Genome-wide profiling of Tbx6myc binding during zebrafish somitogenesis
ABSTRACT: Identification of Tbx6 genomic binding sites during zebrafish somitogenesis in order to identify Tbx6 targets genes Duplicate anti-myc ChIP samples with associated input for heat shocked Tg(hsp70l:tbx6Myc)v8 embryos, plus single paired anti-myc ChIP and input in wild type embryos - 3x ChIP samples; 3x input samples
Project description:Study of Smad2 and Eomesa genomic binding in zebrafish blastulas, their relationship to eachother and the relaptionship between genomic binding and Ndr1 and Eomesa responsive genes as identified by microarray and RNA-seq. Replicate ChIP samples with associated input for Smad2 and Eomesa - 4x ChIP samples (2 per factor); 2x input samples
Project description:Contemporary high throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including chromatin immunoprecipitation sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF co-occupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous co-occupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional, newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide new insights into the transcriptional codes regulating muscle gene expression, and offer a generalizable approach for similar studies in other systems. Examination of Lmd occupancy to genomic DNA from sorted mesodermal cells
Project description:The Myc-Max heterodimer has been thought of as a sequence specific DNA binding protein that regulates transcription of a large number of genes. We demonstrate here that the positions of the human genome occupied by Myc-Max correlate with the RNA polymerase II (Pol II) transcription complex rather than to the canonical DNA binding sequence element CACGTG. The heterodimer is positioned slightly upstream of essentially all promoter proximal paused polymerases and is found throughout the transcribed regions of genes. Using a multi-genome analysis of promoter regions we show that the heterodimers are oriented with respect to the direction of transcription with Myc downstream of Max. In strong support of a model in which the Myc is recruited by transcription complexes rather than specific DNA sequences, we found that the difference in affinities of Myc-Max heterodimers for CACGTG versus non-specific DNA is not great enough to drive the pattern of genome occupancy exhibited. A total of 2 ChIP-Seq data for Myc and Max in human HeLa cell.
Project description:Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics (the study of the whole protein complement of a microbial community) can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Here we present a systematic investigation of variables concerning database construction and annotation, and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. Taxonomic and functional results were revealed to be strongly database-dependent, especially when dealing with mouse samples. As a striking example, in mouse the Firmicutes/Bacteroidetes ratio varied up to 10-fold depending on the database used. Finally, we provide recommendations regarding metagenomic sequence processing aimed at maximizing gut metaproteome characterization, and contribute to identify an optimized pipeline for metaproteomic data analysis.
Project description:The trasncription factor cMyc is an essential transcription factor that establishes a metabolically active and proliferative state in T cells after antigen priming. However, its expression is transient. To date, it remains unknown how T cell activation is maintained after cMyc down-regulation. Here, we identify AP4, encoded by the gene Tfap4, as the transcription factor that is induced by cMyc and sustains activation of antigen-specific CD8+ T cells. Despite normal priming, Tfap4–/– CD8+ T cells fail to continue transcription of a broad range of cMyc gene targets necessary for sustained proliferation. Genome-wide analysis suggests that many activation-induced metabolic genes are shared targets of cMyc and AP4. Thus, AP4 maintains Myc-initiated cellular activation programs in CD8+ T cells to control microbial infections. Naive CD8+ T cells from C57BL6 mice were activated with anti-CD3 and anti-CD28 stimulation in vitro for two days and genome-wide occupancy of Myc, AP4 and Ser2 or Ser5 phipsphorylated RNA polymerase II was profiled by chromatin immunoprecipitation and high-throughput sequencing.
Project description:The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes. Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome. Here, we use a systematic approach that integrates transcriptome-wide differential expression analysis, genome-wide p53 binding profiles, chromatin state maps, and additional genomic features to characterize the global regulatory roles of p53 in response to DNA damage in both human and mouse fibroblast models. In addition to known p53 targets, we identify many previously unappreciated mRNAs and long noncoding RNAs that are regulated by p53. Moreover, we find that p53 binding events occur predominantly within enhancer elements in both human and mouse systems. The ability to modulate enhancer activity offers an additional layer of complexity to the p53 network and greatly expands the diversity of genomic elements that are directly regulated by p53. Human and Mouse fibroblasts cultured in the presence or absence of doxorubicin followed by RNA-Seq (Human:2 cell lines, each condition in duplicate; Mouse:MEF cell line,each condition in triplicate) and p53 ChIP-Seq (Human:2 cell lines, input and IP for each; Mouse:MEF cell line, input and IP)
Project description:KNOTTED1(KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. Despite their importance for plant development, the genomic network targeted by KNOX proteins is poorly understood. Using ChIP-seq, we defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci. To understand how these binding occupancies correlate with changes in transcriptional regulation, we performed RNA-seq on immature ears and tassels, and compared expression profiles between normal and loss-of-function kn1 plants, in addition to immature leaves from normal and gain-of-function Kn1 plants. We found that 643 of the KN1 targets were modulated in one or multiple tissues, with a strong enrichment for transcription factors (including other homeobox genes) and genes participating in several hormonal pathways, most significantly auxin, implicating KN1 at the crossroads of plant hormone signaling. The loss-of-function kn1 phenotype is reminiscent of auxin mutants and kn1 mis-expression in leaves correlates with increased auxin signaling. Our results demonstrate that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function. ChIP-seq was performed using ear primordia and tassel primordia. Input DNA from each sample was used as a normalization control
Project description:ChIP-seq was performed to compare binding the genome-wide binding profile of the CLAMP transcription factor in two different Drosophila species. ChIP seq experiments compare the binding profile of CLAMP in female larvae to identify conservation of its binding sequence.
Project description:Genomic DNA from 192 Col x Ler F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Rowan et al; Yelina et al), with the following modifications. DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37C with 0.4U of DNA Shearase (Zymo research). The barcoded adapters used for library construction are listed in Rowan et al. Each set of 96 libraries was sequenced on one lane of an Illumina NextSeq500 instrument (300-cycle Mid Output run). Sequencing data was analysed to identify crossovers as previously reported, using the TIGER pipeline (Rowan et al; Yelina et al). Rowan BA, Patel V, Weigel D, Schneeberger K. Rapid and Inexpensive Whole-Genome Genotyping-by-Sequencing for Crossover Localization and Fine-Scale Genetic Mapping. G3 (Bethesda). 2015;5: 38598. Yelina NE, Lambing C, Hardcastle TJ, Zhao X, Santos B, Henderson IR. DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Genes Dev. 2015;29: 2183202.
Project description:We sequenced DNA isolated from performing ChIP of full-length KLF6 and an Input sample in an HCC cell line. The goal is to determine KLF6 binding sites in a mouse-derived HCC cell line. Determination of KLF6 binding sites in an HCC cell line using 2 control input libraries and 2 KLF6-ChIP libraries