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CSHL Team Publishes New Details on Heterochromatin Assembly, Gene Silencing in Yeast

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By Doug Macron

Researchers from Cold Spring Harbor Laboratory this week published data from a study in fission yeast revealing new details on the roles played by the various domains of the RNA-Induced initiation of transcriptional gene silencing, or RITS, complex to support heterochromatin assembly and both RNAi-dependent and RNAi-independent gene silencing.

“Tightly controlled establishment and maintenance of heterochromatin is crucial for proper chromosome segregation, transcriptional control, and many other chromosome-associated processes,” the paper's authors wrote in Nature Structural & Molecular Biology.

In Schizosaccharomyces pombe, the RNAi pathway is central to the formation of heterochromatin, and the RITS complex “is the bridge between the chromatin factors and the RNAi machinery,” senior author Leemor Joshua-Tor, a CSHL and Howard Hughes Medical Institute investigator, told Gene Silencing News.

RITS comprises the Argonaute family protein and RNAi component Ago1, the chromodomain protein Chp1, the GW protein Tas3, and a small RNA.

In 2009, the investigators showed that Chp1 binds to the heterochromatic histone H3K9 methyl mark with high affinity, while other groups have reported that Ago1 uses siRNAs to target specific RNA transcripts.

“The cooperation of these two activities mediated through assembly of the RITS complex is essential for the establishment and maintenance of heterochromatin,” the team wrote in this week's paper, to which researchers from St. Jude Children's Research Hospital contributed.

Tas3, meantime, acts as the “backbone” of RITS, binding to both Chp1 through its N-terminal domain and to Ago1 through its GW domain.

“We and the [scientific] community thought that [RITS] was a compact, solid complex,” Joshua-Tor said. However, when she and her colleagues expressed the proteins and analyzed them by proteolysis and X-ray crystallography to better understand the structural and functional role of RITS, “we realized that it's not quite like that.”

“Based on our and other’s work, this complex is likely a collection of well-structured modules that are strung together by long flexible linkers, spreading over long distances,” Thomas Schalch, a research investigator in Joshua-Tor's lab and first author of the paper, added.

In this week's paper, the investigators describe how they discovered a structured core in RITS. Specifically, they found that the interface between Chp1 and Tas3 forms a “tight, elongated complex, resembling the hull of a ship,” according to the paper. They also identified an “unexpected” C-terminal PIN domain in Chp1.

A comparison of the PIN domain to all known protein structures deposited in the protein databank suggested that it was associated with telomeres, one of the areas, along with the centromeres, where heterochromatin is located in fission yeast.

To investigate further, the researchers deleted DNA sequences encompassing the domain in the yeast and found that centromeric heterochromatin was “unaffected by the loss of the PIN domain.” In contrast, removing the PIN domain prevented heterochromatin formation on telomeres, suggesting that RITS functions differently at the two sites.

"RITS might be exerting its effect at centromeres through Ago1 and the RNAi machinery, but might be enforcing its function at the telomeres through Chp1 and its PIN domain,” Joshua-Tor noted in a statement.

In the end, “the identification of a PIN domain in Chp1 provides important clues for the unique function
of the Chp1-Tas3 complex in RNAi-independent silencing of telomeric transcripts,” the researchers wrote in Nature Structural & Molecular Biology.

“The PIN domain may be involved in recruiting nuclease activities to subtelomeric regions,” they noted. “In addition, it has been reported that subtelomeric [telomere-linked helicase] transcripts are regulated by the S. pombe TRAMP complex and the exosome. We think it will be interesting to determine whether the PIN domain of Chp1 is similarly involved in recruitment of the exosome to subtelomeric regions for processing of [these] transcripts.”

Joshua-Tor said that she and her colleagues are also interested in examining the other components that assemble on top of RITS, and gaining better insight into how Argo1 “fits into the picture [since] we don't quite know how Argonaute binds to this complex,” she said.


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