be reached after a phase of sensitivity, a phenomenon called ‘recovery’. This means that silent state is not pre-established and only the transcription by virus triggers silencing and resistance.

The EMBO Journal (1997) 16, 4738–4745. Systemic acquired silencing: transgene- specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions

Transgene-induced gene silencing in plants can occur at the transcriptional or post-transcriptional level. TGS occurs mainly when multiple repeats of a transgene are inserted in the genome of transgenic plants. It correlates with condensation of chromatin and with methylation. The transfer of methylation and silencing from one locus to another clearly indicates that independent parts of the genome communicate and exchange information. This transfer may occur through direct DNA-DNA pairing. Alternatively, it could involve the production of diffusible RNA by one locus, leading to inactivation of homologous targets via an RNA-DNA interaction

Epigenetic modifications of duplicated sequences may be an important biological process because it might prevent pairing-mediated recombination. Either intra- or interchromosomal recombination between transgene repeats may be deleterious for the cell. Interactions between epigenetically induced methylated and structurally altered DNA molecules may not be recombination-proficient, thus preventing deleterious events.