Genetic variation in regulatory sequences that alter transcription factor (TF) binding is a major cause of phenotypic diversity. Brassinosteroid is a growth hormone that has major effects on plant phenotypes. Genetic variation in brassinosteroid-responsive cis-elements likely contributes to trait variation. Pinpointing such regulatory variations and quantitative genomic analysis of the variation in TF-target binding, however, remains challenging. How variation in transcriptional targets of signaling pathways such as the brassinosteroid pathway contributes to phenotypic variation is an important question to be investigated with innovative approaches.Here, we use a hybrid allele-specific chromatin binding sequencing (HASCh-seq) approach and identify variations in target binding of the brassinosteroid-responsive TF ZmBZR1 in maize. HASCh-seq in the B73xMo17 F1s identifies thousands of target genes of ZmBZR1. Allele-specific ZmBZR1 binding (ASB) has been observed for 18.3% of target genes and is enriched in promoter and enhancer regions. About a quarter of the ASB sites correlate with sequence variation in BZR1-binding motifs and another quarter correlate with haplotype-specific DNA methylation, suggesting that both genetic and epigenetic variations contribute to the high level of variation in ZmBZR1 occupancy. Comparison with GWAS data shows linkage of hundreds of ASB loci to important yield and disease-related traits.Our study provides a robust method for analyzing genome-wide variations of TF occupancy and identifies genetic and epigenetic variations of the brassinosteroid response transcription network in maize.The online version contains supplementary material available at 10.1186/s13059-023-02909-w. Efforts such as the human ENCODE project have developed genome-wide maps of variants affecting TF binding for mammalian systems and used them to link cis-regulation to key traits such as disease risk [Linking genetic variation to phenotypic variation is the ultimate goal of genetic and genomic studies. While most studies focus on variation in protein-coding sequences, it recently became clear that variation in transcriptional regulation is a major cause of phenotypic diversity . One of ase risk . CurrentArabidopsis has identified thousands of BZR1 target genes, which mediate BR regulation of cell elongation, development of roots, shoots, organ boundaries, reproductive development, and photomorphogenesis and disease resistance [Plant growth and biomass accumulation are controlled mainly by hormones, among which brassinosteroid (BR) has a major growth-promoting effect and diverse functions in development and physiology in all higher plants. BR acts through a receptor kinase (BRI1) signaling pathway to activate the BZR1 family of TFs. Research in sistance .Arabidopsis, BR-deficient maize mutants are extreme dwarfs with severely reduced organ sizes. In maize, BR plays an additional unique role in sex determination, and BR-deficient maize develops female florets and sets seeds on the tassel [Arabidopsisbri1 mutant, demonstrating the conserved function in monocots [Arabidopsis increased organ size [Arabidopsis, rice, and tomato [1 hybrids.Genetic studies indicate that BR acts through a similar signal transduction mechanism and regulates similar and unique developmental processes in crops such as rice and maize. Similar to e tassel –13. BR se tassel , 13–15. monocots . Geneticgan size , 14, 15.gan size . Such BRd tomato , 18, 19.brd1 (BR6ox2) seedlings treated with and without brassinolide (BL), the most active BR. We identified a total of 2743 BR-responsive genes, including 1354 BR-induced and 1389 BR-repressed genes G) and the G-box (CACGTG), which were previously identified as BZR1 binding sites in Arabidopsis [C/ACCA) was also observed, similar to the observation in Arabidopsis [cis-elements and their combination with other factors are conserved in Arabidopsis and maize.To characterize the BR-regulated transcriptional network in maize, we performed RNA-seq on shoot tissue of wild-type as well as BR biosynthesis-deficient rd1 BR6ox seedlingrd1 BR6ox seedlingbidopsis   were previously identified as AtBZR1 targets [The ZmBZR1 binding targets include 38.5% of the BR-responsive genes, including 580 and 469 BR-activated and -repressed genes, respectively, identified in our RNA-seq analysis  and “ma1 hybrid plants. Genetic variations that affect BZR1 binding will show a shift of the allele frequency after ChIP from the expected 1:1 ratio in an F1 . We chose B73 and Mo17 as they are among the most diverse maize inbred lines and their hybrid is one of the most studied [p < 0.001), without an overall bias to either genome  in their surrounding region in the ChIP-input data  as the most enriched Fig. f. This e1 and parental lines  higher portion than genes without an ASB in their promoter   which was altered in the B73 allele (TGTGTG). This is consistent with the weaker BZR1 binding  and lower BR/PPZ response  of an ARF binding motif, which was altered in Mo17. ARF TFs have been shown to directly interact with BZR1 [VP14 promoter allele could be the reduced binding of an ARF/BZR1 heterodimer. Taken together, these results suggest that variation of BZR1 binding contributes to variation of gene expression.To determine whether variations of BZR1 binding contribute to differential gene expression, we compared our ASB data with the transcript levels of B73 and Mo17 alleles in both parents and B73xMo17 hybrid lines. About 37.5%  of all expressed genes with orthologs in both inbred lines showed allele-specific mRNA differences between B73 and Mo17 plants  for 21 of the traits, compared to the bgSNPs with the same average genomic distribution and allele frequency. The largest fraction  were traits related to growth and yield, as expected based on the main functions of BR known from extensive studies in Arabidopsis. Interestingly, other known phenotypic variations between B73 and Mo17, such as tassel branching [To assess the relationship between variations of BZR1 binding and trait variations, we quantified the enrichment of 4015 GWAS hits across 41 traits  within 2ranching  (13 assoranching  (11 assoranching . We usedPGIP2 (Zm00001eb034870), was observed  flagellin receptor FLAGELLIN SENSITIVE 2  [ZmFLS2 expression is affected by Pseudomonas syringae , Fusarium graminearum  [Puccinia polysora (southern corn rust) tolerant line [ZmFLS2 showed multiple BZR1 binding sites in the promoter  in inbred and F1 lines, respectively  with NLB/SLB GWAS hits [ kb with ved Fig. b. PGIPs sistance , 39, 40 sistance . BZR1 bib070510)  located b070510) . Upon flb070510) , 45. ZmFalk rot) , and in ant line . B73 andHormones have major effects on plant growth and development. Genetic variations in plant hormone pathways have great potential for improving crop yield. For example, genetic variations in hormone synthesis and signaling pathways contributed to the green revolution . As a maArabidopsis [cis-elements in the genome and thus the target genes of BZR1 is highly variable between maize and Arabidopsis and even between two maize inbred lines.Our results suggest that BR, through ZmBZR1, is involved in the regulation of thousands of target genes in maize, analogous to findings in bidopsis . Previoubidopsis , raisingtrans-cis interface of signaling pathways, constituting numerous binding sites of TFs, is a major target of evolution to fine-tune or reshape cellular regulatory pathways. Nearly half of quantitative trait variations in maize are explained by accessible, non-coding regions that may contain regulatory elements, e.g., TF binding sites [Our finding of large numbers of ASBs is consistent with the notion that the ng sites , 5. Pinpng sites . Howeverng sites . Our stuWhile promoters are defined by their proximity to genes, distant enhancer regions, although important for gene regulation, are more difficult to pinpoint, in particular in plants, as they frequently lack the specific marks often associated with enhancers in animals . Oka et cis-elements cannot be distinguished. By performing HASCh-seq analysis of allele-specific BZR1 binding in F1 hybrid plants, we avoided both sample-to-sample variation and trans effects. Another approach is DNA Affinity Purification and sequencing (DAP-seq), which analyzes in vitro TF binding to genomic sequences. While DAP-seq analysis can also identify variations in DNA sequence or methylation that affect TF-DNA binding, many in vivo binding events involve interaction between TFs or heterodimerization of TFs and are not identified by DAP-seq using a single TF. The DAP-seq analysis of Arabidopsis BZR1 identified only about 8.5% of the targets identified by ChIP-seq [While molecular approaches such as traditional ChIP-seq have long been used to identify TF binding sites, quantitative analyses of the effects of genetic and epigenetic variations on TF binding often suffer from sample-to-sample variations. Furthermore, when TF-DNA binding is analyzed in separate genetic samples, the contributions of variation in TF abundance and ChIP-seq , 51. IndChIP-seq , 30. Suc1 reflect the variations of DNA sequence and methylation between the two haplotypes. The effect of a motif variation on BZR1 binding is likely similar in the F1 and parental lines, as the DNA-binding domain of ZmBZR1 has an identical sequence in B73 and Mo17. However, other trans-acting factors may not be identical in F1 and either parent, and thus the impact on gene expression and phenotype could be confounded by other factors that are different in the F1 and the two parents. This is especially expected in plants which show strong heterosis, such as maize. Similarly, phenotype data used for GWAS predominantly comes from inbred lines. While the DNA sequence and most DNA methylation status are maintained in the F1, some epigenetic differences such as chromatin status between parental lines might be lost in the F1 and thus missed by HASCh-seq but detected in the parental line.ASBs identified in the FArabidopsis BES1/BZR2, with their target sequence [1 lines as well as with or without exogenous BR treatment. Our results thus suggest that the DNA methylation contributes to variation in ZmBZR1 binding in vivo. The remaining ASBs that displayed neither changes in BZR1 binding motifs nor in DNA methylation may be due to variation in non-canonical BZR1 binding sites, binding sites of TFs that interact/recruit BZR1, or other epigenetic mechanisms such as histone modifications. Our results are consistent with an allele-specific TF binding analysis in human cell cultures, which also found only a minority of putative causative variants in the canonical motif [We identified 1400 variations of canonical BZR1-binding sites, of which ~90% were associated with a decreased BZR1 occupancy. These represent only 23% of all the cases of differential ZmBZR1 occupancy. However, this is a stringent dataset restricted to the lead SNPs of ChIP-seq peaks. There may be additional variants in BZR1 binding sites if all biased SNPs in ChIP-seq peaks were included. Another possible explanation is that DNA methylation, which in vitro has been shown to reduce the interaction of most TFs, including sequence , may consequence , the redsequence . Consistal motif .Setosphaeria turcicum). B73 and Mo17 plants were either treated with the BR inhibitor PPZ, or mock prior to inoculation with S. turcicum spores. Resistance to NLB was scored as incubation period, the number of days following inoculation when the first necrotic lesion appears. B73, but not Mo17, plants treated with the PPZ showed significantly longer incubation peroids (more resistant) to NLB than mock-treated plants , were particularly prominent. In addition, our results hint at a potential role for ZmBZR1 in disease resistance. We also explored a potential role of BR in the NLB  plants were grown side by side in greenhouses, under long-day conditions , and in the 2013–2016 Carnegie Institution for Science summer fields .Construction of the ZmBES1/BZR1-YFP transgenic line was previously described  and obta1 hybrid plants and their non-YFP-carrying siblings (as negative control) were grown under greenhouse conditions for 26 days. The oldest 2 leaves were removed and 2 cm of meristem-enriched tissue was used  and treated the same way (including BL treatment) as described for ChIP but without crosslinking. Tissues were homogenized in liquid nitrogen, and DNA was isolated with the DNeasy Plant Mini Kit (Qiagen). Libraries were prepared using the NEBNext Enzymatic Methyl-seq Kit (NEB) following the protocol for large DNA inserts. Therefore, 200ng genomic DNA was combined with 0.002 ng CpG methylated pUC19 DNA and 0.04 ng unmethylated lambda DNA. Fragmentation was done by using the Diagenode Bioruptor NGS in three rounds, 30s on, 90s off. Agilent Technologies 4200 Tape Station was used to determine the size distribution and concentration of the libraries.Leaf tissue from BZR1-YFP/B73xMo17 FQuality-filtered ChIP-seq reads were aligned to the B73 AGPv4 genome using bwa-mem (v. 0.7.16a)  with defTo analyze the HASCh-seq data, we created a diploid genome concatenating the recently released B73 V5 genome with the Mo17 CAU genome , 56. PotBZR1 binding peaks were determined using the GEM (v3.4) pipeline described using IP samples against the negative control obtained from ChIP on non-YFP sibling plants. First high-quality peaks were called using the merged file of all ChIP-seq replicas . To obtain enough coverage for GEM peak calling in the replicates, we combined the 6 into 3 replicates (1x B73xMo17 and 1x Mo17xB73 replicate) . Only high-quality peaks that overlap with peaks in all 3 replicates were retained using bedtools (v2.29.0). Although our focus was to perform allele-specific analysis, we also include a peak file of the merged hybrid BZR1-YFP ChIP-seq data compared to the negative control in all replicates that contains not only unique, but also shared peaks between the B73 and Mo17 genome . Of the remaining 429,236 SNPs, we determined significant variation of median allele frequency of 0.494 using a binomial test with a p-value cutoff of ≤ 0.001 adjusted for multiple testing using Bonferroni correction . To focus on ASBs with potential biological relevance, we further restricted ASBs to those located in high-confidence BZR1-binding peaks reproducible in all the biological replicates . While TFs usually bind small DNA regions of ~10 bp [n=7817). Finally, we excluded ASBs that showed a significant bias (p<0.05) in their surrounding region (+/− 1 kb) in the ChIP-input data , to avoid potential artifacts . To measure bias significance of ASBs and establish an empirical significance threshold, a specific set of control background SNPs were proportionally sampled (excluding ASBs) per chromosome in order to establish a distribution of biases in their surrounding regions (+/− 1000 bp per background SNP). Hence, ASBs whose biases were beyond the upper and lower 5 percentiles  were excluded (n=6143 ASBs) from further analysis.ZmBZR1-YFP ASBs were determined using custom R (v. 3.3.2) scripts. In order to accurately access allele frequencies of all homozygous SNPs, we set a minimum read coverage cutoff of ≥ 1 reads for both alleles and ≥ 25 for at least one of the alleles, neglecting SNPs located on scaffolds . Arabidopsis identifiers of Maize homologs were used to avoid any bias of the annotation state of the two species.Functional enrichment analysis for Functional GWAS variants have been shown to be significantly enriched in gene proximal regions . TherefoHeterozygous BZR1-YFP plants were grown in the dark at RT for 10 days in vermiculite with and without 10 µM PPZ. Prior to imaging, 1-cm root tip segments were removed from the mock and PPZ-treated seedlings. The root tip of PPZ-treated plants again was treated for 15 min with 10 µM PPZ with or without 1 µM 24epi-BL.brd1 mutant siblings were grown in soil under greenhouse conditions for 26 days as described above. The oldest 2 leaves were removed, and 2 cm of meristem-enriched tissue  were harvested and total RNA extracted using the RNeasy kit . The NEB directional Ultra II RNA library kit was used to construct poly-A enriched, barcoded libraries. The default fragment insert size was increased to ~400 bp + adapters, to enhance the yield of reads containing B73/Mo17 variants. Sequencing reads were mapped to the concatenated B73 and Mo17 genome described above using STAR . The maizegdb pan-gene dataset was used to determine orthologous B73 and Mo17 genes. In contrast to B73v5, almost all Mo17 CAU gene models lacked 5′ and 3′ UTRs. In addition, the increased use of PacBio long-read technology for the B73v5 compared to the Mo17 CAU annotation may explain the B73 bias in our initial allele-specific transcript quantification. To reduce this bias, we standardized 5′ and 3′ UTRs of both B73 and Mo17 genes to 500 bp from the translation start/stop and removed orthologous transcripts with > 50 bp cds length differences from the analysis. Only reads mapping uniquely to B73 or Mo17 and only those overlapping with a single gene model and at least 20 reads in total and at least one read per allele were considered for further analysis. For comparison with ASB genes, genes with 1.5-fold variations in transcript allele frequencies were considered. To further avoid annotation differences in Mo17 and B73 due to the missing UTR annotations in Mo17, ASB genes were annotated to the B73 allele only in this case and then their mRNA abundance in the respective B73/Mo17 pair was considered.For the analysis of gene transcript differences between B73 and Mo17, two parallel data sets were analyzed. First, a previously published RNA-seq data set was used  includinhttps://github.com/FelixKrueger/TrimGalore), respectively. Both inbred and hybrid methylation data was mapped to the respective genomes  using Bismark (v0.22.3) [Inbred methylation levels for CG, CHG, and CHH for B73 and Mo17 were extracted from Regulski et al. . Low-quav0.22.3)  with bowv0.22.3)  as mappev0.22.3) . MethylaFor local motif enrichment analysis Fig. E, we extTo identify ASBs overlapping with motif variation Fig. h, we ext1 hybrids or inbreds, depending on the analysis, of one allele was ≥ 70% while the level of the corresponding allele was ≤ 10%.To identify ASBs which overlapped with significant variation in either CG, CHG, or CHH methylation between B73 and Mo17, we first, per ASB, assigned averaged methylation levels of Mo17 and B73 methylation levels  within a given window of +/− 20 bp around the ASB position. Differentially methylated alleles were defined as described previously . AccordiPutative B73 enhancer Fig. a regionsWe tested association of ASBs with the curated 4041 significant GWAS hits for 41 different phenotypes of the NAM population . Per trawww.panzea.org). In the analysis, we mapped ASB SNPs and randomly sampled bgSNPs that shared the similar genomic patterns to upstream 5 kb - TSS, within the CDS, TTS - 5 kb downstream of genes as well as intergenic regions. Since the Mo17 CAU annotation often lacks 5′ and 3′ UTRs, both the ASB and bgSNP positional annotation for VCAP was based on their B73 coordinates. The bgSNPs were resampled based on the ASB distribution for a total of 254,017 VCAP-bgSNPs. For each SNP subset, we calculated an additive kinship matrix using the variance component annotation pipeline (VCAP) implemented in TASSEL5 [We estimated the variance components explained by different ASB SNP subsets and the remaining SNPs using the maize NAM population . To cond TASSEL5 . We then TASSEL5 .Co-localization of candidate genes within joint linkage QTLs for resistance to NLB and SLB was represented graphically using R version 3.2.3 . The start and end sites of NAM QTL were identified in AGPv2 for NLB  and SLB Setosphaeria turcica isolate NY001 (race1), containing 4000 spores/ml, into the whorl during the late afternoon. An overhead mister provided water for 10 s every 10 min for approximately 15 h. Disease phenotype was measured as the incubation period (IP), the number of days following inoculation when a necrotic lesion was first observed. Forty-eight days following inoculation, the disease measurements were concluded; a survival analysis was performed in JMP® Pro Version 13.1.0  to test for differences within each genotype for the effect of PPZ on IP using a log-rank test statistic and visualized in a Kaplan-Meier plot.A 2 × 2 factorial experimental design included three replications, B73 and Mo17 genotypes, and two treatments (with or without PPZ) within each replication. B73 and Mo17 were planted (n>7) in 10 cm pots (1 plant per pot) in the greenhouse. At the V6-stage, the plants were treated with either 400 µM PPZ or mock solution through soil fertilizer drenching every 3 to 4 days. Four days after the initial PPZ treatment, the plants were inoculated with a 500-µl liquid spore suspension of Additional file 1: Table S1. List of 2743 BR responsive genes in Maize determined by RNA-seq.Additional file 2: Supplementary figures. Fig. S1. Additional QC data for BZR1-YFP and HASCh-seq. Fig. S2. Multiple sequence alignment of BZR1 DNA-binding domain. Fig. S3. IP-Input allelic bias control for HASCh-seq. Fig. S4. ASBs of ZmBZR1 overrepresentation at enhancer sites. Fig. S5. Differential NBL resistance of B73 and Mo17 in response to BR inhibitor PPZ treatment. Fig. S6. ASBs overlapping with complex differences in ZmBZR1 binding pattern. Fig. S7. ASBs and bgSNPs share similar minor allele frequency.Additional file 3: Table S2. List of 17463 putative ZmBZR1 binding sites of the B73 inbred line.Additional file 4: Table S3. List of 6371 ZmBZR1 target genes. Target genes were defined if the gene CDS +5 kb upstream or -1 kb downstream overlapped with a ZmBZR1 peak.Additional file 5: Table S4. List of 2058 conserved BRZ1 target genes in Maize with orthologs in Arabidopsis.Additional file 6: Table S5. List of GO-slim plant terms found enriched in Maize only BZR1 target genes, Arabidopsis only BZR1 target genes or both. Significantly over-enriched terms in the respective category are marked by a green background, non-enriched ones in either Arabidopsis only or maize only are marked in red, under-enriched terms are marked by red font color of the p-values. For comparison, p-values and enrichment of shared BZR1 target genes are displayed for the same categories.Additional file 7: Table S6. List of 52765 ZmBZR1 binding sites reproducible in all 6 replicas of B73xMo17 F1 hybrids and genes annotated to these regions. Genes with significant BZR1 binding sites from 5 kb upstream to 1 kb downstream of the TSS and TTS, respectively, were considered target genes.Additional file 8: Table S7. List of ZmBZR1 target genes in B73xMo17 hybrids. Genes with significant BZR1 binding sites from 5 kb upstream to 1 kb downstream of the TSS and TTS, respectively, were considered target genes. Gene names are given in B73 and Mo17 coordinates where a homologous gene exists, otherwise only one gene is listed.Additional file 9: Table S8. List of 33267 SNPs with significant allele-specific bias in linkage groups.Additional file 10: Table S9. Genomic location of the 6143 lead SNPs of allele-specific BZR1 binding site, genomic locations of ASBs and association with genes at the high-affinity allele.Additional file 11: Table S10. List of putative ASB ZmBZR1 target genes. Genes with significant BZR1 binding sites from 5 kb upstream to 1 kb downstream of the TSS and TTS, respectively, were considered target genes. Gene names are given in B73 and Mo17 coordinates where a homologous gene exists, otherwise only one gene is listed.Additional file 12: Table S11. ASBs overlapping with variations in motifs and/or DNA methylation.Additional file 13: Table S12. Raw RNA-seq counts in the B73xMo17 hybrids for genes with homologs in both genome annotations. Fold-changevalues were calculated for each replicate and an average FC was calculated to be employed for further analysis. For comparison with ASB genes, only genes with differences in CDS length <=50bp between B73 and Mo17 were employed.Additional file 14: Table S13. ASBs co-locolized within 2 kb of the 4015GWAS hits from Wallace et al. 2014Additional file 15: Table S14. Primers employed in this study.Additional file 16: Table S15. F1 samples sequencing statistics.Additional file 17: Table S16. MACS3 HASCh-seq peaks of reads that map uniquely to one or both parents. BZR1-YFP replicates vs. non-transgenic negative control were used for the peak calling.Additional file 18. Review history.


Early research efforts focused on PTH and 1,25(OH)2D3 and more recently on FGF23 have revealed that each of these hormones regulates the expression of the other two. Despite early suggestions of transcriptional processes, it has been only recently that research effort have begun to delineate the genomic mechanisms underpinning this regulation for 1,25(OH)2D3 and FGF23; the regulation of PTH by 1,25(OH)2D3, however, remains obscure. We review here our molecular understanding of how PTH induces Cyp27b1 expression, the gene encoding the enzyme responsible for the synthesis of 1,25(OH)2D3. FGF23 and 1,25(OH)2D3, on the other hand, function by suppressing production of 1,25(OH)2D3. PTH stimulates the PKA-induced recruitment of CREB and its coactivator CBP at CREB occupied sites within the kidney-specific regulatory regions of Cyp27b1. PKA activation also promotes the nuclear translocation of SIK bound coactivators such as CRTC2, where it similarly interacts with CREB occupied Cyp27b1 sites. The negative actions of both FGF23 and 1,25(OH)2D3 appear to suppress Cyp27b1 expression by opposing the recruitment of CREB coactivators at this gene. Reciprocal gene actions are seen at Cyp24a1, the gene encoding the enzyme that degrades 1,25(OH)2D3, thereby contributing to the overall regulation of blood levels of 1,25(OH)2D3. Relative to PTH regulation, we summarize what is known of how 1,25(OH)2D3 regulates PTH suppression. These studies suggest that it is not 1,25(OH)2D3 that controls PTH levels in healthy subjects, but rather calcium itself. Finally, we describe current progress using an in vivo approach that furthers our understanding of the regulation of Fgf23 expression by PTH and 1,25(OH)2D3 and provide the first evidence that P may act to induce Fgf23 expression via a complex transcriptional mechanism in bone. It is clear, however, that additional advances will need to be made to further our understanding of the inter-regulation of each of these hormonal genes.The regulation of mineral homeostasis involves the three mineralotropic hormones PTH, FGF23 and 1,25-dihydroxyvitamin D We also review existing support for the mechanisms through which 1,25(OH)2D3 has been proposed in early studies to regulate PTH transcription, although advances here are limited. Finally, we discuss the results of our recent studies of the regulation of Fgf23 transcription using an approach involving gene modification via CRISPR-Cas9 gene editing followed by loss of Fgf23 regulation studies in vivo. These efforts attempt to identify the genomic control regions within the Fgf23 gene locus that mediate the regulatory actions of 1,25(OH)2D3, PTH and phosphate (P) at this gene and ultimately to identify unique factors involved in these transcriptional mechanisms. While our focus is on hormonal inter-regulation, it is worth noting that calcium (Ca) and P do not appear to directly control the expression of the renal Cyp27b1 and Cyp24a1 genes, but rather that Ca directly regulates PTH expression whereas P controls FGF23 levels; these in turn then control the production of 1,25(OH)2D3. For this reason, we also include a brief discussion of Ca and a direct consideration of P, with a particular emphasis on P regulation of Fgf23 expression. It will also be evident that it has been the application of several new technologies that helped advance our understanding of this inter-regulation, particularly that regarding regulation of PTH and FGF23. This paper does not represent a thorough review of these subjects, but rather a summary of the recent work that we have done aimed at understanding the molecular mechanisms of this regulation.The maintenance of mineral metabolism is regulated predominantly by 1,25-dihydroxyvitamin Dnd FGF23 Figure 12D3 which represents the primary endocrine source of this hormone, followed by parathyroid gland-produced PTH and bone-derived FGF23, we begin this paper by discussing the new approaches that we have taken to identify the mechanisms involved in gene regulation. Indeed, the development of chromatin-immunoprecipitation (ChIP) and other genomic procedures coupled to next-generation DNA sequencing technics  and their applications have fully changed the way in which gene regulation is explored explored . ChIP-sein vivo indicate several thousand binding sites for the vitamin D receptor (VDR), a number consistent with other regulatory factors; the number and location of these sites for transcription factors in general are, however, dependent upon the factor involved, and are affected by species, the type of target cell studied, and nutritional status, among other variables , the VDR represents a ligand-dependent DNA binding factor  of two hexameric DNA half-sites separated by three base pairs. This DNA structural organization represents a typical VDRE now observed at hundreds of genes and in all species examined. Interestingly, however, VDR binding at sites known to function in repression are more diverse, and often contain novel DNA motifs, suggesting that the VDR may be bound to these sites indirectly via an association with other DNA or non-DNA binding proteins serving additional regulatory functions. These observations at genome-wide levels both confirm and extend our understanding of several key features of vitamin D action via the VDR at target genes and are generally consistent with features of other transcription factors of this type. Perhaps the most important discovery arising from unbiased genome-wide analyses of not only VDR/RXR binding sites, but other transcription factor regulatory sites termed enhancers is the observation that these regulatory sequences are not located exclusively near target gene promoters as previously suspected. Rather, they can be found at sites that are commonly distributed within intergenic regions both upstream and downstream of the genes they regulate, often with unregulated genes interspersed, and frequently within the introns of the target gene itself or in unregulated genes located adjacent to the regulated gene or both in vitro and particularly in vivo, the latter at molecular levels equivalent to those heretofore achievable only in cultured cells. Details now extend to the coupling of changing epigenetic architecture in response to genetic and epigenetic action at regulatory loci to the recruitment of additional transcription factors that play direct roles in promoting or inhibiting transcriptional output 2D3 production by the kidney and FGF23 production in bone were conducted in wildtype and mutant mice in vivo.The technological advances as above have led to new methods to characterize the details of gene regulation both l output . These i2D3 production by the kidney. This hormone plays a central role in the regulation of mineral metabolism by virtue of its unique function to induce Ca and to a lesser extent P absorption from the diet via the intestinal enterocyte 2D3 production in the kidney is PTH which induces the expression of the Cyp27b1 gene whose enzymatic product 25(OH)-1α-vitamin D3 hydroxylase (CYP27B1) is responsible for the synthesis of 1,25(OH)2D3 2-24-vitamin D3 hydroxylase (CYP24A1) is responsible for the initial degradation of 1,25(OH)2D3 2D3 and raising the levels of 1,25(OH)2D3. That the regulation of Cyp24a1 is transcriptional in nature has only recently been defined in molecular terms, as will be discussed herein. Together, these two enzymes regulate the level of 1,25(OH)2D3, which is secreted into the blood where it functions as an endocrine hormone. Thus, the coordinated regulation of both Cyp27b1 and Cyp24a1 expression in the kidney by PTH is essential to both the production as well as the maintenance of appropriate levels of circulating hormonal 1,25(OH)2D3. Since the elaboration of PTH from the parathyroid glands is controlled by decreasing blood levels of Ca, an inverse relationship emerges between blood Ca and PTH, and thus control of circulating 1,25(OH)2D3 2D3, while Cyp24a1 is coordinately induced by each of these hormones.We focus first on the regulation of 1,25(OH)terocyte . Althoug5(OH)2D3 . As impo5(OH)2D3 . Accordi5(OH)2D3 . As will2D3, it has been only recently that the mechanisms associated with the induction of Cyp27b1 and suppression of Cyp24a1 by this hormone have emerged. These findings were facilitated by the technics discussed above, including both ChIP-seq analysis and CRISPR-Cas9 mediated gene editing. To further the relevance of their application as well as our results, we applied these techniques to mice and conducted each of these analyses in vivo. As PTH is known to stimulate cAMP, we explored using ChIP-seq analysis the possibility that PTH might induce CREB at specific sites across the Cyp27b1 locus in tissue derived from the kidney cortex of normal wildtype mice  and discovered that pCREB could be detected at several sites upstream of the Cyp27b1 gene, one located within an intron of the adjacent Mettl1 gene and three located further upstream in an intron of the Mettl21b (Eef1akmt3) gene  resulted in a profound mouse phenotype insensitive to exogenous PTH and consistent with a previously prepared mouse containing a mutation in Cyp27b1 that prevented the global expression of the active CYP27B1 enzyme  in the genome of mice resulted in a much more modest phenotype comprised of only slightly reduced sensitivity to PTH, and normal levels of Ca, P, and 1,25(OH)2D3 , it was not responsive to PTH in any of these tissues ype mice . Followit3) gene Figure 2periment . PTH als1 enzyme , 21. The5(OH)2D3 . These a5(OH)2D3 . Importaed mouse , 22. Mor2 and S3 . While C tissues . Thus, wCyp27b1 activation. Thus, activation of Cyp27b1 in this case is mediated largely via the PTH mediated upregulation of two known coactivators of DNA-bound pCREB. The distinct role of each of these coactivators remains to be determined, however. Although widely expressed CBP retains multiple functions, one of which may be in the acetylation of histone H3K27; a second hypothetical function in the case of Cyp27b1 may be to participate in the 3D repositioning of the four distal CREB enhancer sites at or near the Cyp27b1 promoter 2D3 thereby facilitating the overall upregulation of renal 1,25(OH)2D3 that raises the hormones levels in the blood. While it has long been known that PTH suppresses Cyp24a1 production, the mechanism of this decrease has evaded discovery; indeed, studies during the past decade suggested a post-transcriptional mechanism involving Cyp24a1 transcript stabilization , supporting the concept that PTH is only capable of downregulating Cyp24a1 in the kidney 2D3.While PTH induces 1 levels . This relization . Interesene body Figure 3e to PTH . These lCyp27b1 regulation, we also explored the role of FGF23 and 1,25(OH)2D3 itself in renal Cyp27b1 expression. Interestingly, while PTH speaks to the unique and direct upregulatory control of renal 1,25(OH)2D3 production in response to an increased need to elevate blood Ca levels, both FGF23 and 1,25(OH)2D3 itself are negative regulators that seek to downregulate the production of 1,25(OH)2D3 in response to unique physiologic/metabolic determinants 2D3 to reciprocally induce Cyp24a1, thus collectively reducing blood 1,25(OH)2D3, as discussed in the next section 2D3 likely exerts its effects exclusively in the kidney via a short negative feedback mechanism. Nevertheless, despite an early understanding of the actions of 1,25(OH)2D3 and more recently of FGF23 to reduce 1,25(OH)2D3 production in the kidney via a downregulation of Cyp27b1, a delineation of the mechanisms involved have generally resisted research efforts. Importantly, while both FGF23 and 1,25(OH)2D3 suppress Cyp27b1 expression in wildtype mice, deletion of either the M1 or M21enhancer regions using CRISPR/Cas9 gene editing in vivo reduced but did not eliminate the ability of either to suppress Cyp27b1 expression 2D3 as measured by changes in residual pCREB abundance, or through striking suppression in the levels of residual CBP or CRTC2 at the pCREB sites within these M1 or M21enhancers, or in the ability of FGF23 to confer a differential profile of histone acetylation, these studies did not advance our understanding 2D3 is more effective 2D3 appear to prevent both residual actions as well as the upregulation of Cyp27b1 by PTH. These results provide novel mechanistic evidence for how these three hormones dynamically regulate the expression of renal Cyp27b1 and therefore the production of circulating 1,25(OH)2D3.Returning to our investigation of renal rminants –30. Desp section . It is apression . Deletioar sites Figure 2standing . Finallystanding . We alsoffective . These rCyp24a1 expression has long been known to be positively regulated by 1,25(OH)2D3 in tissues that include not only the kidney but in most VDR containing NRTCs as well. In contrast, while the more recent discovery that FGF23 regulates Cyp24a1 has suggested that this may occur in several tissue types, the bulk of evidence suggests that FGF23 regulation of Cyp24a1 is restricted to the kidney where FGF23 is biologically active 2D3 has long been known to induce Cyp24a1 expression via a specific couplet of VDREs located immediately upstream of the Cyp24a1 promoter  using CRISPR/Cas9 gene editing in the mouse genome and assessed the ability of FGF23 and 1,25(OH)2D3 to induce Cyp24a1 following hormonal injection 2D3 to induce Cyp24a1. Loss of the DS2 region also resulted in a modest decrease in 1,25(OH)2D3 induction in the kidney and other tissues as well. While these results may appear to conflict, it is worth noting that retention of the apparently dominant VDRE located near the promoter remained active in vivo and thus likely underlined the limited loss of 1,25(OH)2D3 response seen in the DS2-deleted mouse. This complexity suggests that additional experiments are needed to dissect the contributions of the promoter proximal VDREs in the Cyp24a1 gene. However, we conclude that the sites of action of FGF23 and 1,25(OH)2D3 across this gene locus are likely distinct, with FGF23 sites located within the DS1 region and the 1,25(OH)2D3 inducible sites located within the DS2 region as well as at the promoter. Importantly, however, further analysis using ChIP-seq analysis revealed additional relevant information. Although both FGF23 and 1,25(OH)2D3 are strong agonists at the Cyp24a1 gene, 1,25(OH)2D3 is a much more robust inducer of Cyp24a1 than FGF23 2D3 exhibited more activity at DS2 and at the promoter proximal region. Interestingly, we noted that while both hormones exerted only nominal action on CRTC2, 1,25(OH)2D3 was much more effective at the Cyp24a1 promoter. This surprising finding suggests that in addition to PTH, 1,25(OH)2D3 may also be able to influence CRTC2 migration to the nucleus. While the actions of FGF23 and 1,25(OH)2D3 are not entirely clear at the levels of pCREB downstream factors, the two hormones’ actions are largely similar at the level of epigenetic modification and the consequences that lead to RNA upregulation. In short, both hormones induce H3K27ac across similar regions of the gene, although 1,25(OH)2D3 is unique in that it also strongly induces H3K9ac. Finally, both hormones induce the recruitment of BRD4, although FGF23 is most active at DS1 whereas 1,25(OH)2D3 is most active at DS2, yet both are active at the promoter. Finally, RNA polymerase II is strongly recruited to the Cyp24a1 gene under both hormone treatments, and this is reflected in the appearance of H3K36me3 across the gene body itself. Thus, there are both common as well as unique properties of both hormones, although it is clear 1,25(OH)2D3 is much more active at the Cyp24a1 gene than FGF23, a feature that can be seen in the relative ability of the two hormones to induce Cyp24a1 RNA transcripts. These data provide significant insight into the mechanisms through which FGF23 and 1,25(OH)2D3 regulate Cyp24a1, although additional work will be required to place the two hormones in perspective, not the least of which will be the identification of the transcription factor that mediates FGF23 action.y active . While Ftor EGR1 –36. Imporeported . And notpromoter –40. Theserodimer . This dian FGF23 . Thus, wCyp27b1 and Cyp24a1 expression in the kidney that is central to the production and maintenance of endocrine 1,25(OH)2D3 by PTH, FGF23 and 1,25(OH)2D3 itself. These events are summarized in in vivo level in mice when the M1 and M21 enhancers are deleted in the mouse, preventing the production of 1,25(OH)2D3 from being synthesized by CYP27B1. Under these conditions, the circulating levels of 1,25(OH)2D3 are strikingly reduced, but not eliminated. This level is insufficient to maintain the skeleton due to high PTH levels and reveals a striking Cyp27b1 null-like phenotype 2D3 synthesized in the kidney to be sustained. We ultimately provide evidence of this hypothesis by rescuing this M1/M21-deleted mouse with a diet rich in Ca and P, which brings down PTH and raises FGF23 to normal levels thereby increasing renal Cyp24a1 expression. Under these conditions, the levels of endocrine 1,25(OH)2D3 in the blood are reduced to undetectable concentrations, indicating the importance of the regulation of both renal Cyp27b1 and Cyp24a1 levels in the maintenance of circulating 1,25(OH)2D3.The above discussion defines the reciprocal co-regulation of 2D3 synthesis by renal Cyp27b1 and likewise retards the hormone’s degradation via suppression of Cyp24a1, it should not be surprising that circulating 1,25(OH)2D3 might be a primary feedback suppressor of PTH expression. In fact, early studies suggested that was indeed the case. Accordingly, both cells in culture and rodents treated with exogenous 1,25(OH)2D3 exhibited a striking downregulation at the level of PTH mRNA in response to the hormone. Response was uncharacteristically slow in both, however, requiring 48 hours to achieve baseline levels suggesting additional mechanisms (2D3 and indeed most effective agonists of this hormone is to suppress PTH levels in various disease states that include chronic kidney disease (CKD) and perhaps other diseases characterized by inflammation 2D3 chanisms –44. Manychanisms . One of ammation –48. Zemp5(OH)2D3 , 50.2D3 might suppress PTH gene expression was explored several decades ago, when it was revealed that the VDR suppressed an artificial construct comprised of a reporter linked to the proximal region of the PTH gene. Accordingly, the VDR was found to bind to a sequence described as a negative VDRE located immediately upstream of the PTH start site art site . This mePTH gene Figure 52D3 on PTH may be secondary to a hormone induced rise in blood Ca which likely reduces PTH levels.Perhaps most interesting are recent studies in which a mouse model was examined wherein the VDR was selectively deleted from the parathyroid glands using the PTH gene promoter driving CRE . SurprisOver the past decades, the discovery of the G protein-based calcium sensing receptor that modulates PTH regulation via Ca sensitivity has been the focus of research on PTH regulation . The litThe literature is replete with studies suggesting that FGF23 regulates PTH. These studies support both positive as well as negative effects and are almost always conditional . Given tThis short summary outlines what is largely known historically of PTH gene regulation. It is not surprising that key details remain to be explored, as useful parathyroid cells are generally not available thereby restricting the use of current techniques described earlier in this manuscript.Cyp27b1 and induces Cyp24a1 gene expression as detailed earlier, thereby reducing the synthesis of 1,25(OH)2D3 and thus the potential intestinal uptake of both Ca and P. The mechanism of action of FGF23 is dependent upon the exceptionally high affinity of this hormone for the renal FGF receptors by virtue of their interaction with α-klotho, a renal co-receptor that functions to elevate the subsequent binding affinity of FGF23 for the appropriate FGFRs  FGFR1c and FGFR3 in the kidney, decreasing P reabsorption in the proximal tubules via the positional regulation in the brush border of Na,-P transporters NAPI2A and NAPI2C , 30, 55.te FGFRs . Activat pathway .Fgf23 by not only the primary mineral regulating hormones such as PTH and 1,25(OH)2D3, but factors that participate in this regulation of Fgf23 at the genomic level. Indeed, early studies suggested the locations of VDRE-like sequences within the upstream region of the Fgf23 gene using traditional transfection assays 2D3 and P at the Fgf23 gene, with the supposition that the identification of the precise genomic regions that mediate such regulation might provide clues as to the nature of transcription factors involved. While certain factors seem likely to participate in the induction of Fgf23 by PTH and 1,25(OH)2D3, for example CREB and the VDR, there do not appear to be likely candidates for a pathway(s) that might mediate P regulation, necessitating an unbiased search. As will be seen in the following description of our current efforts, the presumption of involvement and the mechanisms of such involvement of anticipated specific targets in these early studies has been highly misleading. Indeed, for example, while it is known that the VDR mediates the actions of 1,25(OH)2D3 at numerous target genes, it appears that the pathway whereby Fgf23 is induced by 1,25(OH)2D3 may be indirect and involve the activity of secondary factors. CREB activation represents a natural mediator of PTH. However, although many of the upstream components of PKA induction by PTH are now well known, the molecular mechanisms through which this pathway triggers controls of SIK inhibition and regulates CREB activity remain to be clarified, as highlighted in our discussion of PTH regulation of coregulators of CREB at the Cyp27b1 gene  deleted mouse, no effect was seen in any of the other mouse mutants. Further ChIP-seq analysis of osteocytes revealed the presence of pCREB exclusively at this distal enhancer. Interestingly, recent studies suggest that several SIK inhibitors that promote the recruitment of the CREB receptor transcriptional coactivator 2, (CRTC2) to the Cyp27b1 gene in mouse kidney also induces Fgf23 in the bones of those same mice 2D3 in rat UMR bone cells revealed that the pattern of temporal induction of NURR1, as well as NURR2 and NURR3 and other immediate early genes such as EGR1, 2 and 3 are also upregulated ). These studies allow us to conclude that it is unlikely that the mediator of PTH action at Fgf23 in bone is NURR1. In mice, the rapid actions of exogenous PTH resulted in PTH induction of Fgf23 bone mRNA, and prevented the delayed but coordinated detection of upregulated iFGF23 in the blood, as will be seen for 1,25(OH)2D3 and P.Early exploration of the function Figure 6function . Two sitame mice . These r2D3 upregulates Fgf23 expression at the transcriptional level and is known to require the presence of VDR in both mice and in cells in culture. In cells, this induction is rapid and reminiscent of most other genes that are induced by this hormone 2D3, we were unable to identify the presence of the VDR at any sites across the Fgf23 gene locus, despite its presence at numerous known vitamin D target genes in these bone cells 2D3 might be indirect, and mediated by the ability of this hormone and its receptor to rapidly induce one or more secondary transcription factors that might interact directly with the Fgf23 gene. This result prompted further exploration of the Fgf23 gene and revealed that deletion of only the promoter proximal (PPE) but not the DE16K region of Fgf23 in mice using CRISPR/Cas9 gene editing was essential for loss of 1,25(OH)2D3 induction of Fgf23 2D3 but to P (see below) and other activators as well. Accompanying the quantification of bone Fgf23 mRNA, we also determined whether any changes in the level of mRNA resulted in a change in the circulating levels of intact (i) FGF23 (iFGF23); a change in both was necessary for us to conclude that the deletion resulted in a loss of function. The loss of response to 1,25(OH)2D3 occurred in mutant mice in which deletions were created downstream of the VDRE-like sequence proposed by Liu et al. 2D3. A recent alternative evaluation of this region appears to concur, speculating in detail that this site and transcription factors other than NURR1 may be involved 2D3 sensitive genomic space that appears to be functionally relevant in vivo in an unbiased search for potential transcription factor candidates that are amenable to technical confirmation as mediators of 1,25(OH)2D3 action. Importantly, these data suggest a novel, yet transcriptionally consistent, genomic mechanism whereby 1,25(OH)2D3 induces the expression of an important gene involved in mineral homeostasis.1,25(OH) hormone . Neverthne cells . Our mosof Fgf23 . The unuu et al.  and upstal.  in the measurement of P response, including the upregulation of Fgf23 and prevented exploration of mechanisms whereby P might induce Fgf23. To overcome this issue, we developed a method whereby elevated dietary P could be rapidly delivered to mice and the upregulation of Fgf23 mRNA evaluated within several hours as described previously; importantly, both the upregulation of Fgf23 mRNA as well as blood iFGF23 could be detected within that time frame 2D3. As with the steroid hormone, a loss of P response was identified both upstream of the NURR1/VDRE-like site. Deletion of the NURR1/VDR-like site also had no effect on loss of Fgf23 mRNA or Fgf23 induction. Thus, a single upstream DNA segment identical to that for 1,25(OH)2D3 induction was apparent 2D3 target the same DNA sequence(s) or utilize the same transcription factor(s). Studies are in progress with additional mutant mice to refine the sites of action of P at the Fgf23 gene and to identify functional motifs useful for further exploration of the mechanism of acute P action.To further explore the site of acute action of P at the apparent . It is w2D3 and P, we also explored the general response in bone via RNA-seq analysis Our working hypothesis is that a genome wide response as determined by an RNA-seq analysis and subsequent analysis of regulated RNAs might provide information regarding not only a relationship between the two inducers, but might reveal additional useful pathways for P in particular, and possible reveal novel transcription factor candidates as well. Of course, the actions of each of these inducers may not involve the induction of regulators via transcription but rather the activation of other signaling pathways as well. One pathway that is common to both 1,25(OH)2D3 and P is an upregulation of Wnt/β-catenin signaling, observed via an increase in the expression of numerous Wnts and the downregulation of numerous Wnt inhibitors OH)hibitors . However2D3, and FGF23. While these hormones coordinate the regulation of both Ca and P in multiple organs including the intestine, bone, and kidney, they also exert additional hormone-dependent actions in additional tissues that are unrelated to the control of mineral metabolism. Relative to mineral metabolism, numerous diseases are associated with the actions of these hormones; altered mineral metabolism also emerges due to a wide variety of human disease states. Importantly, each of these hormones coordinate actions that include the regulation of the other two. While this has been widely known, the molecular mechanisms responsible for this inter-regulation are only now emerging, due in part to the development of new technologies that have enable the studies described herein.The regulation of mineral metabolism is highly complex and involves three dominant hormones that includes PTH, 1,25(OH)JP drafted and edited the manuscript; MM edited the manuscript; SL edited the manuscript. All authors contributed to the article and approved the submitted version.


Nitrogen (N) is a crucial nutrient that plays a significant role in enhancing crop yield. Its availability, including both supply and deficiency, serves as a crucial signal for plant development. However, excessive N use in agriculture leads to environmental and economic issues. Enhancing nitrogen use efficiency (NUE) is, therefore, essential to minimize negative impacts. Prior studies have investigated the genetic factors involved in N responses and the process of low-nitrogen (LN) adaptation. In this review, we discuss recent advances in understanding how epigenetic modifications, including DNA methylation, histone modification, and small RNA, participate in the regulation of N response and LN adaptation. We highlight the importance of decoding the epigenome at various levels to accelerate the functional study of how plants respond to N availability. Understanding the epigenetic control of N signaling and adaptation can lead to new strategies to improve NUE and enhance crop productivity sustainably. N is a primary macronutrient for plant growth, and it is a vital component of DNA, RNA, and proteins. Conventionally, farmers have used synthetic fertilizers to provide sufficient N levels to the crops . HoweverPCF (TCP)-DOMAIN FAMILY PROTEIN 20 (TCP20) [TGACG MOTIF-BINDING FACTOR 1 (TGA1) [The uptake and assimilation of N are essential processes that ensure optimal nutrient acquisition, storage, and metabolism in plants ,5. These (TCP20)  and TGAC1 (TGA1) . The int1 (TGA1) . This guTranscriptional regulation in plants is not only controlled by genetic factors, but also in an epigenetic manner. Epigenetics refers to the process where chemical or structural modifications alter the chromatin state, impacting gene expression without altering the underlying DNA sequence. DNA methylation, histone modifications, enhancer-promoter 3D structural interactions, and noncoding RNA-mediated regulation are examples of epigenetic mechanisms that can affect gene expression ,15. In rIn this review, we summarize current knowledge of epigenetic regulation under different N conditions and propose decoding the epigenome at various levels provides an opportunity to accelerate the functional study of how plants respond to N availability, and thus improving NUE in crops.Plants possess the remarkable ability to promptly perceive nutrient availability, initiating a cascade of responses to maintain nutrient homeostasis . N, a viArabidopsis [N metabolism in plants encompasses processes such as uptake, transportation, and assimilation, all of which are subject to precise and dynamic regulation by NMGs ,10. Notabidopsis . Alterinbidopsis ,33,34,35bidopsis ,23,36.Arabidopsis, the H3K36me3 ‘writer’ SET DOMAIN GROUP8 (SDG8) plays a significant role in regulating genes related to energy metabolism, including NMGs and photosynthesis, through H3K36me3 modification [SDG8 is induced by N treatment in roots, particularly in the lateral root cap and pericycle [SDG8 mediates N-triggered gene-expression reprogramming, including NRT2.1, NRT1.5, and GLUTAMINE SYNTHETASE 1;4  [sdg8 mutants have shown lower nitrate acquisition than wild-type plants under high N conditions [Solanum lycopersicum), the homologs of SDG8, namely SlSDG33 and SlSDG34, regulate SlNRT1.1 in an N-dependent manner that is associated with the root response to N treatments [AtNRT2.1 expression in Arabidopsis [AtNRT2.1 locus to repress its transcription [AtHNI9 control reactive oxygen species (ROS) homeostasis under conditions of excess N. The mutation of AtHNI9 leads to elevated ROS levels and ROS-dependent upregulation of AtNRT2.1, indicating the role of HNI9 in balancing N uptake and ROS levels in response to N supply [In dicots, histone modification, such as tri-methylation of lysine 36 or lysine 27 on histone H3  are implicated in the regulation of N uptake process. In fication . SDG8 isericycle . Recent    encodes a subunit of the ATP-dependent chromatin remodeling complex known as the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) complex [ZmCHB101 has been found to govern the expression of nitrate transport genes, including ZmNRT2.1 and ZmNRT2.2, in response to nitrate supply [ZmCHB101-RNAi lines showed significantly reduced nucleosome densities at the −1 and +1 nucleosome regions of ZmNRT2.1 and ZmNRT2.2 as compared to WT under nitrate treatment. These low nucleosome densities resulted in enhanced ZmNLP3.1 binding to the promoter regions of ZmNRT2.1 and ZmNRT2.2, further regulating their expression [In monocots, N uptake was regulated by changes in chromatin rearrangement.  complex ,41. In me supply   on key TFs involved in root development in monocots. One such TF is ARABIDOPSIS NITRATE REGULATED 1 (ANR1), a member of the MIKC-type MADS (MCM1/AGAMOUS/DEFICIENS/SRFl) box family of TFs, which mediates the root response to external N . It stimANR1   [AUXIN RESPONSE FACTOR 8 (ARF8) in pericycle cells in Arabidopsis [T5120, a long non-coding RNA modulated by NLP7 and NRT1.1, promotes nitrate assimilation and root growth  in Arabidopsis [ARF8 being induced by an N metabolite produced during N reduction, coordinating plant growth and development in response to both external and internal N availability [In dicots, in particular 3 (AFB3) . Similarbidopsis   and SQUAMOSA-promoter binding protein-like 14 (SPL14)) through H3K27me3 modification [TEOSINTE BRANCHED1 (OsTB1), which is a branching-inhibitory gene, by recruiting PRC2 and promoting bud outgrowth [N supply exerts an epigenetic influence not only on root development but also on plant architecture, particularly for tillering in monocots. In rice, the application of N fertilizer induces changes in the genome-wide H3K27me3 pattern through NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5 (NGR5). This mechanism facilitates the recruitment of PRC2, thus promoting the repression of branching-inhibitory genes  through interactions with auxin transport ,64. Notabidopsis . Furtherbidopsis  , coupled with a reduction in H3K27me3 modifications, work in synergy to enhance the expression of genes associated with root development under LN conditions , the auxin biosynthesis gene [TAR2 has also been reported to be a target of RNA-directed DNA methylation (RdDM) in Arabidopsis [Regulation of auxin biosynthesis in the roots involves DNA methylation. Under LN conditions, the biosynthesis of auxin in the roots of eedlings  and wheaeedlings  is inducsis gene . Moreovebidopsis   to inherit adaptive traits to cope with N-deficiency stress. In rice, for instance, modified cytosine methylation patterns have been identified as the key mediators enabling the stable transmission of adaptive traits under N-deficiency stress to the progeny . The offArabidopsis. The differential expression of specific miRNAs, such as miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857, has been observed in Arabidopsis in response to N starvation [Arabidopsis in response to N starvation [NUCLEAR FACTOR Y A-B1 (NFYA-B1) has been identified as playing a pivotal role in promoting root development and enhancing the expression of nitrate transporters in response to LN conditions [TaNFYA-B1 by cleaving its mRNA [For adaptation to LN environment, recent studies have shed light on the intricate involvement of miRNAs in the regulation of root architecture in arvation . Notablyarvation . In wheanditions . Interesits mRNA  , and DNA methylations, to orchestrate morphological developmental changes and adapt their N metabolism in response to environmental N availability. Recent evidence emphasizes the functional significance of PRC2-TF regulatory modules as potential targets for enhancing NUE in crops. Nevertheless, several questions remain unanswered. Firstly, it is crucial to elucidate how N signaling triggers chromatin-level responses. Additionally, understanding the specific “mediators,” including bjective .Both plants and animals heavily rely on nutrients and energy for their developmental processes, with nutrient signaling directly impacting the epigenome ,74. A recis-elements within these regions, the trans-factors involved can be identified through DNA-protein interaction assays. Additionally, protein interaction assays, as demonstrated in Arabidopsis [Rather than inducing global alterations in chromatin modification, the availability of N tends to affect specific genomic regions and subsets of genes, playing a crucial role in shaping the developmental programs of particular tissues ,67. Thisbidopsis , can helFurthermore, the response of different root parts to N availability exhibits cellular specificity, suggesting unique functions for each cell type . Single-The use of GWAS analysis is widespread in identifying factors that regulate important agronomic traits and mining elite allelic variations in key regulators . HoweverIn maize, a distal enhancer located approximately 63.9 kb upstream of the transcription start site (TSS) regulates the expression of teosinte branched1 (TB1), influencing shoot branching . MoreoveUnderstanding the molecular connections between N signaling and chromatin response can enhance our knowledge of developmental regulation. By targeting specific chromatin modifiers and factors involved in the response to N availability, we can precisely control gene expression, ultimately increasing crop yields. Exploring the differential DNA methylation region, identification of distal regulatory regions and their interactions with genes through population genetics, and 3D genomics provides valuable insights for optimizing N utilization in crops. With these advancements, CRISPR-based genetic and epigenetic editing that targets genes, regulatory regions, or epigenetic marks have the potential to revolutionize crop improvement, promoting sustainable agriculture practices and minimizing environmental impacts.


However, the role of three-dimensional (3D) chromatin structure in orchestrating changes in transcriptional regulation during direct cell reprogramming is not fully understood.Long-range interactions between promoters and Here, we performed integrated analyses of chromosomal architecture, epigenetics, and gene expression using Hi-C, promoter Capture Hi-C (PCHi-C), ChIP-seq, and RNA-seq during trans-differentiation of Pre-B cells into macrophages with a β-estradiol inducible C/EBPαER transgene. Within 1h of β-estradiol induction, C/EBPα translocated from the cytoplasm to the nucleus, binding to thousands of promoters and putative regulatory elements, resulting in the downregulation of Pre-B cell-specific genes and induction of macrophage-specific genes. Hi-C results were remarkably consistent throughout trans-differentiation, revealing only a small number of TAD boundary location changes, and A/B compartment switches despite significant changes in the expression of thousands of genes. PCHi-C revealed widespread changes in promoter-anchored loops with decreased interactions in parallel with decreased gene expression, and new and increased promoter-anchored interactions in parallel with increased expression of macrophage-specific genes.Overall, our data demonstrate that C/EBPα-induced trans-differentiation involves few changes in genome architecture at the level of TADs and A/B compartments, in contrast with widespread reorganization of thousands of promoter-anchored loops in association with changes in gene expression and cell identity.The online version contains supplementary material available at 10.1186/s12915-023-01766-0. Cis-regulatory elements such as enhancers and promoters of genes they control can be separated by large genomic distances [istances . Howeveristances . Gene existances , 3. DeciGenome-wide contact frequencies determined by Hi-C have revealed higher-order chromatin structural features. Topologically associated domains (TADs) are large megabase-size genomic regions of increased self-interaction that are partially insulated from contacts with neighboring domains , 5. VariAnalysis of Hi-C heatmaps has revealed a “plaid pattern” of increased or decreased contact frequencies between domains which corresponds with their activity state. Active domains tend to contact other active domains, while inactive domains tend to contact other inactive domains, indicating that the nuclear genome is organized into two nuclear compartments referred to as A (active compartment) and B (inactive compartment) . Here agHi-C has been instrumental in discovering and defining the principles of the above aspects of higher-order genome organization. However in the absence of billions of read-pairs, Hi-C lacks the resolution to identify significant interactions between individual promoters and their long-range regulatory elements. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between individual promoters and their long-range regulatory elements in an unbiased manner –21. PCHiPre-B cells are precursors of B lymphocytes, which are adaptive immune cells of the lymphoid lineage , 28. MacIn this study, we used ChIP-seq, RNA-seq, Hi-C, and PCHi-C data to investigate higher-order chromatin organization and promoter interaction dynamics associated with gene expression changes during C/EBPαER-induced trans-differentiation of mouse Pre-B cells into macrophages over a 48-h period , 37. OurTo study the role of promoter-enhancer interactions during Pre-B cell trans-differentiation into macrophages, we employed direct lineage conversion of the C10 mouse Pre-B cell line which harbors an estradiol-inducible rat C/EBPα fused to the estrogen hormone receptor binding domain (C/EBPαER) , 38. UpoH3f3a and H3f3b) genes , macrophage scavenger receptor (Msr1), myeloid restricted serine protease C (Ctsc), and the myeloid cytokine-dependent chemokine 6 (Ccl6) were upregulated,  levels increased in the whole cell extracts through Pre-B trans-differentiation Fig. b. To ananes Fig. d. H3.3 ines Fig. d, consisnes Fig. e. The encis-contacts greater than 10Kb, approximately 15% cis-contacts < 10 Kb, and ~ 14% trans contacts, indicating very high-quality Hi-C libraries . GO analysis of coding genes (n = 6937) within TADs that show boundary changes shows enrichment for immune processes, such as response to chemokine, response to dsRNA, and response to pheromone . PC1 value of PCA shows a higher correlation between biological replicates than between different time points  libraries at each time point. PCHi-C data quality was checked using HiCUP analysis  did not change compartment status over the 48h period. A recent study that used C/EBPα to trans-differentiate a human B cell line to macrophage also shows conservation of TAD boundaries, and observed only a small percentage of dynamic compartments associated with gene expression changes . It is iOur results suggest that the rewiring of promoter-anchored interactions plays a major role in controlling differential gene expression and cell fate transition compared to the relatively minor role played by changes in higher-order organization of TADs and A/B compartments. We show that PIRs are significantly enriched in histone modifications associated with gene regulatory activity. Genes whose promoters are contacted by PIRs with the active enhancer mark, H3K27ac, are highly expressed, while promoters contacted by the repressive H3K27me3 modification are not. Thus, transcription factor-mediated differential loop formation between promoters and their distal regulatory elements is the major driver of the cell-type-specific, promoter interactomes  and is tOur integrated analyses of gene expression, histone modifications, transcription factor binding, and various levels of three-dimensional chromosome conformation during trans-differentiation have revealed that widespread changes in gene expression that characterize the transition from a Pre-B cell to a macrophage phenotype involve surprisingly few changes in TAD structure or A/B compartment status. Instead, we found reorganization of thousands of promoter-anchored loops, in conjunction with transcription factor binding and histone modification dynamics, occurring at thousands of differentially expressed genes suggesting that changes in chromatin interactions at the sub-TAD level are a major mechanism of differential regulation of gene expression that drives cell state transitions.6 C10 cells were plated in 10-cm cell culture dishes, followed by adding100 nM of β-estradiol , 10 nM IL-3 , and CSF-1 . The cell culture medium was replenished every 24h. The Pre-B cell trans-differentiation efficiency was measured by flow cytometry analysis and qPCR gene expression analysis of Pre-B cell-specific genes and macrophage-specific genes. For flow cytometry analysis, C10 cells at various differentiation stages were stained using PE anti-mouse/human CD11b  antibody and APC anti-mouse CD19 antibody . The percentage of Pre-B cells and macrophage population was analyzed on FACSCanto II analyzer (BD Biosciences). Flow cytometry data was analyzed by FlowJo  software. PE Rat IgG2b  and APC Rat IgG2a  antibodies were used as isotype controls to exclude the background fluorescence.C10 cells were cultured in RPMI 1640  medium supplemented with 10% heat-inactivated FBS , 1% Penicillin–Streptomycin , and 50 μM 2-Mercaptoethanol . For C10 cells trans-differentiating into macrophages, 5.0 × 10For western blots, cells were lysed by CelLytic buffer . The protein concentration was quantified by the Micro-BCA assay reagent . A 10 μg protein from each sample was loaded into 4–12% SDS–polyacrylamide  for electrophoresis. The following antibodies were used for Western blot experiments: Anti-C/EBPα , Anti-H3 , Anti-β-Tubulin . For immunofluorescence, C10 cells were first fixed in 4% PFA for 5 min, washed using cold PBS twice, followed by attaching cells to fibronectin and pre-coating the glass slides. Next, the cells were treated using blocking buffer  for an hour at room temperature, followed by primary antibody treatment overnight at 4 °C and secondary antibody treatment at room temperature for an hour. The primary and secondary antibodies were diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in 1X PBS), and DAPI  was used for nuclear staining. Anti-C/EBPα  and Goat anti-Mouse IgG (H + L) Alexa Fluor 488  antibodies were used for immunofluorescence staining. All the immunofluorescence images were taken by LSM 710 confocal microscope (Zeiss) and processed using Zen black (Zeiss).Gapdh using the 2−ΔΔCT method.RNA was prepared from C10 cells at various trans-differentiation stages using RNeasy mini kit  followed by on-column DNase I treatment. Total RNA (1 μg) was used for reverse transcription to generate cDNA by using iScript™ Reverse Transcription Supermix for RT-qPCR , followed by the real-time PCR amplification using Power SYBR Green PCR Master Mix . The real-time PCR was performed with QuantStudio 7 Flex Real-Time PCR System (Thermofisher SCIENTIFIC) for 40 cycles, 95°C for 15 sec, and 60°C for 1 min. Gene expression was normalized to the housekeeping gene For RNA-seq libraries preparation, 1μg of total RNA (RIN > 7) was used for rRNA depletion by using NEB rRNA Depletion Kit (E6310). Next, the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (E7770S) and NEBNext NEBNext® Multiplex Oligos for Illumina® (E7335) were used for library construction. RNA-seq library concentration was quantified by using the TapeStation and KAPA Library QANT Kit , followed by single-end (1 × 100bp) sequencing on the Novaseq S2 flow cell. The sequencing depth is described in Additional file  g for 5 min at 4°C. Next, the cells were lysed in 20 mL cold lysis buffer  on ice for 30 min, followed by centrifuging at 760 g for 5 min to remove the supernatant. The pellet was washed in 1.25X NEB buffer2  and resuspended in 358 μL 1.25X NEB buffer2, followed by adding 11 μL of 10% SDS and incubating at 37 °C with shaking at 950 rpm for 30 min. Lastly, the SDS was quenched by adding 75μL of 10% Triton X-100 and incubating at 37 °C for 15 min with shaking. To digest chromatin using HindIII restriction enzyme, we added 12 μL of 100 U/μL HindIII  to each reaction, followed by shaking at 37 °C and 950 rpm overnight, followed by adding 5 μL of 100 U/μL HindIII  per reaction at 37 °C for 2 h on the following morning. To repair the digested overhangs, we added 6.1 μL 10X NEB buffer2, 25 μL molecular-grade water, 15.3 μL of 1 mM biotin-14-dATP , 1.56 μL of 10 mM dCTP, 1.56 μL of 10 mM dGTP, 1.56 μL of 10 mM dTTP, and 10.2 μL of 5U/μL DNA polymerase I, Large (Klenow) Fragment  and incubated the reaction at 37 °C for 1 h. In-nucleus ligation mix was prepared by adding 102μL 10X T4 DNA ligase buffer, 10.2 μL BSA , 350.9 μL molecular-grade water, and 25.5 μL 1U/ μL T4 DNA ligase , followed by incubation at 16 °C for 4 h in a thermomixer (shaking at 700 rpm for 5 s in every 2 min). Next, the nuclei were pelleted at 2500 g for 5 min and resuspended in 300μL cross-link reversal buffer , followed by adding 5 μL 10 mg/ml RNase A  at 37 °C for 30 min and 20 μL of 20 mg/ml Proteinase K  at 55 °C for 1 h and 68 °C overnight. The genomic DNA was extracted using cold ethanol and sodium acetate (pH 5.2) and resuspended in 130μL of 10 mM Tris–HCl. DNA was transferred to a microTUBE AFA fiber Pre-Slit Snap-Cap  and fragmented using a ME220 Covaris sonicator . The sonicated DNA was transferred to a 1.5-ml tube, and the total volume was brought to 200 μL by adding 70 μL of molecular-grade water. DNA size was double size selected using AMPure XP beads . First, 120 μL of beads were added to each reaction (ratio of AMPure beads to DNA: 0.6 to 1) and incubated for 5 min. The clear supernatant was transferred to a fresh tube through a magnetic separation stand. Next, 30 μl of fresh AMPure XP beads were added to the clear supernatant and incubated for 5 min at room temperature. Beads were separated on a magnetic separation stand and washed twice using 800 μL of 70% ethanol and dried at 37 °C, followed by eluting DNA in 300 μL 1X Tris buffer. DNA was used for library preparation with a normal size distribution between 300 and 500 bp and was visualized by TapeStation.Hi-C and Promoter Capture Hi-C (PCHi-C) library preparation was described in our previous publication with minor modifications . Ten milProcedures for Biotin/Streptavidin pull-down of the Hi-C ligation products, end repair, removal of biotin at the non-ligated DNA ends, adaptors ligation, Hi-C libraries amplification, and Hi-C size selection were described in our recent publication . The Hi-https://github.com/FelixKrueger/TrimGalore). FastQC program was used to check the quality of the data (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmed reads were mapped to mouse reference genome (mm10) using Hisat2 (2.1.0) with default parameters [RNA-seq libraries were prepared for each time point. We included three biological and four technical replicates for each time point. Technical replicates were merged, and Trim Galore (0.6.2) pipeline was used to remove adapters and short reads from the data . JuiceBox was used to visualize Hi-C matrix heatmap and TAD structure [Hi-CFindTADs –correctForMultipleTesting fdr –thresholdComparisons 0.01 –delta 0.01) [Hi-C data were processed using HiCUP (v0.7.2) pipeline  with mm1tructure . Homer ptructure . The cortructure . Hi-C fitructure . TADs anta 0.01) .https://epigenomegateway.wustl.edu/) [peakEnrichemnt4Features function in CHICAGO) [PCHi-C mouse raw reads were processed by the HiCUP pipeline and were mapped to mm10 genome . Hi-CReptl.edu/) . To estiCHICAGO) .BEDTools (v2.26.0) “intersect” function was used to find PIR regions that overlap with H3K27ac peaks or H3K27me3 . To invefindPeaks function of HOMER package was used to call histone modification peaks by setting peak size 1000 bp and the remaining parameters were set to the default values [peakEnrichmen4Features [ p-value < 0.05 and fold change > 1.5 were used as threshold to identify induced C/EBPα binding sites. TSS of all genes were downloaded from Ensembl biomart (v100) [ChIP-seq data C/EBPα along with histone modification ChIP data were downloaded from GEO . Raw reat values . Peak cat values . To visut values . The enrFeatures . FurtherFeatures . p-valuet (v100) . Bedtoolt (v100) .t-test, Mann–Whitney U statistical test, and chi-squared test were performed using GraphPad Prism 8 and R. p-value < 0.05 was considered as significant unless specified and indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001.Unpaired Additional file 1: Fig. S1. Transcriptional profile changes during Pre-B transdifferentiation. a Principal component analysis (PCA) RNAseq of each replicate from different time points. b SNPsplit identifies the percentage of total RNA reads that align to Cebpa gene region that contain Mus musculus specific SNP (mouse specific), Rat specific SNP (Rat specific), regions that do not contain any SNP (Unassignable), conflicting SNP information (Conflicting). c Volcano plots show upregulated and downregulated differentially expressed genes (DEGs). d GO ontology 5 Fuzzy c-means clusters of DEGs e qPCR analysis of Pre-B cell and macrophage marker gene expression during Pre-B cell trans-differentiation. f Gene expression level (TPM) of Pre-B cell-specific genes , and macrophage-specific genes  during Pre-B trans-differentiation. Fig. S2. Hi-C data analysis. aHiCUP pipeline analysis HiC and PCHi-C data, including the following three categories: Cis < 10Kbp, Cis > 10Kbp, and trans-interactions across four-time points. b Heatmaps show Hi-C matrix at 100kb and 25 kb resolution. Fig. S3. Altered TAD and A/B compartment during Pre-B transdifferentiation. a Number of TADs and size distribution at each time point. b GO enrichment analysis of genes changed TAD boundaries from 0h to 48h. c Pearson correlation coefficient of PC1 values (50kb resolution) of the entire genome. d Gene expression level (TPM) in the A and B compartments at four different time points. A two-sided unpaired t-test was performed for the significance test. ePercentage of compartment change for DEGs .  f Log2 fold change of gene expression in dynamic compartments. stable (n= 19157), A to B (n= 450), and B to A (n= 474). g Gene ontology analysis of 2105 genes at 48h that switch from A to B and 1552 genes at 48h that switch from B to A. A two-sided unpaired t-test was performed for the significance test. Fig. S4. PCHi-C data analysis. a HiCUP pipeline analysis of PCHi-C data: Cis <10Kbp, Cis >10Kbp, and trans-interactions across four-time points. b Proportion of promoter-promoter (P-P) and promoter-other interactions (P-O) interactions as deduced from PCHi-C. c Expression values (log2(TPM+1)) of genes/promoters classified into 4 groups (at least one PIR overlaps with histone markers). H3K27ac: interaction between gene/promoter and PIRs only with H3K27ac marks. 0h (n=4829); 1h (n=5710);12h (n=5257); 48h (5735). H3K27Ac+H3K27me3: interaction between gene/promoter and PIRs with both H3K27ac and H3K27me3 marks. 0h (n=8075); 1h (n=7044); 12h (n=7926); 48h (n= 7727). H3K27me3: interaction between gene/promoter and PIRs only with H3K27me3. 0h (n=2757);1h (n=2286); 12h (n= 2545); 48h (n=2165). No marks: interaction between gene/promoter and PIRs without any histone modification. 0h (n=3667), 1h (n=4805); 12h (n=4403); 48h (n= 4291). The box plot represents 25 and 75 percentiles with the median. An unpaired two-sided t test is performed for the significance test. d Boxplot shows gene expression values (log2(TPM +1)) of promoters that do not contact any PIRs with H3K27ac categorized into four groups according to the number of PIRs marked by H3K27me3 at four different time points.  0: Promoters without any PIRs marked by H3K27me3. 0h (n=2551), 1h (n=2991); 12h (n=2711); 48h (n=2682).  1: Promoters with only one PIR marked by H3K27me3 0h (n=1247), 1h (n=1100); 12h (n=1188); 48h (n= 955). 1-5: Promoters with one to five PIRs marked by H3K27me3. 0h (n=749), 1h (n=672); 12h (n=697); 48h (n=567).  >5: Promoters with more than five PIRs marked by H3K27me3. 0h (n=80), 1h (n=63); 12h (n=103); 48h (n=95).  An unpaired two-sided t-test is performed for the significance test. The box plot represents 25 and 75 percentiles with the median. Fig. S5. Integrated analysis of promoter interactions and epigenetic features. a Maf1 and Ssbp1 region that shows promoter interaction changes accompanied by H3K27me3(green), H3K27ac (yellow), and H3K4me1 at interacting regions, along with gene expression TPM of Maf1 and Ssbp1 during Pre-B transdifferentiation. Multiple Maf1 promoters in two HindIII digested fragments b Log2 fold change of gene expression based on the number of H3K4me1 peaks on the increased PIRs (48h vs 0h). The number of histone mark peaks is classified into three groups.  <=1: Genes that gain chromatin interactions with less than one histone mark peaks (n=360). 1-5:  Genes that gain chromatin interactions with one to five histone mark peaks (n=313). >5: Genes that gain chromatin interactions with greater than five histone mark peaks (n=69). The unpaired two-sided t-test was performed to test significant differences between groups. The box plot represents 25 and 75 percentiles with the median. Fig. S6. Decreased promoter interactions at 48h. a GO term for genes that lost promoter interactions at 48h. b Examples of significantly decreased B cell-specific gene promoter interactions  across time points. Fig. S7. C/EBPα ChIPseq data analysis. a The number of identified C/EBPα ChIP peaks at 1h, 1h, 12 h and 48h. b Comparison of 1h, 12,h or 48h C/EBPα ChIP peaks signal to 0h. M value represents log2 fold change of read density between two samples, and A value represents the average signal intensity. The red dots represent induced C/EBPα binding sites at 1h, 12h, and 48h time points. The blue dots represent C/EBPα binding sites at 0h. The grey dots represent unchanged C/EBPα binding sites at four time points. Fig. S8. Uncropped blots.Additional file 2: Table S1.Additional file 3: Table S2.Additional file 4: Table S3.