Project description:In higher eukaryotes, enhancers and promoters share many properties, including binding of transcription factors, existing in open chromatin, and bidirectional transcription. Yet the structural features that distinguish enhancers and promoters are unclear. Genome-wide micrococcal nuclease (MNase) studies previously interpreted MNase hypersensitivity to indicate that active enhancers and promoters are nucleosome-free, yet other studies found histone variants and post-translational modifications at active enhancers. We find that prior MNase genomic studies have an overdigestion bias and that low-level MNase digestion, coupled with mapping core histones, reveals two classes of MNase-hypersensitive sites: at active promoters, which are nucleosome depleted, and at tissue-specific enhancers, which retain core histones and co-bound transcription factors substantially more than promoters. Hypersensitivity of active enhancer nucleosomes may reflect their preferential exposure in chromatin and can be maintained by pioneer transcription factors such as FoxA. These findings unveil fundamental differences in the chromatin structure of active enhancers and promoters.
Project description:In higher eukaryotes, enhancers and promoters share many properties, including binding of transcription factors, existing in open chromatin, and bidirectional transcription. Yet the structural features that distinguish enhancers and promoters are unclear. Genome-wide micrococcal nuclease (MNase) studies previously interpreted MNase hypersensitivity to indicate that active enhancers and promoters are nucleosome-free, yet other studies found histone variants and post-translational modifications at active enhancers. We find that prior MNase genomic studies have an overdigestion bias and that low-level MNase digestion, coupled with mapping core histones, reveals two classes of MNase-hypersensitive sites: at active promoters, which are nucleosome depleted, and at tissue-specific enhancers, which retain core histones and co-bound transcription factors substantially more than promoters. Hypersensitivity of active enhancer nucleosomes may reflect their preferential exposure in chromatin and can be maintained by pioneer transcription factors such as FoxA. These findings unveil fundamental differences in the chromatin structure of active enhancers and promoters.
Project description:In higher eukaryotes, enhancers and promoters share many properties, including binding of transcription factors, existing in open chromatin, and bidirectional transcription. Yet the structural features that distinguish enhancers and promoters are unclear. Genome-wide micrococcal nuclease (MNase) studies previously interpreted MNase hypersensitivity to indicate that active enhancers and promoters are nucleosome-free, yet other studies found histone variants and post-translational modifications at active enhancers. We find that prior MNase genomic studies have an overdigestion bias and that low-level MNase digestion, coupled with mapping core histones, reveals two classes of MNase-hypersensitive sites: at active promoters, which are nucleosome depleted, and at tissue-specific enhancers, which retain core histones and co-bound transcription factors substantially more than promoters. Hypersensitivity of active enhancer nucleosomes may reflect their preferential exposure in chromatin and can be maintained by pioneer transcription factors such as FoxA. These findings unveil fundamental differences in the chromatin structure of active enhancers and promoters.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. RNA-seq S2 cells Drosophila melanogaster
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. Mnase-seq, Mnase-ChIP-seq of Drosophila melanogaster embryo and S2 cells chromatin
Project description:Enhancers act to regulate cell type specific gene expression by facilitating the transcription of target genes. In mammalian cells active or primed enhancers are commonly marked by monomethylation of Histone H3 at lysine 4 (H3K4me1) in a cell-type specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals has been unclear. In the present study, we conducted SILAC Mass-spec experiments with mono-nucleosomes and identified multiple H3K4me1 associated proteins, including proteins involved in chromatin remodeling. We demonstrate that H3K4me1 augments the association of the chromatin remodeling complex BAF to enhancers in vivo. Furthermore we show that in vitro, H3K4me1 nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of a BAF component BAF45c further reveal that monomethylation, but not trimethylation, is accommodated in this protein’s H3K4 binding site. Our results suggest that H3K4me1 plays an active role at enhancers by facilitating the binding of the BAF complex and possibly other chromatin regulators.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect.