ABSTRACT: H3K4me1 binding in murine pre-B cells detected by ChIP-seq. For the ChIP-seq, input and immunoprecipitated DNA was given to the TSRI Next Generation Sequencing Core (the Scripps Research Institute, La Jolla, CA, US), where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
Project description:Genome-wide assessment of protein-DNA interactions binding by ChIP-seq is a key technology to study transcription factor (TF) localization and regulation of gene-expression. In ChIP-seq, signal-to-noise-ratio as well as signal specificity depend on many variables including antibody quality, and efforts to improve ChIP-seq data thus far focused mostly on generating better reagents. Here we introduce KOIN (KO implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout-mice as a critical control for ChIP-seq. We tested our new peak calling strategy (KO implemented normalization = KOIN) on different ChIP-seq datasets to increase signal specificity and reduce noise.
Project description:According to current models, transcription factors (TFs) activated by extracellular stimuli operate in the context of a pre-established enhancer repertoire induced and maintained by lineage-specific TFs. Here, we uncovered the existence of latent enhancers, defined as regions of the genome that in terminally differentiated cells are poorly accessible and lack the histone marks characteristic of enhancers, but readily acquire these features in response to extracellular cues. Stimulation of resting macrophages caused simultaneous binding of stimulus-activated TFs and lineage-determining TFs to these regions, enabling deposition of enhancer-specific features. Once unveiled, these enhancers did not return to a latent state even when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the available cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents. Formaldehyde-Assisted Isolation Of Regulatory Elements (FAIRE) followed by multiparallel sequencing was performed in untreated murine bone marrow-derived macrophages.
Project description:The transcription factor IRF8 is a critical regulator of plasmacytoid dendritic cell (pDC) and classical dendritic cell (cDC) development in both mouse and man. Yet the downstream molecular targets that regulate DC homeostasis and development are largely unknown. A recent study using gene expression analysis of IRF8-deficient myeloid and lymphoid progenitors identified the Myc paralog Mycl1 as a potential transcriptional target of IRF8. We report here that Mycl1 is a mediator of DC homeostasis at steady state and during inflammation, and its expression is regulated by IRF8 in multiple DC lineages. We have further validated these observations with ChIP-Seq of IRF8 binding to the Mycl1 locus. Notably, IRF8 binding to Mycl1 locus is independent of an interaction with the AP1 factor, BATF3. Additionally, our genome-wide survey of IRF8 binding identified both EICE and AICE motifs. Examination of IRF8 binding in dendritic cells
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5’-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
Project description:Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput data sets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif data sets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well- defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks. Bone marrow-derived macrophages were stimulated with lipid A for 0, 15, 30, 60, and 120 minutes. Chromatin was immunoprecipitated with antibodies against RelA, SRF, or IRF3.
Project description:We previously demonstrated by genomic and bioinformatical approaches that human macrophage (MΦ) activation is best described by a spectrum model (Xue et al, Immunity, 2014). MΦ integrate exogenous input signals on transcriptional level in a unique fashion to generate specific functional programs, enabling the plasticity in disease-related pathophysiologies. Such versatile responsiveness requires fast changes of transcription mediated by transcriptional regulators (TRs) or epigenomic changes. To better understand the principles of this regulation during human MΦ activation, we assessed histone modifications including H3K4me1, H3K4me3, H3K27me3, and H3K27Ac by ChIP-sequencing allowing us to characterize the functional state of promoters (active, poised, repressed) and enhancers (active, inactive, intermediate). Using transcriptome data from our MΦ spectrum model, we generated a co-regulation network of all TRs. Next, we overlaid epigenomic information and transcriptional changes of major TRs over time onto the TR network. We observed that input signals like IFNγ or TNFα induce a specific network of TRs that are transcriptionally regulated themselves, the combination of regulated TRs changes over time with a boost of transcriptional regulation of dozens of TRs 4 to 12 hrs post input signal exposure, almost all TRs within the network show active promoters, even if the TR itself is not expressed, and similar results are obtained for enhancers with open or at least intermediated states. These findings strongly suggest that in MΦ, the TR-defined cellular ‘switch panel’ is always accessible thereby allowing MΦ to quickly respond to the diverse input signal repertoire from the environment. Epigenetic analysis of promoter and enhancer sites in primary human macrophage subtypes and correlation to RNA-seq expression data
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:We report the genome wide binding sites of BAP1, HCF1 and OGT in bone marrow derived macrophages. De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor–1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans. For BAP1, bone marrow derived macrophages were used differentiated from bone marrow cells of BAP1-3X Flag Tagged KI mice we generated. For OGT and HCF1, bone marrow derived macrophages were used from BAP1 WT mice.