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.

An ancient clade of Penelope-like retroelements with permuted domains is present in the green lineage and protists, and dominates many invertebrate genomes

“…Notably, we also identified putatively nonautonomous Chlamys elements, which produced pLTRs (and often multi-copy headto-tail insertions) and generally exhibited sequence similarity to autonomous families at their 3' ends. The nonautonomous elements include MRC1, which was previously described as a nonautonomous LTR (Kim, et al 2006) and may be the most active TE in C. reinhardtii laboratory strains (Neupert, et al 2020). Further supporting recent activity, Chlamys copies exhibited minimal divergence from their respective consensus sequences (Fig.…”

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology

“…Therefore the UV strain displays a high transgene expression capacity, comparable to that of strain UVM4, a strain which along with UVM11 was for many years the only option, if high level transgene expression in C. reinhardtii was required. The nuclear genomes of both strains, UVM4 and UVM11 carry mutations in a gene encoding the Sir2-type histone deacetylase SRTA [ 36 ], which were shown to be causative for the high transgene expression phenotype. Sequencing of the SRTA gene in CC-1690 *** confirmed that UV mutagenesis did not cause mutations in its coding sequence, pointing at mutations in other genes as the cause for higher transgene expression in this strain.…”

“…6 B), showing the typical thick cell wall [ 39 ]. The cell-wall deficiency and immotility of UVM4/11 precluded mating experiments [ 11 , 36 ] representing a major drawback for their use a bioproduction chassis strain. Only recently, UVM11 could be crossed with wildtype strain CC-124 and the progeny retained the capacity to express transgenes at a high level [ 36 ].…”

“…The cell-wall deficiency and immotility of UVM4/11 precluded mating experiments [ 11 , 36 ] representing a major drawback for their use a bioproduction chassis strain. Only recently, UVM11 could be crossed with wildtype strain CC-124 and the progeny retained the capacity to express transgenes at a high level [ 36 ]. The mating-competence of the UV strain will facilitate future map-based cloning or whole genome re-sequencing experiments [ 40 ] to identify the causative mutation.…”

The epigenetic origin of life history transitions in plants and algae

“…DNA methylation is confined to repeats in Chlamydomonas and remains stable across its life cycle (Lopez et al 2015) but is altered during environmental adaptation (Kronholm et al 2017), while it is linked with transgene silencing in Volvox (Babinger et al 2001). Epigenetic silencing of exogenous transgenes is also reported in Chlamydomonas, which has been used to isolate causal silencing pathways (Jeong et al 2002;Neupert et al 2020). One such screen identified SET3p, a protein homologous to the Arabidopsis SUVH family that functions in vitro as a specific H3K9 monomethyltransferase (Casas-Mollano et al 2007).…”