Identification of genes preventing transgenerational transmission of stress-induced epigenetic states

Abstract:

Examples of transgenerational transmission of environmentally induced epigenetic traits remain rare and disputed. Abiotic stress can release the transcription of epigenetically suppressed transposons and, noticeably, this activation is only transient. Therefore, it is likely that mechanisms countering the mitotic and meiotic inheritance of stress-triggered chromatin changes must exist but are undefined. To reveal thesemechanisms, we screened for Arabidopsis mutants impaired in the resetting of stress-induced loss of epigenetic silencing and found that two chromatin regulators, Decrease in DNA methylation1 (DDM1) andMorpheus’Molecule1 (MOM1), act redundantly to restore prestress state and thus erase “epigenetic stress memory”. In ddm1 mutants, stress hyperactivates heterochromatic transcription and transcription persists longer than in the wild type. However, this newly acquired state is not transmitted to the progeny.
Strikingly, although stress-induced transcription in mom1 mutants is as rapidly silenced as in wild type, in ddm1mom1double mutants, transcriptional signatures of stress are able to persist and are found
in the progeny of plants stressed as small seedlings. Our results reveal an important, previously unidentified function of DDM1 and MOM1 in rapid resetting of stress induced epigenetic states, and therefore also in preventing their mitotic propagation and transgenerational inheritance.
Intro
Although environmentally induced traits and their transgenerational transmission in plants have been described repeatedly, trait stability and the involvement of epigenetic mechanisms in their generation remain controversial (1–3). In contrast, it has been well documented that environmental challenges such as elevated temperature can transcriptionally activate chromosomal loci normally silenced by repressive chromatin (4–7). However, this release of epigenetic silencing, unaccompanied by changes in DNA methylation or histone modifications, is only transient (4, 5). Such noncanonical release of transcriptional gene silencing (TGS) is similar to alterations in epigenetic regulation observed in the mom1 mutant, where release of TGS occurs without major changes in epigenetic marks (8–13). Although, molecular mechanisms used by MOM1 in TGS regulation are not well understood, genetic studies have linked MOM1 activity to small interfering RNAs (14) and RNA processing (15). In addition, structure/function studies have suggested that a conserved domain ofMOM1 forms a homodimer, which is possibly required as a binding platform
for additional silencing factors (13, 16). The rapid resilencing of heterochromatic transcription induced by heat stress seems to involve changes in nucleosome occupancy and resilencing is delayed in mutants with impaired chromatin assembly (5). These observations suggest that suppressive chromatin has certain plasticity in response to stress, but also a robust buffering system that resets its prestress state. This counters the persistence of stress-induced epigenetic alterations during subsequent development and thus their transmission to the progeny.
Results and Discussion
To identify factors involved in the erasure of “epigenetic stress
memory,” we performed a genetic screen using Arabidopsis line
LUC25 carrying a transcriptionally silenced transgene encoding
firefly luciferase (LUC) (14), which as an endogenous chromosomal
TGS target loci can be transiently activated after heat
stress. First, we introduced the mom1 mutation into LUC25 (mom1
LUC25). The mom1 mutation partially releases silencing of LUC25,
producing weak luciferase signals in roots but not aerial parts, where
the LUC transgene remains silenced. Importantly, the LUC transgene
in mom1 LUC25 is strongly activated by heat stress, similar to
LUC25 (Fig. S1A). We presumed that the introduction of the mom1
mutation would enhance stress-induced luciferase signals, increasing
clarity and thus the efficiency of the mutant screen. Moreover,
although themom1mutation does not directly influence the kinetics
of stress-induced TGS release, MOM1 is involved in buffering
epigenetic states of chromatin (11).We hypothesized, therefore,
that any deficiency in such buffering would facilitate phenotypic
detection of additional epigenetic regulators involved in the
rapid restoration of TGS after stress and, thus, in the erasure of
epigenetic stress memory.
M2 seedlings of mutagenized mom1 LUC25 were germinated
for 5 d and individuals showing enhanced luciferase signals before
stress treatment were removed, because these plants release TGS
constitutively. The remaining seedlings were subjected to heat stress
and plants showing significantly stronger and/or longer-lasting
luciferase signals were selected and grown to maturity (Fig. S1B).
We examined their M3 progeny to determine whether selected

Nada que ver

Upon facing stress conditions the budding yeast Saccharomyces cerevisiae is able to choose among different cellular fates such as unicellular or multicellular organization, with each organism being able to undertake growth modes accordingly to the different needs as differential growth modes can confer the cell protection and enhance its dissemination and substrate colonization. When either haploid or diploid Saccharomyces cerevisiae cells are subject of such stress like during starvation conditions, they will develop two different types of growth: adhesive/invasive for the first and pseudohyphae for the latter. For both of these phenomena, yeasts will experience an increment in cell length, change of polarity and an augmentation of cell to cell adhesion; in pathogens, this kind of development is fundamental for host-cell attachment, virulence and tissue invasiveness.

It has been identified that Flo11 have a central role in pseudohyphal (filamentous growth) growth: when FLO11 is deleted, diploids of Saccharomyces cerevisiae strain Σ1278b do not form pseudohyphae and there is no agar invasion in haploids. In nitrogen starvation conditions, wild type diploids but not haploids will accumulate FLO11 transcripts. (Lo and Dranginis, 1998) FLO11also has a particularly long promoter expanding for about 3 kilo bases (kb) and intensive studies by Baumgarner et al. have unveiled the presence of a paradigmatic epigenetic toggle switch controlled by two cis-interfering long noncoding RNAs. The region upstream the FLO11 ORF encodes ICR1, an ncRNA of about 3.2 kb long whose transcription inhibits that of FLO11 trough the mechanism of promoter occlusion. The second ncRNA, PWR1 is transcribed from the complementary, Watson strand, blocking ICR1 transcription and in consequence, promoting that of FLO11. These two transcripts, IRC1 and PWR1, are at the same time subjected to the control of two transcription factors, Sfl1 and Flo8 and chromatin remodeler Rpd3L, an HDAC with the paradoxical effect of promoting FLO11 transcription. In the model proposed by the authors, Rpd3L localizes to the FLO11 promoter and contributes to the repression of ICR1 transcription by facilitating Flo8 binding and PWR1 transcription together with FLO11.

In addition to nitrogen depletion, amino acid starvation has been reported to induce an increase of FLO11 expression. When transcriptional coactivators Rsc1 and Gcn5 are deleted, FLO11 transcription is impaired together with the concomitant changes on invasive growth, but in the case of amino acid starvation such deletion have a different effect. FLO11 transcription is very low, but rsc1Δ and gcn5Δ still show invasive growth and the FLO11^prom-lacZ-encoded β-galactosidase activities increase importantly. ( rewrite to point out the function of Gcn5) 

It has been demonstrated that Gcn5, a histone acetyltransferase (HAT) that has functions as coactivator in transcriptional regulation, contribute largely to transcriptional activation and particularly to enhance substrate specificity in combination with other histone modifications. (Clements et al., 2003) Gcn5p is the catalytic subunit for three chromatin modifying complexes of the ADA, SAGA and SILK/SALSA that act regulating a wide arrange of genes either in a positive and negative fashion. 

Additionally, it was shown by Fischer et al. that amino acid starvation induced FLO11-dependent adhesive growth of the rsc1Δ and gcn5Δ strains although FLO11transcription remained very low. The double deletion strain rsc1Δflo11Δ, however, does not grow adhesively, suggesting that the adhesion of the rsc1Δ strain at amino acid starvation is FLO11-dependent. Hence, it was resolved that very low FLO11 transcripts are essential and sufficient for derepression of FLO11 expression and adhesive growth during amino acid starvation. (Fischer et al., 2008)

Results

The histone acetyl transferase (HAT) Gcn5 is involved in pseudohyphal development.

We tested the haploid and diploid strains for phenotype changes caused by the deletion of GCN5 (Figure 1A). In the case of the haploid, it was shown through wash tests that GCN5 is required to develop invasive growth; the same was true for snf1 and FLO11. In the case of the diploid, the mutant strain didn’t develop pseudohyphae similarly to the pseudo-diploid; an α haploid containing a single MATa1 plasmid whose WT presents the same behavior than natural diploids. FLO11 mRNA levels have different pattern of expression in WT diploids than that in WT haploids during amino acid starvation (Figure 1B); the first have an striking increase peaking at around 1.5h, but clearly decreasing until getting back to basal levels after two more hours. Haploids, on the other hand, have mRNA levels that increase gradually and only reach that equal half the maximum detected for the diploid strain, however such expression is stably maintained for about four hours, until a final sharp drop. Nevertheless, FLO11 expression is heavily impaired in both haploid and diploid gcn5 mutants (Figure 1C) under amino acid starvation conditions, with just very low levels still remaining. To study if this effect is due to the histone acetyl transferase (HAT) activity of GCN5, we also tested a strain that expresses full length GCN5 carrying a catalytic impairing mutation, E173Q. The level of expression are similar to those of the deletion mutant, so we can infer that the HAT activity is required for FLO11 expression during amino acid starvation.

Invasive and filamentous growth phenotypes are restored in deletion mutant strains carrying an episomal GCN5.

FLO11 expression is required for haploid invasive growth, and flo11 strains are totally washed away when tested (Figure 2A). The lower levels of FLO11 expression on gcn5 strains have a repercussion in that phenotype and less than 20% of the total growth remains after the wash test. However, when the yeast are transformed with a plasmid containing GCN5, the invasive growth increases up to the double than the deletion mutant on 50% the level of the WT strain, but when the transformation was done with a plasmid carrying the non-catalytic, E173Q GCN5,  the invasive growth levels are similar to the gcn5 strain. A similar outcome can be seen in diploids, where filamentous development in the gcn5 and the E173Q strains is heavily impaired, but is significantly rescued with the GCN5 carrying plasmid (Figure 2B).  

The transcription factor Gcn4 binds to FLO11 promoter in response to 3-AT induction.

Amino acid starvation is sensed into the cell trough diverse pathways, and microarray data characterized GCN4 as a positive regulator during that phenomenon. Being a broad gene activator, it’s well arguable that an interaction may exist between the FLO11 promoter and this transcription factor, so we analyze that possibility using chromatin immunoprecipitation with 8 primers located at different regions along the extensive upstream control region (Figure 3A). For haploid strains, we measured the interaction at 0, 2 and 4 hours (Figure 3B) and time 0 and 1 hour for diploid (Figure 3C). Our results shows that the regions covered by primers NR2 and NR5 exhibit a notable enrichment for both kind of strains, with a striking multifold increase for the diploid. To our understanding this is the first report concerning such direct interaction between Gcn4 and the FLO11 promoter.

Gcn5 regulate the transcription of an ncRNA (ICR1) and a flocculin gene (FLO11) in response to 3-AT induction.

In the extensive promoter region upstream of FLO11 two long non-coding RNAs are found controlling an aforementioned epigenetic toggle switch (Figure 4A) where FLO11 expression is halted via the promoter occlusion mechanism when ICR1 transcription takes place. For the haploid strain, the pattern of IRC1 RNA expression turned out remarkably similar among the WT and gcn5 strains when they were subject of 3-AT induction and also when they were not starved and nevertheless FLO11 RNA levels are significantly different (Figure 4B). The low levels of FLO11 RNA expressed are consistent and correlate to the phenotype shown in Figure 1A, were a big percentage, but not all, of the invasive growth is lost. Noticeably the only difference in the pattern can be found at about -1 Kb from the FLO11 transcription start site, and while small it seems to be significant.

We found a radically different story told by the pattern of expression from the diploid strains; even in normal nutritional status the gcn5 strain has a significantly higher level of ICR1 transcriptional activity around -2 and -1 Kb upstream the FLO11 transcriptional start. Under amino acid starvation conditions, this difference is magnified several fold and the FLO11 profile shows a silencing of the FLO11 transcription that supports the observations annotated in Figure 1B, no pseudohyphal development is observed for gcn5. This results indicate that GCN5 exerts a different control, at least in magnitude, among haploid and diploids. It should also be noted that the regions with a higher ICR1 expression correspond to those two regions enriched by Gcn4 binding.

Gcn5 is a negative regulator of ICR1 in diploid strains.

ICR1 is a long non coding transcript whose expression inhibits that of FLO11,  

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