Nucleosomes are non-uniformly distributed across eukarytic genomes, with stretches of ‘open’ chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and damaged genomic material. Here we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

Nucleosome positioning plays an important role in crucial biological processes like replication, transcription, and gene regulation. It has been widely used to predict the genome’s function and chromatin organisation. So far, the studies of patterns in nucleosome positioning have been limited to transcription start sites, CTCFs binding sites, and some promoter and loci regions. The genome-wide organisational pattern remains unknown. We have developed a theoretical model to coarse-grain nucleosome positioning data in order to obtain patterns in their distribution. Using hierarchical clustering on the auto-correlation function of this coarse-grained nucleosome positioning data, a genome-wide clustering is obtained for Candida albicans. The clustering shows the existence beyond hetero- and eu-chromatin inside the chromosomes.

The nucleosome is a major modulator of DNA accessibility to other cellular factors. Nucleosome positioning has a critical importance in regulating cell processes such as transcription, replication, recombination or DNA repair. The DNA sequence is a major factor influencing the position of nucleosomes on genomes. Different sequence motifs can promote or inhibit the nucleosome formation, thus influencing the accessibility to the DNA. Sequence-encoded nucleosome positioning having functional consequences on cell processes can then be selected or counter-selected during evolution. We review the interplay between sequence evolution and nucleosome positioning evolution. We first focus on the different ways to encode nucleosome positions in the DNA sequence, and to which extent these mechanisms are responsible of genome-wide nucleosome positioning in vivo. Then, we discuss the findings about selection of sequences for their nucleosomal properties. Finally, we illustrate how the nucleosome can directly influence sequence evolution through its interactions with DNA damage and repair mechanisms. This review aims to provide an overview of the mutual influence of sequence evolution and nucleosome positioning evolution, possibly leading to complex evolutionary dynamics.

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA near the superhelical location (SHL) -5.5/5.5. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in vitro and in vivo inspired an intrinsic nucleosome exclusion mechanism in vivo. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. 1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. 2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. 3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and 4) actively maintained in vivo. 5) S. cerevisiae promoters evolved a biased poly(dA) versus poly(dT) distribution. 6) Nucleosome depletion over poly(dA) is directional in vivo. 7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5’ of poly(dA). Especially bias and directionality would not be expected for an intrinsic mechanism. Together, this argues for a mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome occupancy.

The natural flavonoid epigallocatechin gallate (EGCG) has a wide range of biological activities including binding to nucleic acids; however, mechanisms of interaction of EGCG with DNA organized in chromatin have not been systematically studied. In this work, interaction of EGCG with chromatin in cells, and with nucleosomes and chromatosomes in vitro was studied using fluorescent microscopy and single particle Förster resonance energy transfer approaches, respectively. EGCG effectively penetrates into the nuclei of living cells and binds to DNA there. Interaction of EGCG with nucleosomes in vitro induces large-scale, reversible uncoiling of nucleosomal DNA that occurs without dissociation of DNA or core histones at sub- and low-micromolar concentrations of EGCG. EGCG does not reduce the catalytic activity of poly(ADP-ribose) polymerase 1 (PARP1), but causes modulation of the structure of the PARP1-nucleosome complex. EGCG significantly changes the structure of chromatosomes, but does not cause dissociation of the linker histone. Reorganization of nucleosomes and chromatosomes by EGCG might facilitate access of protein factors involved in DNA repair, replication and transcription to DNA and thus might contribute to the modulation of gene expression by EGCG that was reported earlier.

Background: Liquid biopsies are revolutionary tools to detect tumor-specific genetic alterations in body fluids, and cell-free DNA (cfDNA) can be used for molecular diagnosis in cancer patients. In brain tumors cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: CSF cfDNA of pediatric patients with medulloblastoma (n=18), ATRT (n=3), ETMR (n=1), CNS NB FOXR2 (n=2) and pediatric EBT NOS (n=1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES, and/or a targeted sequencing approach, to determine single nucleotide variations (SNVs)/copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n=15), a mean of 83 (range 1-160) shared SNVs was observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were observed suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% of cases (15/25), with WES feasible in 83% (15/18). These results pave the way for implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins that work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases resulting in abasic sites. Many DNA glycosylases are product inhibited by binding to the abasic site more avidly than the damaged base. Traditionally apurinic/apyrimidinic endonuclease, APE1, was believed to help turnover the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three-to-five-fold. Moreover, we have shown UV-DDB can assist chromatin decompaction facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.