Shade-induced transcription of PIF-Direct-Target Genes precedes H3K4-trimethylation chromatin modification rises

The phytochrome (phy)-PIF (Phytochrome Interacting Factor) sensory module perceives and transduces light signals to Direct-Target Genes (DTGs), which then drive the adaptational responses in plant growth and development, appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness, and the change in color of the light that is filtered through, or reflected from, neighboring vegetation (‘shade’). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs. Using RNA-seq and ChlP-seq, time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally-coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

Direct Target Genes (DTGs) that are directly, transcriptionally regulated by PIFs (Zhang et al., 68 2013;Pfeiffer et al., 2014). 69 The relative abundance of the Pfr and Pr forms of the phyB molecule, and by extension 70 the accumulation and activity of the PIFs, is determined by the ratio of red to far-red light in the 71 immediate environment. The active Pfr form is favored under white-light illumination where the 72 R/FR ratio is high, whereas the inactive Pr form is favored in the dark and in conditions where 73 the R/FR ratio is low, such as under vegetative shading (Quail et al., 1995). As a consequence of 74 the photoreversible nature of the phyB molecule, PIF accumulation and activity is high in 75 darkness and in the shade. The transcriptional responses of many PIF DTGs, however, do not 76 exhibit a photoreversible pattern (Leivar et al., 2012). 77 In a previous study, we were able to categorize the transcriptional responses of PIF DTGs 78 in tothree distinct patterns: those that respond during the transition from the etiolated dark-grown 79 state to R, those that respond during the transition from white light into simulated shade or those 80 that respond during both transitions (Leivar et al., 2012). The differential responsiveness of these 81 three broad sets of PIF DTGs, indicates that PIF abundance is not the sole determinant of PIF 82 DTG expression. Core components of the plant circadian oscillator have been implicated in 83 modulating some of these changes in gene expression (Martín et al., 2018;Zhang et al., 2020). 84 Most recently, changes in the chromatin environment have been shown to be directly involved in 85 triggering shade-induced transcription (Willige et al., 2021). 86 One form of chromatin remodeling that can modulate the transcriptional output of light-87 regulated genes involves the enzymatic modification of histones (Fisher and Franklin, 2011;88 Perrella conditions. The accumulation of one particular mark, histone 3 lysine 4 trimethylation 94 (H3K4me3), at the transcriptional start site (TSS) of genes has long been known to strongly 95 correlate with transcriptional activity of those genes (Bernstein et al., 2002), but the biological 96 function of this mark remains relatively less-well defined (Fiorucci et al., 2019). Proposed roles 97 include facilitating transcriptional elongation (Ding et al., 2012) or serving as "transcriptional 98 memory" (Liu et al., 2014) 99 Here, we have refined the list of PIF DTGs by integrating previously published ChIP 100 binding and RNA-seq data for the PIF quartet, with newly obtained RNA-seq data from both 101 wild-type and a mutant lacking six of the PIFs (PIF1, 3, 4, 5, 6 and 7). Using this system, we 102 have explored the potential role of the epigenetic mark H3K4me3 in mediating the observed 103 differential patterns of expression of PIF DTGs. Our data suggest a possible functional role for 104 H3K4me3 in stabilizing the expression levels of DTGs in established green plants, against the 105 rapidly switching light/shade transitions that occur naturally in leaf canopies. 106 107 108

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Characterization of pifqpif6pif7 sextuple mutant 110 The pif1pif3pif4pif5 quadruple mutant (hereafter pifq) displays a constitutively 111 photomorphogenic phenotype when grown in darkness, indicating that these four PIFs are 112 necessary and sufficient to control de-etiolation in response to R (Leivar et al., 2008;Leivar et 113 al., 2009). The pifq mutant does not, however, exhibit a complete lack of responsiveness to 114 simulated shade (Figure 1), supporting the hypothesis that additional factors are required for the 115 complete shade avoidance response (Leivar et al., 2012). PIF7 has been implicated in playing a 116 major role in regulating this process (Li et al., 2012;de Wit et al., 2015;Mizuno et al., 2015) 117 with the quintuple pifqpif7 mutant reported to show no statistically-significant shade avoidance 118 response (Zhang et al., 2020). 119 However, when we measured the shade avoidance response in the pifqpif7 mutant under 120 slightly different conditions to Zhang et al. (Zhang et al., 2020), we were still able to detect a 121 small, yet statistically-significant (p < 0.05) residual shade avoidance response (Figure 1). A 122 possible reason for this small difference is that the results presented here were obtained on 2-123 day-old seedlings exposed to simulated shade, whereas our previous experiments were 124 performed on 3-day-old seedlings exposed to simulated shade. Alternatively, this minor residual 125 shade-avoidance response observed under our conditions could be due to the presence of yet 126 other members of the PIF-subfamily, such as PIF8 or PIL1 (PIF2) (Leivar and Quail, 2011;127 Pham et al., 2018), or to other light-responsive transcription factors. Nevertheless, we then tested 128 whether PIF6 might be responsible for this residual response by generating a sextuple 129 pifqpif6pif7 (pifS) mutant and measuring its hypocotyl length in response to simulated shade. 130 This sextuple mutant displayed significantly shorter hypocotyls than the wild-type in response to 131 shade, but no significant decrease relative to the pifqpif7 quintuple mutant (Figure 1). These 132 results suggest that PIF6 plays no significant role in mediating the shade-avoidance response, 133 consistent with its proposed role in seed dormancy and development (Penfield et al., 2010). 134 135 Generation of a high-confidence list of PIF DTGs and subcategorization into E, ES and S 136 classes 137 Many PIF direct target genes (DTGs) have been previously observed to be upregulated in 138 the presence of the PIFs while others are downregulated. For the purposes of this study, we 139 focused only on PIF-induced genes (i.e. those genes which appear to require the PIFs for high 140 levels of transcription) because PIFs have been shown to have intrinsic activating activity (Huq 141 et al., 2004;Al-Sady et al., 2008;de Lucas et al., 2008;Dalton et al., 2016). 142 In brief, we first integrated the data from a previously published RNA-seq experiment on 143 dark-grown seedlings exposed to 1h of R light (Pfeiffer et al, 2014) with a new RNA-seq time-144 course experiment of white light (WL)-grown seedlings exposed to 3h of simulated shade 145 (shade-light). We then combined previously published RNA-seq data from the pifq mutant 146 grown in darkness (Pfeiffer et al., 2014), with new RNA-seq data, that were obtained using the 147 pifqpif6pif7 mutant (pifS) grown in WL and exposed to 3h shade-light. Lastly, we used 148 previously published data to identify those genes whose promoters were found to be bound by 149 PIF1, PIF3, PIF4, PIF5 and/or PIF7 (no genome-wide binding data are available for PIF6) 150 (Hornitschek et al., 2012;Oh et al., 2012;Zhang et al., 2013;Pfeiffer et al., 2014;Chung et al., 151 2020). By selecting only the genes that met all three of our criteria (light-responsiveness, PIF-152 dependence and PIF-binding), we obtained 169 candidate PIF-induced, red-light repressed and/or 153 shade-light-induced DTGs (Table 1). 154 As described in Leivar et al. (Leivar et al., 2012), PIF DTGs may be broadly classified 155 into one of three classes: re-labeled here as E, ES and S (E for Etiolation-induced only; ES for 156 both Etiolation-and Shade-induced; and S for Shade-induced only) (Figure 2). We therefore 157 subdivided our combined 169 shade-light-induced and red-repressed PIF DTGs into these classes 158 based on their patterns of expression during the D to R, and WL to shade-light transitions. Using 159 these criteria, our initial list of 169 genes was found to contain 24 E genes, 17 ES genes and 128 160 S genes ( Table 1). Upon further analysis, we removed 25 genes that exhibited various 161 anomalous expression profiles and resorted the remaining 144 genes using relaxed cutoff criteria. 162 This resulted in a redistribution between the classes so that the final numbers of genes in each 163 class were: 17 E genes, 56 ES genes and 71 S genes (Table 1).

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Examination of potential epigenetic regulation of DTGs 166 We next tested our hypothesis that the variation in transcriptional responses of the PIF-167 activated DTGs to darkness and shade might be due to differences in histone tail modifications. 168 One histone mark, H3K27me3, has already been linked to light-mediated transcriptional 169 repression (Charron et al., 2009). Because we were focused on loci at which PIFs act as 170 transcriptional activators, we sought to examine the levels of a histone mark associated with 171 active transcription. One such mark, H3K4me3 is both correlated with actively transcribed genes 172 (Bernstein et al., 2002) and inversely correlated with H3K27me3 levels (Zhang et al., 2009). We 173 therefore chose to assay H3K4me3 levels at the transcriptional start sites (TSS) of E, ES and S 174 genes by ChIP-seq. We measured H3K4me3 levels in dark-grown seedlings and in WL-grown 175 seedlings after exposure to 0, 30, 60, 120 and 180 min of simulated shade, and after 180 min of 176 further retention in WL. We also measured H3K4me3 levels in WL-grown pifS seedlings after 0 177 and 180 min of simulated shade and after 180 min of continued WL. 178 As expected, H3K4me3 levels for E class genes were higher in D than in WL and 179 simulated shade (Figure 3). On average, H3K4me3 levels for ES and S class genes increase over 180 the course of the shade treatment and this increase is attenuated in the pifS mutant ( Figure 4). In 181 both classes, however, the increase only occurs after 60 minutes of FR, while an increase in 182 transcript level abundance is already visible after 30 minutes of FR. Both classes also exhibit a 183 transient reduction in H3K4me3 levels after 30 minutes of FR. Collectively, these data indicate 184 that the shade signal induces a transcriptional response prior to the induction of increased H3K4 185 trimethylation in these DTGs. 186 187 188

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As a prelude to exploring the role of epigenetic factors in light/shade-regulated gene 190 expression, we generated a set of 144 "high-confidence", PIF-induced DTGs, that we identified 191 by integrating our newly obtained data with previously published analyses. This provided three 192 subclasses of PIF-DTGs, displaying three contrasting patterns of transcriptional responsiveness 193 to light and shade signals (E, ES and S) during young seedling development. By focusing on the 194 shade-responsiveness of these gene sets, we were able to concurrently assess whether differences 195 in the epigenetic landscape might be associated with the observed transcriptional pattern 196 differences, and whether comparison of the temporal patterns of shade-induced transcript and 197 H3K4me3 changes might indicate the potential sequence of such changes. 198 Broadly speaking our data are consistent with previous studies reporting that high 199 H3K4me3 levels are correlated with actively transcribing genes. However, comparison of our 200 integrated RNA-seq and ChIP-seq analyses over time following shade exposure, showed no clear 201 temporal coincidence of transcript and H3K4me3 levels. On the contrary, for the shade-induced 202 PIF DTGs, we found that, on average, transcript levels rise before their corresponding H3K4me3 203 levels rise (Figure 4). These results indicate that H3K4me3 plays little or no role in causing or 204 priming the rapid, shade-induced transcriptional responsiveness of these genes. Instead, the data 205 are more consistent with previous reports indicating that high levels of transcription from a given 206 locus leads to trimethylation of H3K4 (Le Martelot et  the binding of PIF7 to the promoter of the ATHB2 gene, and similarly rapidly triggers ejection of 212 the histone variant H2A.Z, as well as increasing H3K9 acetylation (H3K9ac). These findings 213 indicate that PIF7 occupancy of target gene promoters can shape the local chromatin status in 214 response to shade. These changes preceded changes in gene expression, leading to the conclusion 215 that chromatin remodeling is not a consequence of transcriptional activation. Given, firstly, that 216 our data indicate, conversely to those of Willige et al. (Willige et al., 2021), that the shade-217 invoked, PIF-mediated induction of target gene expression appears to precede the increases in 218 H3K4me3 levels at those genes; and secondly, that these H3K4me3 increases are considerably 219 slower than both ( canopies, as a result of breeze-induced movement under natural conditions. The mechanism by 227 which PIF binding activates H3K4 trimethylation remains to be determined. 228 Collectively, these changes in chromatin landscape add another dimension of complexity 229 to the multilayered network of mechanisms and pathways that regulate and intersect with the 230 phy-PIF module.

Generation of PIF DTG list and subcategorization into E, ES and S classes 280
To identify PIF DTGs, we first imposed strict statistically-significant two-fold (SSTF) 281 cutoffs and selected all 764 genes whose expression levels decreased in response to red light 282 (Pfeiffer et al., 2014) and/or increased in response to shade-light (this study). We then further 283 narrowed our list to include only those genes that show a dependence on PIFs for their 284 expression by combining the previously published RNA-seq data from the pifq mutant grown in 285 darkness (Pfeiffer et al., 2014), with our newly obtained RNA-seq data, obtained using the 286 pifqpif6pif7 mutant (pifS) grown in WL and exposed to 3h shade-light. We selected only those 287 genes that were SSTF induced in WT relative to their levels in the corresponding pif mutant. By 288 filtering out those genes that were not among the 764 light-responsive genes identified above, we 289 were left with 278 PIF-dependent, light-responsive genes. Selecting only those genes that were 290 found to be bound by one or more PIF (Hornitschek et al., 2012;Oh et al., 2012;Zhang et al., 291 2013;Pfeiffer et al., 2014;Chung et al., 2020) yielded 169 genes ( Table 1). 292 We subcategorized genes into E, ES and S classes as in Leivar et al, 2012. Class E 293 (formerly Class L) represents genes whose dark-grown wild-type transcript levels are both (a) 294 SSTF higher than those in dark-grown pifq and (b) SSTF repressed by the initial red light (R) 295 signal in WT. Although some Class E genes show a degree of re-induction in the shade, this is 296 weaker (i.e. non-SSTF), and the PIF-dependency is less, than initially in the dark (Figure 2). 297 Conversely, Class S (formerly Class R) represents genes that do display SSTF induction by 298 shade-light, as well as PIF-dependent SSTF induction in the shade, but that do not exhibit a 299 SSTF response to either: (a) the PIFs in dark-grown seedlings, or (b) red light exposure ( Figure  300 2). Finally, Class ES (formerly Class M) represents those genes that display SSTF, mutually-301 converse responsiveness to the onset of the light and shade-light signals, respectively, as well as 302 PIF-dependent SSTF induction, both in the dark and in shade-light (Figure 2). 303 A subset of these E, ES and S class genes exhibited anomalous transcription profiles. We 304 manually removed these 25 genes because they were either highly expressed in WL (6 genes), 305 were induced, rather than repressed, by red light (16 genes), were lowly expressed (1 gene) or 306 were otherwise likely to be artifactual (2 genes). The remaining 144 PIF DTGs were then 307 resorted using relaxed cutoffs. Of the non-anomalous genes first categorized as S class, 38 308 showed a R-dependent reduction (p < 0.1) in transcript levels but were excluded from the ES 309 class because they did not show a SSTF reduction in dark-grown pifq mutant relative to WT. 310 These genes were reclassified as ES. Two E class genes were also reclassified as ES genes 311 because they exhibited a statistically-significant upregulation in response to FR despite not being 312 SSTF downregulated in the pifS mutant. Ultimately, we were left with 17 E genes, 56 ES genes 313 and 71 S genes (

R60
log2FC of transcript levels after exposure of 3-day-old dark-grown seedlings exposed to 60 minutes of R light pifQ log2FC of transcript levels in 3-day-old dark-grown pifq mutant seedlings relative to WT FR30 log2FC of transcript levels in 3-day-old WL-grown seedlings exposed to 30 min supplemental FR

FR60
log2FC of transcript levels in 3-day-old WL-grown seedlings exposed to 60 min supplemental FR FR120 log2FC of transcript levels in 3-day-old WL-grown seedlings exposed to 120 min supplemental FR

FR180
log2FC of transcript levels in 3-day-old WL-grown seedlings exposed to 180 min supplemental FR pS log2FC of transcript levels in 3-day-old WL-grown pifS relative to 3-day-old WL-grown WT exposed to 180 min supplemental FR PIF bound confirmed binding by PIF1, PIF3, PIF4, PIF5 and/or PIF7

New class
New class after resorting: E, ES, S or anomalous (ANOM)