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RNAi Abstracts
Click on the PubMed link below the abstract to view the article on PubMed

 

PLoS One. 2009 Jul 8;4(7):e6182
Drosophila RISC component VIG and its homolog Vig2 impact heterochromatin formation.

Gracheva, E.M., Dus, M., and Elgin, S.C.R.

Department of Biology, Washington University, St Louis, MO 63130, USA

Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein null mutations vig(EP812) and vig2(PL470) act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vig(EP812) mutant. The vig2(PL470) mutant strain demonstrates a decreased level of H3K9me2. Pull-down experiments using antibodies specific to HP1 recovered both VIG and Vig2. The association between HP1 and both VIG and Vig2 proteins depends on an RNA component. The above data and the developmental profiles of the two genes suggest that Vig2 may be involved in heterochromatin targeting and establishment early in development, while VIG may have a role in stabilizing HP1/HP2 chromatin binding during later stages.
PubMed

Biochim Biophys Acta. 2009 Jan;1789(1):3-16. Epub 2008 Aug 16.

Small RNA-directed heterochromatin formation in the context of development: what flies might learn from fission yeast.

Huisinga KL, Elgin SC.
Department of Biology, Washington University, St Louis, MO 63130, USA.

A link between the RNAi system and heterochromatin formation has been established in several model organisms including Schizosaccharomyces pombe and Arabidopsis thaliana. However, the data to support a role for small RNAs and the associated machinery in transcriptional gene silencing in animal systems is more tenuous. Using the S. pombe system as a model, we analyze the role of small RNA pathway components and associated small RNAs in regulating transposable elements and potentially directing heterochromatin formation at these elements in Drosophila melanogaster.

PubMed

Genes and Development Vol 21, 2300-2311, 2007.

Drosophila PIWI associates with chromatin and interacts directly with HP1a

Brent Brower-Toland, Seth D. Findley, Ling Jiang, Li Lie, Hang Yin, Monica Dus, Pei Zhou, Sarah C.R. Elgin, and Haifan Lin

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome.

Note added 4/2010:  The PIWI antibody used above (prepared against the C-terminal peptide CSIPQNALEKKFYYL) passed all quality control tests: it specifically recognized a protein of 95 kD on Western blots, and the signal was depleted both on RNAi KD in cells and in mutant larvae (see Supplemental Materials, Figures 1-3).  Unfortunately, our subsequent attempts to prepare additional antibodies against this peptide have not been successful.  We have tested four antibodies prepared against other epitopes, and none have given the distinctive polytene chromosome pattern reported above, albeit a variety of tests indicate a nuclear role for PIWI.  We are continuing to investigate.
PubMed
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Current Biology Vol 16, 2222-27, 2006

Element 1360 and RNAi Components Contribute to HP1-Dependent Silencing of a Pericentric Reporter

Karmella A. Haynes, Amy A. Caudy, Lynne Collins, and Sarah C.R. Elgin

In eukaryotes, distinct regions of the genome are packaged as euchromatin (less condensed, more active) or heterochromatin (condensed, silenced). Studies in yeast, plants, and flies suggest that RNA interference (RNAi) is linked to heterochromatin formation and transcriptional silencing of transposable element (TE) sequences. We previously reported that insertion of a mobile hsp70-white reporter within 10 kb of a 1360 element on chromosome four of Drosophila melanogaster correlates with variegation (silencing). Here, we report small RNAs (23 nt) corresponding to 1360, indicating processing by the RNAi machinery. To directly test the ability of 1360 to silence a nearby gene in vivo, we introduced a P element construct carrying a single copy of 1360 upstream of the hsp70-white reporter into flies. This 1360 element contributes to HP1-dependent variegation at a pericentric insertion site, as demonstrated by a decrease in silencing after FLP-mediated removal of 1360. In euchromatin, 1360 is not sufficient to induce silencing, suggesting that proximity to pericentric heterochromatin and/or a high local TE density contributes to heterochromatin formation. Silencing of the 1360, hsp70-white reporter is sensitive to mutations in RNAi components. Our results implicate 1360 as a target for sequence-specific heterochromatic silencing through an RNAi-dependent mechanism.
PubMed

Science, Vol 303, Issue 5658, 669-672, 30 January 2004.

Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery

Manika Pal-Bhadra,1,3 Boris A. Leibovitch,2 Sumit G. Gandhi,3 Madhusudana Rao,3 Utpal Bhadra,1,3 James A. Birchler,1 Sarah C. R. Elgin2

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants.

1 Division of Biological Sciences, 117 Tucker Hall, University of Missouri, Columbia, MO 65211, USA.
2 Department of Biology, Washington University, St. Louis, MO 63130, USA.
3 Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular Biology, Hyderabad 500007, India.

img img2
Mutations in RNAi components piwi, aubergine and homeless suppress PEV.
Several alleles of each gene were tested for their affect using 118E-10, a line carrying our P element in the fourth chromosome pericentric heterochromatin, and 39C-12, a line carrying our P element in the banded portion of the fourth chromosome. Average eye pigment values for various alleles (alone and in heteroallelic flies) are shown on the right. The loss of silencing suggests that the RNAi pathway plays a role in setting and maintaining heterochromatic domains.

Note added 4/2010:  Cytological studies reported in this paper show a redistribution of HP1 on the polytene chromosomes in stocks carrying homozygous mutations in piwi, aubergine, and spindle-E, with the latter showing the strongest impact.  These results were obtained with the squashing and staining protocol developed by M. Pal-Bhadra (given in the Materials and Methods), and have been repeated by her.  The redistribution is not observed using the protocol developed by our lab described below.  Squashes of the mutant and wild type salivary glands on the same slide produced by M. Pal-Bhadra show a clear contrast, ruling out some trivial reasons for the discrepancy (see Supplemental Online Material, http://www.sciencemag.org/cgi/content/full/303/5658/669/DC1).  Studies with vig, another RNAi component,show that HP1 is less stable in the presence of this mutation (Gracheva et al., 2009; see above), an effect that might explain the redistribution observed in the above case.

PubMed

Methods Enzymol. 2004;376:372-93.

Immunofluorescent staining of polytene chromosomes: exploiting genetic tools

Stephens GE, Craig CA, Li Y, Wallrath LL, Elgin SC.
Biology Department, Washington University in St. Louis, Missouri 63130, USA.

No Abstract available

 

 

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