Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo–electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, ‘histone-like’ protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and repair. In humans, the UV-DDB complex detects ultraviolet light induced pyrimidine dimers throughout the genome, yet it remains unknown how these lesions are recognised in chromatin, where nucleosomes restrict DNA access. Here we report cryo-electron microscopy structures for UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer, and a DNA damage mimic at a variety of positions. We find that UV-DDB binds UV damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. For buried lesions facing the histone core, UV-DDB changes the predominant translational register of the nucleosome, and selectively binds the lesion in an accessible, exposed, position. These findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes. We identify s lide- a ssisted s ite- e xposure (SAsSE) as a mechanism for high-affinity DNA-binding proteins to access otherwise occluded sites in nucleosomal DNA.

Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo , quantitatively and without crosslinking and ligation, we implemented a modified version of DamID named DamC, which combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and biophysical modelling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

Genotype of cell lines was tested at the level of DNA sequence and protein (Western blotting). Mycoplasma contaminationCell lines tested negative for Mycoplasm.

Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S. pombe chromosomes. Known features of genome organization (e.g. the clustering of telomeres and retrotransposon long terminal repeats (LTRs)) were observed throughout the cell cycle. There were clear correlations between transcript levels and chromosomal interactions between genes, consistent with a role for interactions in transcriptional regulation at specific stages of the cell cycle. In silico reconstructions of the chromosome organization within the S. pombe nuclei were made by polymer modeling. These models suggest that groups of genes with high and low, or differentially regulated transcript levels have preferred positions within the S. pombe nucleus. We conclude that the S. pombe nucleus is spatially divided into functional sub-nuclear domains that correlate with gene activity. The observation that chromosomal interactions are maintained even when chromosomes are fully condensed in M phase implicates genome organization in epigenetic inheritance and bookmarking.

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