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Harvard s Danesh Moazed on RNAi and Heterochromatin Assembly

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At A Glance

Name: Danesh Moazed

Position: Associate professor, cell biology, Harvard Medical School

Background: Postdoc, University of California, San Francisco — 1990-1998; PhD, University of California, Santa Cruz — 1989; BS, University of California, Santa Cruz — 1985

After working as a postdoc in two labs at the University of California, Santa Cruz, Danesh Moazed made his way to Harvard Medical School, first as an assistant professor and most recently as an associate professor.

His research focuses on epigenetic mechanisms that control cell identity and chromosome stability in eukaryotes, and has expanded to include the role of RNAi in heterochromatin assembly. Recently, he published a paper in the Proceedings of the National Academy of Sciences describing how he and his colleagues found that RNA-dependent RNA polymerase is both an essential component of a self-enforcing RNAi loop and critical to the siRNA production necessary for heterochromatin formation.

Recently, Moazed spoke with RNAi News about his work.

Could you give an overview of what you do in your lab?

The broad interests of my lab are in understanding how epigenetic chromatin domains are assembled and inherited, and how epigenetics is used to maintain cell identity in various systems.

Our main focus is on the biochemistry of the protein and RNA complexes that assemble the epigenetic chromatin domain. We use yeast as a model system because yeast silent chromatin, or heterochromatin, provides an example of chromatin that is very stably maintained and epigenetically inherited.

In budding yeast, we work on the SIR complex, which contains the Sir2 NAD-dependent deacetylase as well as Sir3 and Sir4, two histone-binding proteins. This complex has the remarkable ability to spread or polymerize along the chromatin fiber, once it is recruited to a nucleation site on DNA, to create a self-propagating chromatin domain.

We also use fission yeast [to investigate how] the RNAi pathway is involved in initiating and maintaining heterochromatin at specific chromosome domains.

Can you get into what your research looks like … on the RNAi front?

On the RNAi front, our goal is to use fission yeast to completely reconstitute the [RNAi] pathway, beginning with how double-stranded RNAs are processed by the Dicer ribonuclease into siRNAs; then how siRNAs go down into specific effector complexes like the RITS complex, which my lab has purified; then what the mechanism of targeting specific chromosome regions is.

Our goal is to identify all the proteins that participate in this process, and develop in vitro assays for how RNAi targets specific DNA regions. Once there, [the goal becomes figuring out] how the components of the RNAi pathway recruit histone-modifying activities so that you can get this conversion to heterochromatin.

At this point, where is the component-identification process?

It’s hard to tell, but I think we’re probably about halfway there.

The RITS complex is a major component in the pathway that uses siRNAs to target specific chromosome domains. This complex provides a direct link between RNAi and heterochromatin as it contains an Argonaute homolog and a structural component of heterochromatin.

In addition to RITS, we have purified a novel RNAi complex that is involved in using siRNAs to make double-stranded RNA, which is then processed by Dicer to make more siRNAs. We call this second complex RDRC, which stands for RNA-directed RNA polymerase complex. RDRC contains the fission yeast RNA-directed RNA polymerase, or Rdp1; a conserved RNA helicase; and a protein from the polyA polymerase family of enzymes. We have shown that RDRC has RNA synthesis activity and physically interacts with RITS. Also, this interaction requires siRNAs.

So, we have proposed that RITS loaded with siRNAs acts as a priming complex for RDRC and directs the synthesis of appropriate double stranded RNAs. This then helps maintain RNAi silencing. Another important component of the pathway is the non-coding RNAs that are transcribed from centromeric repeats and other sites that are assembled into heterochromatin. These RNAs had previously been shown to be the source of siRNAs. Using cross-linking experiments, we have shown that both RITS and RDRC localize to these non-coding RNAs. Again, the localization is directed by siRNAs. Because these non-coding RNAs are chromatin associated, we have proposed that the localization of RNAi complexes to specific chromosome regions involves initial interactions with nascent chromatin-bound RNA transcripts.

What we want to know now is: Once you get this initial targeting, how do RITS and possibly non-coding RNAs recruit other heterochromatin proteins, such as histone modifying enzymes? I think that’s the biggest challenge now.

Another challenge is to figure out how [Dicer] actually makes siRNAs from double-stranded RNA. In vitro, Dicer is very efficient in making siRNA from double-stranded RNA templates, but we suspect that in vivo its activity is highly regulated. We’d like to know exactly where Dicer fits into the pathway, whether it’s recruited to double-stranded RNA by interacting with one of the two complexes we’ve already identified.

Are you collaborating with anybody?

We’ve have been collaborating with Shiv Grewal’s lab at the NIH.

In terms of the overall impact of this, where do you see the field headed once the components are identified et cetera?

There is a relatively sophisticated understanding of how RNAi mediates gene silencing at the post-transcriptional level. Our work provides mechanistic insight into how RNAi can also target specific chromosome regions for inactivation. The new complexes and proteins identified in yeast are likely to have counterparts in other systems and should contribute to a better understating of RNAi in general.

Where do you see the RNAi field headed, say, five years out?

The field has been moving along at an amazing pace. So, I am sure there will be more unexpected discoveries and we will have a better mechanistic understanding of how RNAi regulates gene expression at different levels.

Other than that, I think small RNAs will emerge as major regulators of gene expression with expanding roles in regulation of development and physiology. Perhaps, the efforts towards using siRNAs as therapeutic agents will start to pay off.

 

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