Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade

Abstract

Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genome-wide can be applied to validate dynamic GRN models for any pathway or organism of interest.

Fig. 1. TARGET TF-perturbation assay captures direct targets of NLP7 based on TF regulation or TF binding in isolated root cells.

a Schematic of the cell-based TARGET TF-perturbation system and experimental design (see “Methods”). Root cells isolated from an nlp7 mutant^, transfected with a 35S::NLP7-GR construct were allowed to express the TF–GR fusion protein and sequentially treated with (i) the nitrogen (N) signal transduced by the TF, (ii) cycloheximide (CHX) to block translation, allowing mRNA synthesis of only direct NLP7 targets, (iii) dexamethasone (DEX) to induce NLP7-GR nuclear import. Samples for NLP7 binding to targets as assayed by ChIP using anti-GR antibodies were collected after 0, 5, 10, 30, and 180 min of DEX-induced TF nuclear import. Genes whose expression is affected NLP7 nuclear import were assayed by RNA-seq (steady-state mRNA) or by affinity capture of de novo mRNA using 4tU. b Representative examples of direct targets transcriptionally activated by NLP7. The expression of NRT2.1, LBD37, and CIPK8 is induced by +DEX as compared with −DEX treatment in both steady state and 4tU-enriched RNA-seq experiments (green bars). NLP7 binding was captured by ChIP-seq, and NLP7 peaks were identified by MACS2 using an input sample as a control (red bars) (see “Methods”). NLP7 peaks identified in whole roots by ChIP-chip (orange bars).

Fig. 2. Transient and highly transiently bound NLP7 actively transcribed targets are captured by time-series ChIP and/or DamID.

a Intersection of NLP7 directly regulated genes and NLP7-bound genes captured by time-series ChIP-seq (minutes after DEX-induced nuclear import). Red bars indicate genes that are bound and directly regulated by NLP7. Intersection of these datasets revealed three distinct classes of direct NLP7-regulated targets: (Class I) stable or late, (Class II) transient, (Class III) highly transient. b DamID captures a significant proportion of genes belonging to all three classes, including NLP7–target interactions that were missed by ChIP (Class III). Genes from all three classes are enriched in genes whose active transcription is induced by NLP7 (4tU-labeled). Fisher’s exact test *p-value < 0.001. c The profile of DamID, indicated by the number of normalized DamID sequencing reads in the 1000-bp upstream regions of TSS to the 1000-bp downstream regions of TTS. The DamID profile is similar to the ChIP profile, indicated by the normalized ChIP-seq reads. d LBD37 is an example of active NLP7-initiated transcription (4tU) where binding is captured by both DamID (orange) and ChIP (red). CDF1 is an example of active NLP7-initiated transcription in which TF binding is captured by DamID, but missed by ChIP.

Fig. 3. NLP7-direct targets are enriched in early N-responsive genes including secondary TFs.

a Intersection of NLP7-direct and indirect targets detected in root cells with a time-series of N-response genes in whole roots. The time points represent the just-in-time analysis which binned genes based on the first time point at which their mRNA levels were affected by N treatment at 5, 10, 15, 20, 30, 45, 60, and 90 min. The significance (p-value) of the intersection between NLP7 targets with each N-time point was calculated and −log10 (p-value) was graphed. b Intersection of N-responsive secondary TFs regulated by NLP7 and genes belonging to each class of NLP7 binding. Size of overlap is listed in parentheses, and significance is indicated by yellow highlighting and asterisks (Fisher’s exact test, *p-value< 0.05; **p-value < 0.01; ***p < 0.001). c The transcriptional network regulated by NLP7. NLP7 directly regulates the expression of N-responsive secondary TFs enriched in early time points of the N-response in roots. Node color depicts changes of transcript abundance of TFs in response to NLP7 nuclear import (+DEX/−DEX) (Supplementary Data 5). Edge color corresponds to the different NLP7 mode of actions.

Fig. 4. NLP7-regulated secondary TFs mediate downstream effects of NLP7 in the N transcriptional cascade.

a Intersection of direct targets of validated secondary TFs with NLP7 indirect targets (2059 genes). The significance (p-value) of each intersection was calculated and −log10 (p-value) was used for the heatmap. b Intersection of the union of direct targets of secondary TFs identified in root cells, with N-response genes in whole roots. The significance (p-value) of the intersection between direct targets of secondary TFs with each time point was calculated and −log10 (p-value) was graphed. c DREM2 reconstructed RNA expression NLP7-dependent paths 90 min post N treatment. Each path corresponds to a set of genes that are co-expressed. Split nodes (yellow and blue circles) represent a temporal event where a group of genes co-expressed up to that point diverge in expression, most likely due to regulatory events. d The contribution of secondary TFs mediating each DREM2 path were assessed by intersecting genes from each path with genes induced or repressed by each secondary TF.

Fig. 5. Overexpressing early and transient TF targets of NLP7, HAP2C, and CDF1 significantly increases plant biomass and primary root growth.

Col-0, 35S:NLP7, 35S:HAP2C, and 35S:CDF1 plants were grown for 2 weeks on vertical plates containing different concentrations of N. a Plant growth of NLP7, HAP2C, and CDF1 overexpressor lines is enhanced as compared with wild-type Col-0 plants under increasing N concentrations. b Primary root growth of NLP7, HAPC, and CDF1 overexpressor lines is enhanced as compared with wild-type plants under increasing N concentrations. Data are from three independent experiments (n = 21). Different letters indicate mean values differ significantly between genotypes in each condition (t test, p-value < 0.05). Source data are provided as a Source Data file.

Fig. 6. A validated model for NLP7 role in rapidly initiating a cascade that amplifies the downstream N-response.

Transient interactions of NLP7 initiate early N-response genes including genes involved in N-uptake and early N-response TFs. The transient interactions of NLP7 enable it to rapidly activate secondary TFs leading to a transcriptional burst in a short period of time. Secondary TFs amplify the NLP7-initiated cascade by regulating downstream late N response genes enriched in phosphate, carbohydrate, and amino acid processes (Supplementary Data 26). LBD37 and LBD38 primarily mediate transcriptional repression, and both have been shown to have in planta relevance; CDF1 and TGA4 primarily mediate transcriptional activation; and Int-type and HAP2C act as either gene activators or repressors depending on the target downstream of NLP7. Collectively, the direct targets of NLP7 and direct targets of secondary TFs account for 53% of the N response in plant roots.