Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome

Abstract

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. In this study, using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) was expressed from intergenic regions, we found that cryptic promoter activation occurs by at least two different mechanisms: one is the capturing of pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is the entirely new formation of promoter chromatin near the 5’ end of the inserted LUC ORF. To discriminate between these, we denoted the former mechanism as “cryptic promoter capturing”, and the latter one as “promoter de novo origination”. The latter finding raises a question as to how inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter–LUC constructs, we found that the functional core promoter region, where transcription start sites (TSS) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the LUC ORF sequence alters the local distribution of the TSS in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

Plant genome response to incoming coding sequences: stochastic transcriptional activation independent of integration loci

“…Thus, chromatin remodelling at the site of T-DNA integration may account, at least in part, for integration-dependent stochastic transcriptional activation. If this is the case, the expected chromatin remodelling could be the mechanism underlying the de novo promoter origination identified by us previously (Kudo, Matsuo, and Satoh et al ., 2020).…”

“…We previously demonstrated that such unexpected transcriptional activation in promoter-trapping experiments occurred via two different mechanisms: (1) cryptic promoter capturing, in which exogenous DNA was transcribed by trapping a pre-existing promoter-like chromatin configuration; and (2) promoter de novo origination, in which promoter-like epigenetic landscapes were newly formed via chromatin remodelling triggered by the insertion of DNA (Kudo, Matsuo, and Satoh et al ., 2020). However, the experimental throughput of this promoter-trapping experiment was too limited to illustrate the generality and extensiveness of these transcriptional activation mechanisms.…”

De novoactivated transcription of inserted foreign coding sequences is inheritable in the plant genome

“…We speculated that the insertion of exogenous coding sequences might activate local chromatin remodelling to shape the promoter-like epigenetic landscape, resulting in such de novo transcriptional activation. Although the evidence of this type of epigenetic rewiring was not confirmed in the studies of cultured cells, we observed that promoter-like histone species were newly localized in the vicinity of the 5’ end of the inserted promoterless coding sequences under a similar experimental system in transgenic plants (Kudo, Matsuo, and Satoh et al, 2020).…”

De novo activated transcription of inserted foreign coding sequences is inheritable in the plant genome

“…ChIP and MBDIP were performed according to a method previously described [ 27 ], with the following modifications. For the ChIP assay, ~10 ng of solubilized chromatin (median, 200 bp) and antibodies (2.4 μg of an anti-H2A.Z antibody [ 70 ] and 2.0 μg of an anti-H3K36me3 antibody (Abcam: ab9050), respectively) were used for each experiment. For the MBDIP assay, the methylated DNA fraction (mC) was collected from 1.0 μg of sheared DNA (median, 200 bp) using an EpiXplore Methylated DNA Enrichment Kit (Clontech) according to the manufacturer’s instructions.…”