A dynamic interplay of enhancer elements regulates Klf4 expression in naïve pluripotency.

Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.

Filling annotation gaps in yeast genomes using genome-wide contact maps.

MOTIVATIONS: De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. RESULTS: Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. AVAILABILITY AND IMPLEMENTATION: The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. CONTACT: romain.koszul@pasteur.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Normalization of a chromosomal contact map.

BACKGROUND: Chromatin organization has been increasingly studied in relation with its important influence on DNA-related metabolic processes such as replication or regulation of gene expression. Since its original design ten years ago, capture of chromosome conformation (3C) has become an essential tool to investigate the overall conformation of chromosomes. It relies on the capture of long-range trans and cis interactions of chromosomal segments whose relative proportions in the final bank reflect their frequencies of interactions, hence their spatial proximity in a population of cells. The recent coupling of 3C with deep sequencing approaches now allows the generation of high resolution genome-wide chromosomal contact maps. Different protocols have been used to generate such maps in various organisms. This includes mammals, drosophila and yeast. The massive amount of raw data generated by the genomic 3C has to be carefully processed to alleviate the various biases and byproducts generated by the experiments. Our study aims at proposing a simple normalization procedure to minimize the influence of these unwanted but inevitable events on the final results. RESULTS: Careful analysis of the raw data generated previously for budding yeast S. cerevisiae led to the identification of three main biases affecting the final datasets, including a previously unknown bias resulting from the circularization of DNA molecules. We then developed a simple normalization procedure to process the data and allow the generation of a normalized, highly contrasted, chromosomal contact map for S. cerevisiae. The same method was then extended to the first human genome contact map. Using the normalized data, we revisited the preferential interactions originally described between subsets of discrete chromosomal features. Notably, the detection of preferential interactions between tRNA in yeast and CTCF, PolII binding sites in human can vary with the normalization procedure used. CONCLUSIONS: We quantitatively reanalyzed the genomic 3C data obtained for S. cerevisiae, identified some of the biases inherent to the technique and proposed a simple normalization procedure to analyse them. Such an approach can be easily generalized for genomic 3C experiments in other organisms. More experiments and analysis will be necessary to reach optimal resolution and accuracies of the maps generated through these approaches. Working with cell population presenting highest levels of homogeneity will prove useful in this regards.

instaGRAAL: chromosome-level quality scaffolding of genomes using a proximity ligation-based scaffolder.

Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs during genome assembly, resulting in chromosome-level assemblies. Because few robust programs are available for this type of data, we developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. instaGRAAL features a number of improvements over GRAAL, including a modular correction approach that optionally integrates independent data. We validate the program using data for two brown algae, and human, to generate near-complete assemblies with minimal human intervention.

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization.

RNA polymerase II clustering through carboxy-terminal domain phase separation.

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

Contact genomics: scaffolding and phasing (meta)genomes using chromosome 3D physical signatures.

High-throughput DNA sequencing technologies are fuelling an accelerating trend to assemble de novo or resequence the genomes of numerous species as well as to complete unfinished assemblies. While current DNA sequencing technologies remain limited to reading stretches of a few hundreds or thousands of base pairs, experimental and computational methods are continuously improving with the goal of assembling entire genomes from large numbers of short DNA sequences. However, the algorithms that piece together DNA strands face important limitations due, notably, to the presence of repeated sequences or of multiple haplotypes within one genome, thus leaving many assemblies incomplete. Recently, the realization that the physical contacts experienced by a portion of a DNA molecule could be used as a robust and quantitative assay to determine its genomic position has led to the emerging field of contact genomics, which promises to revolutionize current genome assembly approaches by exploiting the flexible polymer properties of chromosomes. Here we review the current applications of contact genomics to genome scaffolding, haplotyping and metagenomic assembly, then outline the future developments we envision.

Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms.

Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis.

High-quality genome (re)assembly using chromosomal contact data.

Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left 'unfinished.' Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm--named GRAAL--generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches.

A predictive computational model of the dynamic 3D interphase yeast nucleus.

BACKGROUND: Despite the absence of internal membranes, the nucleus of eukaryotic cells is spatially organized, with chromosomes and individual loci occupying dynamic, but nonrandom, spatial positions relative to nuclear landmarks and to each other. These positional preferences correlate with gene expression and DNA repair, recombination, and replication. Yet the principles that govern nuclear organization remain poorly understood and detailed predictive models are lacking. RESULTS: We present a computational model of dynamic chromosome configurations in the interphase yeast nucleus that is based on first principles and is able to statistically predict the positioning of any locus in nuclear space. Despite its simplicity, the model agrees with extensive previous and new measurements on locus positioning and with genome-wide DNA contact frequencies. Notably, our model recapitulates the position and morphology of the nucleolus, the observed variations in locus positions, and variations in contact frequencies within and across chromosomes, as well as subchromosomal contact features. The model is also able to correctly predict nuclear reorganization accompanying a reduction in ribosomal DNA transcription, and sites of chromosomal rearrangements tend to occur where the model predicted high contact frequencies. CONCLUSIONS: Our results suggest that large-scale yeast nuclear architecture can be largely understood as a consequence of generic properties of crowded polymers rather than of specific DNA-binding factors and that configurations of chromosomes and DNA contacts are dictated mainly by genomic location and chromosome lengths. Our model provides a quantitative framework to understand and predict large-scale spatial genome organization and its interplay with functional processes.