Active Promoters give rise to false positive 'Phantom Peaks' in ChIP-Seq experiments
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE67323 | GEO | 2015/07/10
SECONDARY ACCESSION(S): PRJNA279510
REPOSITORIES: GEO
Similar Datasets
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.
2015-07-10 | E-GEOD-67323 | biostudies-arrayexpress
Project description:The chromatin remodeling complexes CHRAC and ACF combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
2018-02-05 | GSE106732 | GEO
Project description:The chromatin remodeling complexes CHRAC and ACF combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
2018-02-05 | GSE106731 | GEO
Project description:Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq).We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters.Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. 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 appear as 'Phantom Peaks'. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.
| S-EPMC4538825 | biostudies-literature
Project description:We applied a multiplexed genomic tethering assay (Brueckner, Epigenetics Chromatin, 2016; PMID:27777628) to elucidate the function of ACF1, the large subunit of the ACF nucleosome sliding complex in drosophila Kc167 cells. Targeting a Gal4DBD-ACF1 fusion to several hundred integrated reporters revealed a context-dependent repressive effect which is modulated by gene activity and chromatin state.
2017-11-10 | GSE106759 | GEO
Project description:Acf1 was isolated almost 15 years ago from Drosophila embryo extracts as a subunit of several complexes that possess chromatin assembly activity. Acf1 binds to the ATPase ISWI (SNF2H in mammals) to form the ACF/CHRAC chromatin remodeling complexes. Studies in Drosophila and in human and mouse cell culture implicate Acf1 in many cellular processes including transcriptional repression, heterochromatin formation and replication, and DNA damage checkpoints and repair. However, the in vivo function of Acf1 in mammals is unknown. We generated mice deficient for Baz1a, the mammalian homolog of Drosophila Acf1. In contrast to partially penetrant lethality of the mutation in flies, Baz1a-deficient mice are viable. The mutant mice show no obvious defects in B or T cell lineages, class switch recombination in cultured B cells, or meiotic recombination. Thus, BAZ1A is dispensable in vivo for the survival and differentiation of cells that require the repair of developmentally programmed DNA double-strand breaks (DSBs). However, Baz1a-/- male mice are sterile because of a severe defect in spermiogenesis that results in fewer and non-motile sperm with morphological defects. Baz1a-deficient round spermatids display widespread perturbation of gene expression. The mutant cells faithfully execute the widespread reprogramming of gene expression that normally accompanies the developmental transition from meiosis to postmeiotic differentiation, but overlaid on this normal developmental program is mis-regulation of a large number of additional genes. The inappropriate expression of this eclectic group of genes is likely the cause of the pleiotropic spermiogenesis defects in the mutant. We propose that the dramatic changes in chromatin composition that occur in late meiotic prophase and early spermiogenesis create a window of vulnerability during which BAZ1A-targeted chromatin assembly functions are needed to prevent inappropriate transcription changes. This appears to be the sole essential function of the ACF and CHRAC complexes in mouse. total RNA isolated from FACS-enriched pachytene/diplotene spermatocytes and round spermatids from three littermate pairs of Baz1a-/- and Baz1a-/+ animals. Data from all samples are provided, but expression data for one Baz1a-/- spermatocyte sample (683_PD) was censored because of inferred high degree of contamination with round spermatids
2013-11-12 | E-GEOD-41303 | biostudies-arrayexpress
Project description:Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of repression remains unclear. We used a forward genetic approach to identify novel genes involved in transcriptional silencing of H3K27 methylated chromatin in the filamentous fungus, Neurospora crassa. We found crf4-1Iswi and crf4-2Itc1/Acf1 are required for repression of a subset of H3K27 methylated genes and that their protein products interact to form an ACF-like chromatin remodeling complex. This N. crassa ACF-like complex is promiscuous, yet can target facultative heterochromatin, where it is responsible for pulling the +1 nucleosome in the 5’ direction. H3K27 methylated genes that are up-regulated when crf4-1Iswi or crf4-2Itc1/Acf1 are deleted show a downstream shift of the +1 nucleosome suggesting that proper nucleosome positioning is important for repression of facultative heterochromatin.
2022-03-08 | GSE168277 | GEO
Project description:Despite depression being one of the most prevalent and debilitating disorders worldwide, it has been difficult to understand its pathophysiology and to develop more effective treatments. Maladaptive transcriptional regulation within limbic neural circuits, including reward processing regions such as the nucleus accumbens (NAc), in response to chronic stress is thought to be a major contributor to the development of the syndrome. Epigenetic events?in particular, histone writers and erasers?that alter chromatin structure to regulate programs of gene expression have increasingly been associated with depression-related behavioral abnormalities in animal models and in depressed humans examined postmortem. However, very little is known about the ATP-dependent chromatin remodelers that control nucleosome positioning and the packing state of chromatin. Here we show that the ACF complex, part of the ISWI family of chromatin remodelers, is persistently and selectively upregulated in the NAc of mice that are susceptible to chronic social stress, as well as in the NAc of depressed human. We further establish that ACF induction is both necessary and sufficient for susceptibility to stress-induced depressive-like behaviors. Using ChIP-seq, we demonstrate that altered ACF binding after chronic stress is strongly correlated with altered nucleosome positioning, in particular, around the transcriptional start sites of affected genes. These alterations in ACF binding and nucleosome repositioning are associated with repressed expression of a subset of genes in animals that are susceptible to chronic stress. Together, these findings establish that active ATP-dependent chromatin remodeling by the ACF complex is a key regulator in the repression of genes that mediate susceptibility to social stress, and provide novel candidate targets for improved therapeutics of depression and other stress-related disorders. c57bl/6 mice underwent chronic social defeat stress (CSDS), and social interaction test was used to separate animals into control, susceptible and resilient groups. Nucleus accumbens (NAc) tissue was collected 48 hours after the last defeat session, and then Acf1, SNF2H ChIP-seq or H3 MNase-seq were performed based on the control, susceptible, and resilient groups. Three sequencing replicates were performed on each group.
2015-09-21 | E-GEOD-54263 | biostudies-arrayexpress
Project description:Acf1 was isolated almost 15 years ago from Drosophila embryo extracts as a subunit of several complexes that possess chromatin assembly activity. Acf1 binds to the ATPase ISWI (SNF2H in mammals) to form the ACF/CHRAC chromatin remodeling complexes. Studies in Drosophila and in human and mouse cell culture implicate Acf1 in many cellular processes including transcriptional repression, heterochromatin formation and replication, and DNA damage checkpoints and repair. However, the in vivo function of Acf1 in mammals is unknown. We generated mice deficient for Baz1a, the mammalian homolog of Drosophila Acf1. In contrast to partially penetrant lethality of the mutation in flies, Baz1a-deficient mice are viable. The mutant mice show no obvious defects in B or T cell lineages, class switch recombination in cultured B cells, or meiotic recombination. Thus, BAZ1A is dispensable in vivo for the survival and differentiation of cells that require the repair of developmentally programmed DNA double-strand breaks (DSBs). However, Baz1a-/- male mice are sterile because of a severe defect in spermiogenesis that results in fewer and non-motile sperm with morphological defects. Baz1a-deficient round spermatids display widespread perturbation of gene expression. The mutant cells faithfully execute the widespread reprogramming of gene expression that normally accompanies the developmental transition from meiosis to postmeiotic differentiation, but overlaid on this normal developmental program is mis-regulation of a large number of additional genes. The inappropriate expression of this eclectic group of genes is likely the cause of the pleiotropic spermiogenesis defects in the mutant. We propose that the dramatic changes in chromatin composition that occur in late meiotic prophase and early spermiogenesis create a window of vulnerability during which BAZ1A-targeted chromatin assembly functions are needed to prevent inappropriate transcription changes. This appears to be the sole essential function of the ACF and CHRAC complexes in mouse.
2013-11-12 | GSE41303 | GEO
Project description:We comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Across a total of 30 new and 6 published CUT&Tag datasets we found that no experiment recovers more than 50% of known ENCODE peaks, regardless of the histone mark. We tested peak callers MACS2 and SEACR, identifying optimal peak calling parameters. Balancing both precision and recall of known ENCODE peaks, SEACR without retention of duplicates showed the best performance. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
2022-03-31 | GSE199611 | GEO