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An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

ABSTRACT: Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

SUBMITTER: Neupert J

PROVIDER: S-EPMC7722844 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Neupert Juliane J Gallaher Sean D SD Lu Yinghong Y Strenkert Daniela D Segal Na'ama N Barahimipour Rouhollah R Fitz-Gibbon Sorel T ST Schroda Michael M Merchant Sabeeha S SS Bock Ralph R

Nature communications 20201208 1

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupt ...[more]

PMID: 33293544

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Project description:Algal photo-bio hydrogen production, a promising method for producing clean and renewable fuel in the form of hydrogen gas, has been studied extensively over the last few decades. In this study, microarray analyses were used to obtain a global expression profile of mRNA abundance in the green alga Chlamydomonas reinhardtii at five different time points before the onset and during the course of sulphur depleted hydrogen production. The present work confirms previous findings on the impacts of sulphur deprivation but also provides new insights into photosynthesis, sulphur assimilation and carbon metabolism under sulphur starvation towards hydrogen production. For instance, while a general trend towards repression of transcripts encoding photosynthetic genes was observed, the abundance of Lhcbm9 (encoding a major light harvesting polypeptide) and LhcSR1 (encoding a chlorophyll binding protein) was strongly elevated throughout the experiment, suggesting remodeling of the photosystem II light harvesting complex as well as an important function of Lhcbm9 under sulphur starvation. This study presents the first global transcriptional analysis of C. reinhardtii during hydrogen production using five major time points at Peak Oxygen, Mid Oxygen, Zero Oxygen, Mid Hydrogen and Peak Hydrogen. Keywords: Time course, sulfur deprivation, hydrogen production. Time course microarray analyses were used to analyze the global gene expression in sulfur depleted hydrogen producing C. reinhardtii. When depleted of sulfur, a sealed an illuminated C. reinhardtii culture slowly becomes anaerobic and produces hydrogen gas. Based on the changes in the dissolved O2 level and H2 production rate, the whole course of H2 production from the starting of S deprivation to the end of H2 production can be divided into five phases including an Aerobic Phase (I) in which the dissolved O2 level goes up (I), an O2 Consumption Phase (II), an Anaerobic Phase (III), a H2 Production Phase (IV) and a Termination phase (V). Samples were taken from three different bioreactors (biological replicates) at the following time points after the start of S depletion: 6 h, 16 h, 21 h, 37 h and 52 h corresponding to Peak O2, Mid O2, Zero O2, Mid H2 and Peak H2.

Project description:Swimming suspensions of Chlamydomonas eugametos were pelleted and homogenized, and the metabolism of inositol polyphosphates by cellular homogenates or supernatants was investigated. Ins(1,4,5)P3 was dephosphorylated under physiological conditions to yield a single InsP2, Ins(1,4]2. In the presence of ATP it was phosphorylated to give Ins(1,3,4,5)P3 as the only InsP4. The Ins(1,4,5)P3 3-kinase activity was predominantly soluble, was not detectably affected by calmodulin or Ca2+, and had a Km for Ins(1,4,5)P3 of 50 microM (two orders of magnitude higher than its mammalian counterpart). Ins(1,3,4,5)P4 was dephosphorylated by the cellular supernatants to Ins(1,3,4)P3 and Ins(1,4,5)P3, and could be phosphorylated to Ins(1,3,4,5,6)P4. No Ins(1,3,4)P3 6-kinase activity could be detected, and experiments with [3H]Ins(1,4,[32P]5)P3 revealed that Ins(1,3,4,5,6)P5 is formed from Ins(1,4,5)P3 with little loss of the 5-phosphate, i.e. the predominant route of synthesis is probably by a direct 6-phosphorylation of Ins(1,3,4,5)P4. Similar experiments with an (NH4)2SO4 fraction of turkey erythrocyte cytosol gave essentially the same result, i.e. direct phosphorylation of Ins(1,3,4,5)P4 in the 6 position is the predominant route of synthesis of InsP5 from that InsP4 in vitro. No InsP6 formation was detected in any of these experiments, but labelling of intact C. eugametos with [3H]inositol revealed that the cells do synthesize InsP6. The lipids of C. eugametos cells contain PtdIns, PtdIns(4)P and PtdIns(4,5)P2 [Irvine, Letcher, Lander, Drøbak, Dawson & Musgrave (1989) Plant Physiol. 64, 888-892]. Further examination of 32P-labelled lipids revealed that about 20% of the PtdInsP was the PtdIns(3)P isomer, and about 1% or less of the PtdInsP2 was the PtdIns(3,4)P2 isomer. The overall inositide metabolism of C. eugametos resembles that of a mammalian cell more closely than it does that of a plant cell or slime mould, and this suggests firstly that the known metabolism of inositol polyphosphates arose at an early time in eukaryotic evolution, and secondly that Chlamydomonas might prove a useful organism for genetic and comparative studies of inositide enzymology.

Dataset Information

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Publications

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Similar Datasets

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