Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ‘ruler’ that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

On the role of transcription in positioning nucleosomes

Nucleosome positioning is crucial for the genome’s function. Though the role of DNA sequence in positioning nucleosomes is well understood, a detailed mechanistic understanding on the impact of transcription remains lacking. Using numerical simulations, we investigated the dependence of nucleosome density profiles on transcription level across multiple species. We found that the low nucleosome affinity of yeast, but not mouse, promoters contributes to the formation of phased nucleosomes arrays for inactive genes. For the active genes, a heterogeneous distribution of +1 nucleosomes, caused by a tug-of-war between two types of remodeling enzymes, is essential for reproducing their density profiles. In particular, while positioning enzymes are known to remodel the +1 nucleosome and align it toward the transcription start site (TSS), spacer enzymes that use a pair of nucleosomes as their substrate can shift the nucleosome array away from the TSS. Competition between these enzymes results in two types of nucleosome density profiles with well- and ill-positioned +1 nucleosome. Finally, we showed that Pol II assisted histone exchange, if occurring at a fast speed, can abolish the impact of remodeling enzymes. By elucidating the role of individual factors, our study reconciles the seemingly conflicting results on the overall impact of transcription in positioning nucleosomes across species.

On the role of transcription in positioning nucleosomes

Nucleosome positioning is crucial for the genome’s function. Though the role of DNA sequence in positioning nucleosomes is well understood, a unified framework for studying the impact of transcription remains lacking. Using numerical simulations, we investigated the dependence of nucleosome density profiles on transcription level across multiple species. We found that the low nucleosome affinity of yeast, but not mouse, promoters contributes to the formation of phased nucleosomes arrays for inactive genes. For the active genes, a tug-of-war between two types of remodeling enzymes is essential for reproducing their density profiles. In particular, while ISW2 related enzymes are known to position the +1 nucleosome and align it toward the transcription start site (TSS), enzymes such as ISW1 that use a pair of nucleosomes as their substrate can shift the nucleosome array away from the TSS. Competition between these enzymes results in two types of nucleosome density profiles with well- and ill-positioned +1 nucleosome. Finally, we showed that Pol II assisted histone exchange, if occurring at a fast speed, can abolish the impact of remodeling enzymes. By elucidating the role of individual factors, our study reconciles the seemingly conflicting results on the overall impact of transcription in positioning nucleosomes across species.

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ‘ruler’ that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length

The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we investigate chromatin boundaries in mouse embryonic stem cells, defined by the regions with decreased Nucleosome Repeat Length (NRL) for ∼20 nucleosomes near CTCF sites, affecting up to 10% of the genome. We found that the nucleosome-depleted region (NDR) near CTCF is asymmetrically located >40 nucleotides 5′-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA and the presence of cohesin is correlated with the decrease of NRL near CTCF, and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation, a subset of preserved, common CTCF sites maintains asymmetric nucleosome pattern and small NRL. The sites which lost CTCF upon differentiation are characterized by nucleosome rearrangement 3′-downstream, with unchanged NDR 5′-upstream of CTCF motifs. Boundaries of topologically associated chromatin domains frequently contain several inward-oriented CTCF motifs whose effects, described above, add up synergistically.

CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length

The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we focus on the relationship between CTCF binding and the decrease of the Nucleosome Repeat Length (NRL) for ∼20 adjacent nucleosomes, affecting up to 10% of the mouse genome. We found that the chromatin boundary near CTCF is created by the nucleosome-depleted region (NDR) asymmetrically located >40 nucleotides 5’-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA is correlated with the decrease of NRL near CTCF and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation of embryonic stem cells to neural progenitor cells and embryonic fibroblasts, a subset of common CTCF sites preserved in all three cell types maintains a relatively small local NRL despite genome-wide NRL increase. The sites which lost CTCF upon differentiation are characterised by nucleosome rearrangement 3’-downstream, but the boundary defined by the NDR 5’-upstream of CTCF motif remains.

Nucleosome positioning and spacing: from genome-wide maps to single arrays

The positioning of nucleosomes relative to DNA and their neighboring nucleosomes represents a fundamental layer of chromatin organization. Changes in nucleosome positioning and spacing affect the accessibility of DNA to regulatory factors and the formation of higher order chromatin structures. Sequencing of mononucleosomal fragments allowed mapping nucleosome positions on a genome-wide level in many organisms. This revealed that successions of evenly spaced and well-positioned nucleosomes—so called phased nucleosome arrays—occur at the 5′ end of many active genes and in the vicinity of transcription factor and other protein binding sites. Phased arrays arise from the interplay of barrier elements on the DNA, which position adjacent nucleosomes, and the nucleosome spacing activity of ATP-dependent chromatin remodelers. A shortcoming of classic mononucleosomal mapping experiments is that they only reveal nucleosome spacing and array regularity at select sites in the genome with well-positioned nucleosomes. However, new technological approaches elucidate nucleosome array structure throughout the genome and with single-cell resolution. In the future, it will be interesting to see whether changes in nucleosome array regularity and spacing contribute to the formation of higher order chromatin structures and the spatial organization of the genome in vivo.