Project description:In this study we investigated the developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, and revealed potential mechanisms for repressing branches in distinct stem cell populations in developing maize inflorescences. To identify targets of RA1 and to distinguish direct vs. indirect interactions, we performed Chromatin Immunoprecipitation (ChIP)-seq and compared the results to gene expression data (RNA-seq datasets for Eveland et al., 2013, submitted). We mapped genome-wide occupancy of RA1 and showed that it differently regulates modules of target genes based on spatiotemporal context. Plants expressing complementing RA1 transgenes tagged with HA or YFP were used in parallel experiments. Ear and tassel primordia were collected and tag-specific antibodies were used to pull down RA1 bound to its target loci. Genome-wide analysis of RA1 occupancy revealed thousands of putative binding sites (i.e. peaks significantly enriched (p < 1e-05) compared to input DNA).
Project description:In this study we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. These are the RNA-seq datasets used in this study. We profiled changes in gene expression during normal maize ear and tassel development and in developing maize ear primordia upon genetic perturbation of the RAMOSA branching pathway. For the wild-type ear and tassel developmental series, greenhouse-grown B73 inbred plants were used. 10mm ears were collected and sectioned as follows from tip to base along the developmental gradient: tip 1mm sampled (tip; Inflorescence Meristem/Spikelet Pair Meristem), next 1mm discarded, next 1mm sampled (mid; Spikelet Meristem), next 2mm discarded, next 2 mm sampled (base; Floral Meristem), and immediately frozen in liquid nitrogen. Sections from ~30 sampled ears were pooled for each of 2 biological replicates to represent tip, mid, and base stages. Tassels were hand-dissected, measured, separated by stage: 1-2mm (stg1), 3-4mm (stg2), and 5-7mm (stg3), and immediately frozen in liquid N. For each stage, ~20-30 tassels were pooled for each of 2 biological replicates. For ramosa mutant series, segregating families (1:1) of ra1-R, ra2-R, and ra3-fea1 mutant alleles, all introgressed at least 6 times into the B73 inbred background, were grown at CSHL Uplands Farm. Field-grown plants were genotyped and collected 6-7 weeks after germination (V7-V8 stage). First and second ear primordia were immediately hand-dissected, measured, and frozen in liquid nitrogen. For ra1, ra2 and ra3 mutants and wild-type controls, ears were pooled into two size classes: 1) 1mm class included a range of 0.7-1.5mm sized ears and nine ears were pooled for each of 2 biological replicates; 2) 2mm class included a range of 1.8-2.5mm sized ears and six ears were pooled for each of three biological replicates. Wild-type samples were proportional mixtures of heterozygote siblings segregating in ra1, ra2, and ra3 populations. Variability factors (e.g. ear size within class, ear rank on the plant, and time of collection) were distributed evenly across pooled samples.
Project description:fasciated ear4 (fea4) is a semi-dwarfed mutant with fasciated ears and tassels, and greatly enlarged vegetative and inflorescence meristems. Chromatin Immunoprecipitation-sequencing (ChIP-seq) and expression profiling by RNA-seq suggest that fea4 is required to regulate the auxin response and leaf differentiation programs in the periphery of the meristem, suggesting a new mechanism of meristem size regulation that is spatially and mechanistically distinct from the CLV-WUS model. To gain insight into transcriptional regulation by FEA4, we used RNA-seq to profile genome-wide expression changes in developing ear primordia of fea4 loss-of-function mutants compared to fea4/+ wild-type (wt) siblings. To map genome-wide occupancy of FEA4 and define its putative transcriptional targets, we performed ChIP-seq using the pFEA4-YFP::FEA4 transgenic lines.
Project description:In this study, we present a comprehensive peptidomic atlas of 13 tissues in maize spanning the vegetative and reproductive phases. Using 3-frame translation of canonical coding sequences (CDSs), we identified 6,100 non-redundant endogenous peptides, significantly expanding the known repertoire of peptides in plants. By integrating the peptidomic coexpression profiles with previously reported proteomic profiles, we found that the peptide abundance did not consistently correlate with the abundance of their source proteins, suggesting complex regulatory mechanisms. This integrated peptidomic and proteomic map not only provides a valuable resource for exploring the functional roles of endogenous peptides in maize development, but will also enable researchers to explore the functional relationship among genes, peptides, and proteins in various biological contexts.
Project description:The objective of the current study is to unravel the gene regulatory networks controlled by the nkd genes during maize endosperm development. We compared wild type (B73) vs. nkd mutant (introgressed into B73 background) transcriptomes in aleurone vs. starchy endosperm cell types captured by laser capture microdissection technology. We performed RNA seq analysis of mid-mature (15DAP) endosperm in two cell types [aleurone (A) and starchy endosperm (S)] of wild type B73 (B) and nkd mutant (N) kernels with three independent biological replicates.
Project description:Genome-wide target genes of ZmAN3 were identified through ChIP-seq on the growth zone of the maize leaf, encompassing the division, transition and expansion zone. For ChIP-seq, ZmAN3 was fused to the GSyellow TAP tag and expressed from the ubiquitin promoter (pUBIL). The pUBIL:ZmAN3-GSyellow construct was transformed into the maize inbred line B104. ChIP was performed using anti-GFP antibody (abcam290).
Project description:Maize LEAFBLADELESS1 (LBL1) and Arabidopsis SUPPRESSOR OF GENE SILENCING3 (SGS3) play orthologous roles in the biogenesis of 21 nucleotide trans-acting short-interfering RNAs (tasiRNAs). The phenotypes conditioned by mutation of lbl1 and SGS3 are, however, strikingly different, suggesting that the activities of these small RNA biogenesis components, or the tasiRNAs and their targets might not be entirely conserved. To investigate the basis for this phenotypic variation, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects all major classes of small RNAs, and reveal unexpected crosstalk between tasiRNA biogenesis and other small RNA pathways regulating miRNAs, retrotransposons, and DNA transposons. We further identified genomic regions generating phased siRNAs, including numerous loci generating 22-nt phased small RNAs from long hairpin RNAs or overlapping antisense transcripts not previously described in other plant species. By combining both analyses, we identified nine TAS loci, all belonging to the conserved TAS3 family. Contrary to other plant species, no TAS loci targeted by a single miRNA were identified. Information from target prediction, RNAseq, and PARE analyses identified the tasiARFs as the major functional tasiRNAs in the maize vegetative apex where they regulate expression of ARF3 homologs. As such, divergence in TAS pathways is unlikely to account for the distinct phenotypes of tasiRNA biogenesis mutants in Arabidopsis and maize. Instead, the data suggests variation in the spatiotemporal regulation of ARF3, or divergence in its function, as a plausible basis for the dramatic phenotypic differences observed upon mutation of SGS3/lbl1 in Arabidopsis and maize. An analysis of tasiRNA biogenesis, activity, and contribution to developmental phenotypes in the maize leaf. Data generated includes small RNA sequencing data and mRNA sequencing data. All data was generated in both wild type and lbl1 mutant maize leaf apices. Three replicates were generated for each genotype for the small RNA data. Two of these replicates were also used for the RNA-seq data.
Project description:Genome-wide target genes of PPD2 were identified through ChIP-seq on Arabidopsis cell cultures. For ChIP-seq, PPD2 was fused to the GSyellow TAP tag and expressed from the 35S promoter. The p35S:PPD2-GSyellow construct was transformed into Arabidopsis thaliana PSB-D cell culture. ChIP was performed using anti-GFP antibody (abcam290).
Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize plants of two genetic backgrounds, B73 and Mo17, representatives of two distinct heterotic groups. There are numerous differences in the distribution of H3K27me3 among tissues. In contrast, we found the distribution of H3K27me3 among the two genetic backgrounds to be quite similar. The tissue-specific patterns of H3K27me3 are often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. Many maize genes with important developmental roles, including numerous genes encoding putative transcription factors, are modified with H3K27me3 in at least one of the tissues that were profiled. A comparison of the H3K27me3 targets in rice, maize, and Arabidopsis provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole genome duplication. Genomic profiling of H3K27me3 in loss-of-function mutant stocks for Mez2 and Mez3, two of the three putative H3K27me3 methyltranferases present in the maize genome, suggests partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 requires Mez2 and/or Mez3 and there was limited evidence for functional consequences of H3K27me3 at these targets. This study provides a catalogue of 6,337 genes that are marked by H3K27me3 in five maize tissues. B73 tissues for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. histone modification mutants for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. Mo17 tissues for h3k27 analysis: Samples contain antibody pulldown (IP) and control genomic DNA (input) channels.
Project description:The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRC) resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations, thus can be used as tumor-specific therapeutic target for most CRCs.