RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex
Supplemental File 1
Supplemental File 2
Supplemental File 3
Supplemental File 4
AuthorsCernilogar, Filippo M.
Burroughs, A. Maxwell
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
KAUST Environmental Epigenetics Research Program (KEEP)
MetadataShow full item record
AbstractBackground:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.
CitationCernilogar FM, Burroughs AM, Lanzuolo C, Breiling A, Imhof A, et al. (2013) RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex. PLoS ONE 8: e65740. doi:10.1371/journal.pone.0065740.
PublisherPublic Library of Science (PLoS)
PubMed Central IDPMC3681981
- Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.
- Authors: Cernilogar FM, Onorati MC, Kothe GO, Burroughs AM, Parsi KM, Breiling A, Lo Sardo F, Saxena A, Miyoshi K, Siomi H, Siomi MC, Carninci P, Gilmour DS, Corona DF, Orlando V
- Issue date: 2011 Nov 6
- Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation.
- Authors: Pushpavalli SN, Bag I, Pal-Bhadra M, Bhadra U
- Issue date: 2012 Apr
- RNAi components are required for nuclear clustering of Polycomb group response elements.
- Authors: Grimaud C, Bantignies F, Pal-Bhadra M, Ghana P, Bhadra U, Cavalli G
- Issue date: 2006 Mar 10
- Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex.
- Authors: Lanzuolo C, Roure V, Dekker J, Bantignies F, Orlando V
- Issue date: 2007 Oct
- Role of histone H3 lysine 27 methylation in Polycomb-group silencing.
- Authors: Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS, Zhang Y
- Issue date: 2002 Nov 1
Showing items related by title, author, creator and subject.
Interaction between the triglyceride lipase ATGL and the arf1 activator GBF1Ellong, Emy Njoh; Soni, Krishnakant G.; Bui, Quynh-Trang; Sougrat, Rachid; Golinelli-Cohen, Marie-Pierre; Jackson, Catherine L. (Public Library of Science (PLoS), 2011-07-18)The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.
Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processingIwata, Yuji; Takahashi, Masateru; Fedoroff, Nina V.; Hamdan, Samir (Oxford University Press (OUP), 2013-08-05)Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing. 2013 The Author(s) 2013.
Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String ConfigurationTrujillo, Uldaeliz; Vázquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J.; Vassallo, David A.; Vega, Irving E.; Arold, Stefan T.; Baerga-Ortiz, Abel (Public Library of Science (PLoS), 2013-02-28)The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures. 2013 Trujillo et al.