The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

Proximal-end bias from in-vitro reconstituted nucleosomes and the result on downstream data analysis

The most basic level of eukaryotic gene regulation is the presence or absence of nucleosomes on DNA regulatory elements. In an effort to elucidate in vivo nucleosome patterns, in vitro studies are frequently used. In vitro, short DNA fragments are more favorable for nucleosome formation, increasing the likelihood of nucleosome occupancy. This may in part result from the fact that nucleosomes prefer to form on the terminal ends of linear DNA. This phenomenon has the potential to bias in vitro reconstituted nucleosomes and skew results. If the ends of DNA fragments are known, the reads falling close to the ends are typically discarded. In this study we confirm the phenomenon of end bias of in vitro nucleosomes. We describe a method in which nearly identical libraries, with different known ends, are used to recover nucleosomes which form towards the terminal ends of fragmented DNA. Finally, we illustrate that although nucleosomes prefer to form on DNA ends, it does not appear to skew results or the interpretation thereof.

Chemical map-based prediction of nucleosome positioning using the Bioconductor package nuCpos

Background Assessing the nucleosome-forming potential of specific DNA sequences is important for understanding complex chromatin organization. Methods for predicting nucleosome positioning include bioinformatics and biophysical approaches. An advantage of bioinformatics methods, which are based on in vivo nucleosome maps, is the use of natural sequences that may contain previously unknown elements involved in nucleosome positioning in vivo. The accuracy of such prediction attempts reflects the genomic coordinate resolution of the nucleosome maps applied. Nucleosome maps are constructed using micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq). However, as MNase has a strong preference for A/T-rich sequences, MNase-seq may not be appropriate for this purpose. In addition to MNase-seq-based maps, base pair-resolution chemical maps of in vivo nucleosomes from three different species (budding and fission yeasts, and mice) are currently available. However, these chemical maps have yet to be integrated into publicly available computational methods. Results We developed a Bioconductor package (named nuCpos) to demonstrate the superiority of chemical maps in predicting nucleosome positioning. The accuracy of chemical map-based prediction in rotational settings was higher than that of the previously developed MNase-seq-based approach. With our method, predicted nucleosome occupancy reasonably matched in vivo observations and was not affected by A/T nucleotide frequency. Effects of genetic alterations on nucleosome positioning that had been observed in living yeast cells could also be predicted. nuCpos calculates individual histone binding affinity (HBA) scores for given 147-bp sequences to examine their suitability for nucleosome formation. We also established local HBA as a new parameter to predict nucleosome formation, which was calculated for 13 overlapping nucleosomal DNA subsequences. HBA and local HBA scores for various sequences agreed well with previous in vitro and in vivo studies. Furthermore, our results suggest that nucleosomal subsegments that are disfavored in different rotational settings contribute to the defined positioning of nucleosomes. Conclusions Our results demonstrate that chemical map-based statistical models are beneficial for studying nucleosomal DNA features. Studies employing nuCpos software can enhance understanding of chromatin regulation and the interpretation of genetic alterations and facilitate the design of artificial sequences.

Accurate modeling of DNA conformational flexibility by a multivariate Ising model

The sequence-dependent structure and deformability of DNA play a major role for binding of proteins and regulation of gene expression. So far, most efforts to model DNA flexibility are based on unimodal harmonic stiffness models at base-pair resolution. However, multimodal behavior due to distinct conformational substates also contributes significantly to the conformational flexibility of DNA. Moreover, these local substates are correlated to their nearest-neighbor substates. A description for DNA elasticity which includes both multimodality and nearest-neighbor coupling has remained a challenge, which we solve by combining our multivariate harmonic approximation with an Ising model for the substates. In a series of applications to DNA fluctuations and protein–DNA complexes, we demonstrate substantial improvements over the unimodal stiffness model. Furthermore, our multivariate Ising model reveals a mechanical destabilization for adenine (A)-tracts to undergo nucleosome formation. Our approach offers a wide range of applications to determine sequence-dependent deformation energies of DNA and to investigate indirect readout contributions to protein–DNA recognition.

Structural and dynamic mechanisms of CBF3-guided centromeric nucleosome formation

Accurate chromosome segregation relies on the specific centromeric nucleosome–kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.

Slow motions in A·T rich DNA sequence

In free B-DNA, slow (microsecond-to-millisecond) motions that involve equilibrium between Watson–Crick (WC) and Hoogsteen (HG) base-pairing expand the DNA dynamic repertoire that could mediate DNA–protein assemblies. R1ρ relaxation dispersion NMR methods are powerful tools to capture such slow conformational exchanges in solution using 13C/15 N labelled DNA. Here, these approaches were applied to a dodecamer containing a TTAAA element that was assumed to facilitate nucleosome formation. NMR data and inferred exchange parameters assign HG base pairs as the minor, transient conformers specifically observed in three successive A·T base pairs forming the TAA·TTA segment. The abundance of these HG A·T base pairs can be up to 1.2% which is high compared to what has previously been observed. Data analyses support a scenario in which the three adenines undergo non-simultaneous motions despite their spatial proximity, thus optimising the probability of having one HG base pair in the TAA·TTA segment. Finally, revisiting previous NMR data on H2 resonance linewidths on the basis of our results promotes the idea of there being a special propensity of A·T base pairs in TAA·TTA tracts to adopt HG pairing. In summary, this study provides an example of a DNA functional element submitted to slow conformational exchange. More generally, it strengthens the importance of the role of the DNA sequence in modulating its dynamics, over a nano- to milli-second time scale.

Measuring DNA mechanics on the genome scale

Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions1. However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA – a proxy for DNA bendability – in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors2 by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a ‘mechanical code’ with broad functional implications.

Interplay among ATP-Dependent Chromatin Remodelers Determines Chromatin Organisation in Yeast

Cellular DNA is packaged into chromatin, which is composed of regularly-spaced nucleosomes with occasional gaps corresponding to active regulatory elements, such as promoters and enhancers, called nucleosome-depleted regions (NDRs). This chromatin organisation is primarily determined by the activities of a set of ATP-dependent remodeling enzymes that are capable of moving nucleosomes along DNA, or of evicting nucleosomes altogether. In yeast, the nucleosome-spacing enzymes are ISW1 (Imitation SWitch protein 1), Chromodomain-Helicase-DNA-binding (CHD)1, ISW2 (Imitation SWitch protein 2) and INOsitol-requiring 80 (INO80); the nucleosome eviction enzymes are the SWItching/Sucrose Non-Fermenting (SWI/SNF) family, the Remodeling the Structure of Chromatin (RSC) complexes and INO80. We discuss the contributions of each set of enzymes to chromatin organisation. ISW1 and CHD1 are the major spacing enzymes; loss of both enzymes results in major chromatin disruption, partly due to the appearance of close-packed di-nucleosomes. ISW1 and CHD1 compete to set nucleosome spacing on most genes. ISW1 is dominant, setting wild type spacing, whereas CHD1 sets short spacing and may dominate on highly-transcribed genes. We propose that the competing remodelers regulate spacing, which in turn controls the binding of linker histone (H1) and therefore the degree of chromatin folding. Thus, genes with long spacing bind more H1, resulting in increased chromatin compaction. RSC, SWI/SNF and INO80 are involved in NDR formation, either directly by nucleosome eviction or repositioning, or indirectly by affecting the size of the complex that resides in the NDR. The nature of this complex is controversial: some suggest that it is a RSC-bound “fragile nucleosome”, whereas we propose that it is a non-histone transcription complex. In either case, this complex appears to serve as a barrier to nucleosome formation, resulting in the formation of phased nucleosomal arrays on both sides.

An overview of high-mobility group box 1, a potent pro-inflammatory cytokine in asthma

High-mobility group box 1 (HMGB1), also called amphoterin, HMG1 and p30, is a highly conserved protein between different species that has various functions in nucleus such as stabilization of nucleosome formation, facilitation of deoxyribonucleic acid (DNA) bending and increasing the DNA transcription, replication and repair. It has also been indicated that HMGB1 acts as a potent pro-inflammatory cytokine with increasing concentrations in acute and chronic inflammatory diseases. Asthma is a common chronic respiratory disease associated with high morbidity and mortality rates. One central characteristic in its pathogenesis is airway inflammation. Considering the inflammatory role of HMGB1 and importance of inflammation in asthma pathogenesis, a better understanding of this protein is vital. This review describes the structure, cell surface receptors, signaling pathways and intracellular and extracellular functions of HMGB1, but also focuses on its inflammatory role in asthma. Moreover, this manuscript reviews experimental and clinical studies that investigated the pathologic role of HMGB1.