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University of Missouri s Jim Birchler on RNAi and His Science Paper

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At A Glance

Name: James Birchler

Position: Professor of biological sciences, University of Missouri

Background: Assistant professor, organismic and evolutionary biology, Harvard University —1985-1991; Postdoc, University of California, Berkeley — 1981-1995; Postdoc, Oak Ridge National Laboratory — 1977-1981; PhD, genetics, Indiana University at Bloomington — 1977; BS, botany and zoology, Eastern Illinois University — 1972

After stints at Harvard and UC, Berkeley, Jim Birchler landed at the University of Missouri where his lab conducts research in such areas as gene expression in polyploids, the molecular basis of heterosis, and the role of RNAi in heterochromatin maintenance. He also co-authored a paper — written in collaboration with Sarah Elgin from Washington University and Manika Pal-Bhadra from the Centre for Cellular and Molecular Biology in Hyderabad, India, among other researchers — that has just been published in the Jan. 30 issue of Science. The paper is entitled “Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery.”

Birchler recently spoke with RNAi News about his work and the Science paper.

How did you get involved in RNAi?

We work on both maize and Drosophila, and we had had, and still do, an interest in the dosage sensitivity or haplo-insufficiency of regulatory genes. So, back in the late 80’s we made a promoter-reporter construct from two different fly genes: the white eye color gene and the alcohol dehydrogenase enzyme. Those transgenes did not behave correctly — they showed silencing. Since we work on plants as well, we were aware of the transgene silencing that had been found by Marjori Matzke and Antonius Matzke, and then also by Rich Jorgensen … which turned out in the former case to be transcriptional silencing and then post-transcriptional silencing.

So, through the early 90’s, we continued to work on this silencing of these white-Adh transgenes and a postdoc in the lab, Manika Pal-Bhadra, took up this project in a more serious manner. Then it became obvious that these transgenes and several others in flies were undergoing this co-suppression phenomenon that had been described in the plant kingdom.

At the time, that was the only case in animal species that had been described. Of course, the silencing in plants was by a post-transcriptional mechanism and people … had implied that there was an RNA turnover involved because run-on transcription was normal in the silenced plants and there were some cases of transcriptional silencing that involved transgenes with similar promoters.

We continued to study this in Drosophila and then … it turned out that this was dependent upon the polycomb complex of chromatin proteins and so that implied that it was in fact transcriptional silencing. We later went on to do run-on transcription analysis that confirmed that was indeed the case. But, along the way we also found cases of post-transcriptional silencing in Drosophila involving Adh transgenes. … So, there’s both post-transcriptional and transcriptional silencing that occurs with various transgenes in flies, and the mechanism of the post-transcriptional silencing is similar to the post-transcriptional silencing that occurs in plants in that it involves the production of siRNAs.

Hence, the intersection with RNAi.

Would you talk about the Science paper and the findings?

First of all there’s a long-standing phenomenon in Drosophila of position effect variegation. … There’s heterochromatin and euchromatin, and the heterochromatin is primarily around the centromeric regions of the chromosomes in flies. Whenever a chromosomal aberration is created that brings heterochromatin next to euchromatin, then the active genes in euchromatin are very often silenced in a mosaic fashion. This [was] duplicated a number of years ago … by creating a tandem array of the mini-white cluster in Drosophila. This would mimic position effect variegation, so there was heterochromatin formation by the creation of repetitive sequence clusters. [It was] also mimicked by transgenes that insert into the tiny fourth chromosome of flies, [an insertion] which had been done in Sally Elgin’s lab.

So, we took these systems and determined whether or not the position effect variegation was ameliorated by mutations in the RNAi machinery, which are homozygous viable. In flies, there are three genes that are implicated in RNAi in a variety of systems, and those are piwi, aubergine, and homeless or spindle-E — it goes by two different names.

Aubergine and spindle-E will not support RNAi in embryos that are homozygous — this was determined by Richard Carthew, who first showed that RNAi worked in flies. So, we looked at these, and these mutations, in particular the homeless mutation, would suppress this silencing that was typical of position effect variegation systems. This implied, then, that the RNAi machinery was involved in the formation of heterochromatin.

We had been interested in how the RNAi machinery is involved in transcriptional silencing from our transgene work, and then Shiv Grewal and Rob Martienssen’s lab have published a pair of papers in Science on heterochromatin formation in Schizosaccharomyces pombe, in which they had shown that silencing of repeats in pombe was relieved by mutations in the RNAi machinery.

That finding, together with the finding that we had had previously that the RNAi machinery was required for transcriptional silencing, inspired our groups to ask this question about heterochromatin in a multicellular organism: Drosophila. The early indications were that there was indeed an effect.

Sally Elgin’s lab had a long history of work on heterochromatin in Drosophila — her lab was the one that first documented the presence of heterochromatin protein 1, or HP1, that is primarily associated with heterochromatin and also with the tiny fourth chromosome in flies and a few other places in the nucleus; more recently her lab has documented a partner protein HP2 that follows along and associates with HP1.

So, we examined: What was the effect of these mutations on the distribution of HP1 and HP2? What we found was that in — particularly in the homeless mutation — HP1 and HP2 were dramatically redistributed in the nucleus. Normally, its only found in the centric heterochromatin, as I said, and in the fourth chromosome and a few other places in the euchromatic arms, but not very many. To some degree it’s detectable in piwi and aubergine; there’s some redistribution of HP1 and HP2, as well — but primarily in homeless it basically coats the whole nucleus.

This was an exciting result for us.

HP1 is typically thought to have this localization because of its association with the modification of histone H3, primarily methylation at lysine 9. So, then we examined the levels of H3-mK9 in these various mutations and, of course, the levels of H3-mK9 — which is typical of silenced regions of the genome — was very high in the centric heterochromatin. The levels of the modification are reduced in piwi/aubergine to some degree, but very strongly in homeless.

This particular result was consistent with the effects of HP1 and HP2 because HP1 binds to regions that are associated with the modified form of H3, and therefore if those levels are diminished then HP1 would lose its specificity for localization.

The implication, then, is that the determination of where the silencing domains are set up has an RNAi component and it uses the RNAi machinery. So, one of the future directions will be to try to understand how the targeting of the modification of H3 at lysine 9 occurs using the RNAi machinery as a guide. Presumably, the implication of this work is that the heterochromatin creates an RNase that is then used to have a sequence specificity for the determination of the modification of H3 that creates a silencing domain.

The correlation of the gene expression that we looked at and the molecular analysis of HP1, HP2, and H3-mK9 all suggest that the RNAi machinery is involved in this transcriptional silencing of a different type. We had previously shown that the RNAi machinery was involved in the transgene silencing, which is polycomb dependent. Now, in this work, we show that the RNAi machinery is implicated in transcriptional silencing again, but of the type that involves H3-mK9 modification and subsequent association with HP1.

Would you comment on other future projects involving RNAi you are looking at?

Sally Elgin’s lab is very interested in the heterochromatin aspects of flies. So, she’s interested in trying to understand the mechanism of how the RNAi machinery is involved in targeting heterochromatin.

Our lab had come into this from trying to understand the relationship to repeat-induced silencing, and obviously that is related to heterochromatin silencing, so it’s very good that the three labs worked together on [the Science] project, since there were overlapping interests.

Dr. Bhadra’s lab, I think, is interested in looking at some of the other RNAi genes that are involved in this process and understanding their role in heterochromatin silencing.

We continue to be interested in the role of RNAi in transgene silencing, and we are currently doing some studies to understand what triggers the interrelationship between RNAi — which in its first incarnations was thought to be something that happened in the cytoplasm but obviously has implications for transcriptional silencing — [and] transcriptional silencing using a transgene approach.

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