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Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis - and trans -acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs. 

Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II’s NCAPG2 subunit. MCPH1’s ability to block condensin II’s association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis. 

Abstract Background The helmeted honeyeater (Lichenostomus melanops cassidix) is a Critically Endangered bird endemic to Victoria, Australia. To aid its conservation, the population is the subject of genetic rescue. To understand, monitor, and modulate the effects of genetic rescue on the helmeted honeyeater genome, a chromosome-length genome and a high-density linkage map are required. Results We used a combination of Illumina, Oxford Nanopore, and Hi-C sequencing technologies to assemble a chromosome-length genome of the helmeted honeyeater, comprising 906 scaffolds, with length of 1.1 Gb and scaffold N50 of 63.8 Mb. Annotation comprised 57,181 gene models. Using a pedigree of 257 birds and 53,111 single-nucleotide polymorphisms, we obtained high-density linkage and recombination maps for 25 autosomes and Z chromosome. The total sex-averaged linkage map was 1,347 cM long, with the male map being 6.7% longer than the female map. Recombination maps revealed sexually dimorphic recombination rates (overall higher in males), with average recombination rate of 1.8 cM/Mb. Comparative analyses revealed high synteny of the helmeted honeyeater genome with that of 3 passerine species (e.g., 32 Hi-C scaffolds mapped to 30 zebra finch autosomes and Z chromosome). The genome assembly and linkage map suggest that the helmeted honeyeater exhibits a fission of chromosome 1A into 2 chromosomes relative to zebra finch. PSMC analysis showed a ∼15-fold decline in effective population size to ∼60,000 from mid- to late Pleistocene. Conclusions The annotated chromosome-length genome and high-density linkage map provide rich resources for evolutionary studies and will be fundamental in guiding conservation efforts for the helmeted honeyeater. 

Patterns of genomic architecture across insects remain largely undocumented or decoupled from a broader phylogenetic context. For instance, it is unknown whether translocation rates differ between insect orders. We address broad scale patterns of genome architecture across Insecta by examining synteny in a phylogenetic framework from open-source insect genomes. To accomplish this, we add a chromosome level genome to a crucial lineage, Coleoptera. Our assembly of the Pachyrhynchus sulphureomaculatus genome is the first chromosome scale genome for the hyperdiverse Phytophaga lineage and currently the largest insect genome assembled to this scale. The genome is significantly larger than those of other weevils, and this increase in size is caused by repetitive elements. Our results also indicate that, among beetles, there are instances of long-lasting (>200 Ma) localization of genes to a particular chromosome with few translocation events. While some chromosomes have a paucity of translocations, intra-chromosomal synteny was almost absent, with gene order thoroughly shuffled along a chromosome. This large amount of reshuffling within chromosomes with few inter-chromosomal events contrasts with patterns seen in mammals in which the chromosomes tend to exchange larger blocks of material more readily. To place our findings in an evolutionary context, we compared syntenic patterns across Insecta in a phylogenetic framework. For the first time, we find that synteny decays at an exponential rate relative to phylogenetic distance. Additionally, there are significant differences in decay rates between insect orders, this pattern was not driven by Lepidoptera alone which has a substantially different rate. 

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future. 

Abstract Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution. 

Cells adapt and respond to changes by regulating the activity of their genes. To turn genes on or off, they use a family of proteins called transcription factors. Transcription factors influence specific but overlapping groups of genes, so that each gene is controlled by several transcription factors that act together like a dimmer switch to regulate gene activity. The presence of transcription factors attracts proteins such as the Mediator complex, which activates genes by gathering the protein machines that read the genes. The more transcription factors are found near a specific gene, the more strongly they attract Mediator and the more active the gene is. A specific region on the transcription factor called the activation domain is necessary for this process. The biochemical sequences of these domains vary greatly between species, yet activation domains from, for example, yeast and human proteins are often interchangeable. To understand why this is the case, Sanborn et al. analyzed the genome of baker’s yeast and identified 150 activation domains, each very different in sequence. Three-quarters of them bound to a subunit of the Mediator complex called Med15. Sanborn et al. then developed a machine learning algorithm to predict activation domains in both yeast and humans. This algorithm also showed that negatively charged and greasy regions on the activation domains were essential to be activated by the Mediator complex. Further analyses revealed that activation domains used different poses to bind multiple sites on Med15, a behavior known as ‘fuzzy’ binding. This creates a high overall affinity even though the binding strength at each individual site is low, enabling the protein complexes to remain dynamic. These weak interactions together permit fine control over the activity of several genes, allowing cells to respond quickly and precisely to many changes. The computer algorithm used here provides a new way to identify activation domains across species and could improve our understanding of how living things grow, adapt and evolve. It could also give new insights into mechanisms of disease, particularly cancer, where transcription factors are often faulty. 

Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make two-state transitions between closed conformations and open dumbbell conformations. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories. 

Abstract We introduce the Nucleome Data Bank (NDB), a web-based platform to simulate and analyze the three-dimensional (3D) organization of genomes. The NDB enables physics-based simulation of chromosomal structural dynamics through the MEGABASE + MiChroM computational pipeline. The input of the pipeline consists of epigenetic information sourced from the Encode database; the output consists of the trajectories of chromosomal motions that accurately predict Hi-C and fluorescence insitu hybridization data, as well as multiple observations of chromosomal dynamics in vivo. As an intermediate step, users can also generate chromosomal sub-compartment annotations directly from the same epigenetic input, without the use of any DNA–DNA proximity ligation data. Additionally, the NDB freely hosts both experimental and computational structural genomics data. Besides being able to perform their own genome simulations and download the hosted data, users can also analyze and visualize the same data through custom-designed web-based tools. In particular, the one-dimensional genetic and epigenetic data can be overlaid onto accurate 3D structures of chromosomes, to study the spatial distribution of genetic and epigenetic features. The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu.