BiTS Chromatin Immunoprecipitation of H3K4me3 and H3K27ac in Drosophila melanogaster differentiating cardiomyocytes
ABSTRACT: Here we improved BiTS-ChIP (Bonn et al, Nature Protocols 7, 978-994 (2012)) to identify active enhancer and promoter elements genome wide in the 104 cardiomyocytes that constitute the Drosophila heart tube and represents only ~0.5% of the total cell content of the embryo. A transgenic Drosophila strain expressing nuclear GFP under the control of a cardiac specific enhancer (TinC*>GFP) was used for staged embryo collections at stages 13-14 (10-13h of development). After embryo fixation and dissociation, intact fixed nuclei were fluorescent labelling. Purification of this rare nuclear population was achieved by a two-step sorting procedure, yielding ~98% purity. Chromatin was extracted and used for immunoprecipitation and sequencing (ChIP-seq) to analyze chromatin modifications at promoters (H3K4me3 and H3K27ac) and enhancers (H3K27ac). Two independent biological replicates (from FACS sorting, chromatin preparations and ChIP-Seq) were performed for each mark and sequenced using Illumina HiSeq.
Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all c ...[more]
Project description:We used a Drosophila melanogaster line (a "double balancer") carrying balancer chromosomes for both the second (CyO) and third (TM3) chromosomes, and crossed it to an isogenic wild-type "virginizer" line. Trans-heterozygous adults from the F1 generation were further crossed to the wild-type parental line to obtain the pool of N1 embryos. Allele-specific chromosome conformation capture (Capture-C) was used to measure changes in chromatin organization on both chromosomes.
Project description:Chromosome conformation capture (4C-Seq) in Drosophila Twist-H2B embryos (carrying nuclear tag specifically in the mesoderm) during embryogenesis was performed, anchoring on 107 different viewpoints. Two timepoints (3-4hrs and 6-8hrs after egg laying) and two tissue context (whole embryo and mesoderm) were assayed. Two independent collections were performed at each timepoint.
Project description:We used a Drosophila melanogaster line (a "double balancer") carrying balancer chromosomes for both the second (CyO) and third (TM3) chromosomes, and crossed it to an isogenic wild-type "virginizer" line. Trans-heterozygous adults from the F1 generation were further crossed to the wild-type parental line to obtain the pool of N1 embryos. Allele-specific chromosome conformation capture (Hi-C) was used to measure changes in chromatin organization on both chromosomes.
Project description:DNase-seq over 3 matching developmental time points in Drosophila melanogaster and Drosophila virilis embryos was performed. The aim is to assess conservation of hypersensitive regions between two distantly related species. Samples were sequenced using Illumina HiSeq.
Project description:To analyse the transcriptome landscape of differentiating cardiomyocytes, GFP labeled cardiac cells were purified by fluorescent activated cell sorting using the TinC*>GFP transgenic line, and expression profile analysed by deep sequencing.
Project description:We applied ChIP-seq to map the chromosomal binding sites for two nucleosome remodeling complexes containing the ATPase ISWI, ACF and RSF, in Drosophila embryos. Employing a panel of polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. For further validation we repeated the mapping using chromatin of mutant embryos that do not express ACF1 or RSF1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and give rise to ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Examination of various ACF1 and RSF1 antibodies in Drosophila melanogaster embryos which are wildtype or mutant for the antibody targets.
Project description:A genomic locus tends to form a particular conformational structure in the nucleus, but it is not known how a specific structure is dictated. To understand the mechanism, we made serial genomic rearrangements (deletion and inversion) in the Tfap2c-Bmp7 locus in mice, and analyzed the chromatin conformation in these alleles by 4C. For the wild type allele, we took not only the whole embryo but also different tissues such as the heart, forebrain (lateral and medial part separately), limb buds, and we used only the whole embryos for the mutant alleles.
Project description:ChIP-chip was performed using chromatin isolated from formaldehyde crosslinked, staged embryos, and affinity purified antibodies against 21 transcription factors (including: BCD, HB, KR, KNI, GT, CAD, HKB, TLL, D, FTZ, HRY, PRD, RUN, SLP1, DA, DL, MAD, MED, SHN, SNA, TWI) that belong to 11 DNA binding domain families, specify distinct developmental fates; as well as the general transcription factor TFIIB, and RNA POLII. Affinity purification of the antibodies was performed with E.coli expressed His-tag fusions of the whole protein (designated as 3, e.g. anti-DL 3), the N-terminal portion of the full length protein (designiated as 1, e.g. anti-BCD 1), or the C-terminal portion of the protein (designated as 2, e.g. anti-BCD 2). While for most factors, we only used one antibody in the ChIP-chip study, for some factors, two or more antibodies that recognize non-overlapping parts of each of the proteins were used. The chromatin immunoprecitation and hybridization for each antibody were carried out in duplicates, and in each chromatin immunoprecitation and hybridization series, two mock IP controls and two input DNA controls were also generated. The microarray used in this study is the Affymetrix Drosophila genomic DNA tiling array, which has about 3 million oligo pairs (perfect match-mismatch), covering the whole euchromatic sequences of the fly genome at a resolution of about 35 bp.
Project description:Gene transcription can be regulated by remote enhancer regions through chromosome looping either in cis or in trans. Cancer cells are characterized by wholesale changes in long-range gene interactions, but the role that these long-range interactions play in cancer progression and metastasis is not well understood. In this study, we used IGFBP3, a gene involved in breast cancer pathogenesis, as bait in a 4C-seq experiment comparing normal breast cells (HMEC) with two breast cancer cell lines (MCF7, an ER positive cell line, and MDA-MB-231, a triple negative cell line). The IGFBP3 long-range interaction profile was substantially altered in breast cancer. Many interactions seen in normal breast cells are lost and novel interactions appear in cancer lines. We found that in HMEC, the breast carcinoma amplified sequence gene family (BCAS) 1-4 were among the top 10 most significantly enriched regions of interaction with IGFBP3. 3D-FISH analysis indicated that the translocation-prone BCAS genes, which are located on chromosomes 1, 17 and 20, are in close physical proximity with IGFBP3 and each other in normal breast cells. We also found that epidermal growth factor receptor (EGFR), a gene implicated in tumorigenesis, interacts significantly with IGFBP3 and that this interaction may play a role in their regulation. Breakpoint analysis suggests that the interchromosomal rearrangements seen in the MCF7 cancer cell line involve regions that engage in long-range interactions in normal breast cells. Overall, our data from multiple lines of evidence suggest an important role for long-range chromosomal interactions in the pathogenesis of cancer. Comparison of IGFBP3 interaction profiles in normal breast tissue and 2 breast tumor subtypes