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Transgenes have different states than endogenous genes in cells

Hervé Vaucheret’s team has demonstrated for the first time that the epigenetic system – and particularly histones – differentiates transgenes from endogenous genes. This discovery could explain how certain transgene RNA become silenced.
Hervé Vaucheret, research director at INRA’s Versailles-Grignon site, provides insight on the phenomenon.

In vivo visualisation of the chromatin of a rabbit embryo cell. © INRA, ADENOT Pierre
By Pascale Mollier
Updated on 05/28/2013
Published on 05/02/2013

What do you hope to achieve through this research?

Hervé Vaucheret: We’ve been asking ourselves a fundamental question for twenty years: What happens in a cell when a DNA fragment is inserted in the genome? The issue comes up when studying transgenic plants, but it also relates to physiological events such as the “jump” of a transposable element (1) or the insertion of a virus’s DNA into the host’s genome. In some cases, the cell uses specific mechanisms to silence the expression of the introduced sequence (see Insert 1). During transgenesis experiments, it is almost always possible to find a transformant that escapes silencing, resulting in the stable expression of the transgene. As such, these silencing phenomena are not considered as obstacles to transgenesis. This is why, in terms of biotechnology applications, our research is not very “marketable”, especially considering current political attitudes on GMOs. GMO promoters believe the process works and have little concern about what happens at the cellular level, while those who reject them are even less interested!

We are interested, because beyond what happens during transgenesis, our work contributes to understanding how silencing mechanisms are involved in regulating gene expression and how genomes properly function.

What have you found?

HV: We were particularly interested in PTGS, a form of silencing that does not affect transgene transcription but destroys the mRNA (messenger RNA). We had previously identified the cellular genes that control the degradation of transgenic RNA. However, we did not know why a sequence introduced as a transgene was recognised as foreign and set off PTGS, whereas the same sequence in its natural location on a genome never undergoes PTGS. For the first time, we have demonstrated the role that histones play in this recognition and identified a cellular protein that can differentiate transgenes from endogenous genes: the JMJ14 enzyme. It is a demethylase that acts on histone H3 when it is trimethyled on lysine 4. This form, also called marker H3K4me3 (see Insert 2), is known to be associated with an active state of gene transcription in several types of eukaryotes. The JMJ14 enzyme – which removes this histone’s methyl group – reduced transcription rate, thereby moderating the expression of endogenous genes. What we’ve demonstrated is that this enzyme does not act on transgenes, regardless of their sequences. As a result, it does not moderate transcription. This contributes to increasing the transgene’s transcription rate, which, beyond a certain threshold, sets off PTGS.

What do these results mean?

HV: As we discover these histones, we measure how important they are in regulating gene expression. From a broader perspective, we also look at determining the role of epigenetics – in other words, what lies outside of the DNA’s nucleotide sequence, such as the surrounding histones, the methylation on the DNA’s nucleotides, etc. You have to imagine that each DNA fragment has its own environment, which determines its expression. This environment is a combination of histone “marks”, meaning several histones with one or several groups – methyl, acetyl, etc. – on one or several strategic amino acids (for instance, lysine in position 4, 9, 27 or 36 for histone H3). This shows that this system offers multiple combination possibilities. Each DNA fragment is “indexed” by histone combination around it, which makes its expression strong, weak, or even silenced. This state is dynamic, because the histone markers can be modified at any time (e.g., by demethylation).

A transgene, when introduced into the genome, is a “naked” DNA fragment – without histones – and therefore has no state in the cell. It is probable that the cell attributes epigenetic markers to it by default. Our results show that when the cell does not block a trangene’s transcription by TGS, it actually tends to increase this transcription to encourage PTGS. We will now explore this hypothesis by looking for the epigenetic markers attributed to transgenes and which hinder action by the JMJ14 enzyme.

(1) Transposable elements, or transposons, are DNA sequences that can change position within the genome and sometimes multiply. They can have a mutagenic effect when they are introduced near genes.

Reference: Le Masson I., Jauvion V., Bouteiller N., Rivard M., Elmayan T., Vaucheret H. 2012. Mutations in the Arabidopsis H3K4me2/3 demethylase JMJ14 suppress Posttranscriptional Gene Silencing by decreasing transgene transcription. The Plant Cell 24, 3603-3612.

Scientific contact(s):

Associated Division(s):
Science for Food and Bioproduct Engineering, Plant Biology and Breeding
Associated Centre(s):

A two-tiered surveillance system: TGS and PTGS

Cells have a two-tiered mechanism which silences expression of a DNA sequence that is introduced into the cell’s genome:

- TGS (Transcriptional Gene Silencing) inhibits DNA transcription via epigenetic modifications, such as methylations of DNA or lysine 9 on histone H3.

- In PTGS (Post-Transcriptional Gene Silencing), the DNA is transcribed, but the mRNA is destroyed. This mechanism guides the formation of double-stranded RNA, which is subsequently processed into small RNA. These small RNA bind to the equivalent regions of the mRNA and bring with them a protein complex that causes degradation.

When the introduced DNA is not silenced by TGS, it can still be caught by PTGS. This is generally the case when DNA transcription is strong, which depends in part on its promoter sequence, insertion site and the histone markers that will associate with it.

Histone markers

Histones are small proteins that associate with DNA and influence its expression through its conformation, which conditions the accessibility to transcription factors. There are many different types of histones. They are categorised into four major classifications and several sub-classifications. Each histone presents a great many forms, called “markers” (e.g., methyls, acetyls). They are found at different degrees and located on different amino acids. There are multiple histone markers, but it appears that they are organised in a limited number of active combinations for transcription. Recent research (1) on Arabidopsis identified three major combinations: one stimulates gene transcription, another inhibits it, and the third silences the expression of transposable elements. For example, the first of these combinations is written: H3K4me3 (=trimethylation on lysine 4 of histone 3), H3K9me3, H3K36me3, H3K4me2, H3K56Ac, H2Bub. Modifications to histone markers (demethylation or deacetylation, etc.) can still affect the level of transcription. This system of combinations allows for a regulation of gene expression that is both extremely precise and very dynamic both in time and space.

 (1) François Roudier et al. 2011. Integrative epigenomic mapping defines four main chromatin states in Arabidopsis. The EMBO Journal 30, 1928–1938.