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In eukaryotic genomes, rDNA generally resides as a highly repetitive and dynamic structure, making it difficult to study. Here, a synthetic rDNA array on chromosome III in budding yeast was constructed to serve as the sole source of rRNA. Utilizing the loxPsym site within each rDNA repeat and the Cre recombinase, we were able to reduce the copy number to as few as eight copies. Additionally, we constructed strains with two or three rDNA arrays and found that the presence of multiple arrays did not affect the formation of a single nucleolus. Although alteration of the position and number of rDNA arrays did impact the three-dimensional genome structure, the additional rDNA arrays had no deleterious influence on cell growth or transcriptomes. Overall, this study sheds light on the high plasticity of rDNA organization and opens up opportunities for future rDNA engineering. [Display omitted] • An efficient method for constructing ectopic rDNA arrays is established • As few as eight copies of rDNA repeats are adequate to sustain yeast viability • Two or three DNA arrays on separate chromosomes form a single nucleolus in yeast • Extra rDNA arrays do not disrupt normal cell growth or global transcriptomes Jiang et al. have devised effective techniques for engineering repetitive rDNA arrays. Their research not only unveils that yeast viability necessitates no more than eight copies of rDNA repeats but also showcases the formation of a single nucleolus by three rDNA arrays, emphasizing the considerable adaptability of rDNA organization. [ABSTRACT FROM AUTHOR]

Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function. [Display omitted] • CEBPA drives compartment switching and forms active 3D chromatin "hubs" in B cells • Aromatic residues in the CEBPA-IDR confer phase separation capacity in vitro • CEBPA forms hubs that colocalize with transcriptional co-activators in a native cell context • Findings suggest a mechanistic role for phase separation in CEBPA function Christou-Kent et al. demonstrate that myeloid factor CEBPA undergoes phase separation (PS) through its intrinsically disordered region in vitro. The authors suggest that a PS mechanism may contribute to its capacity to rewire higher-order chromatin and modulate gene expression by bringing chromatin sites together at transcriptionally active hubs. [ABSTRACT FROM AUTHOR]

Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila , the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis -regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B , with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output. [Display omitted] • Boundary elements separate domains of 3D genome organization and histone modifications • Different pairs of boundaries can separate the active Abd-B cis -regulatory domain • Abd-B chromatin dynamics follow a non-canonical inversion of collinearity • Dynamic regulatory hierarchies drive differential Abd-B alternative promoter use Moniot-Perron et al. determine gene-regulatory hierarchies for the Abd-B Drosophila Hox gene. Boundary elements are essential to separate cis -regulatory regions, allowing differential 3D chromatin organization and domains of histone modifications. This multilevel organization creates a non-canonical inversion of collinear chromatin dynamics at Abd-B , which drives differential alternative promoter use. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (NUPs) and is a hallmark of eukaryotes. NUPs have been suggested to be important in regulating gene transcription and 3D genome organization. However, evidence in support of their direct roles remains limited. Here, by Cut&Run, we find that core NUPs display broad but also cell-type-specific association with active promoters and enhancers in human cells. Auxin-mediated rapid depletion of two NUPs demonstrates that NUP93, but not NUP35, directly and specifically controls gene transcription. NUP93 directly activates genes with high levels of RNA polymerase II loading and transcriptional elongation by facilitating full BRD4 recruitment to their active enhancers. dCas9-based tethering confirms a direct and causal role of NUP93 in gene transcriptional activation. Unexpectedly, in situ Hi-C and H3K27ac or H3K4me1 HiChIP results upon acute NUP93 depletion show negligible changesS2211-1247(22)01437-1 of 3D genome organization ranging from A/B compartments and topologically associating domains (TADs) to enhancer-promoter contacts. [Display omitted] • Cut&Run reveals chromatin association of core NUPs at active enhancers and promoters • Acute depletion of NUPs uncovers direct roles of NUP93 in gene transcriptional control • NUP93 acts as an activator for specific enhancers adjacent to the nuclear periphery • Acute depletion shows that NUP93 is largely dispensable for 3D genome organization Acute depletion of human core nucleoporin, particularly NUP93, elucidates its direct and selective role in transcription activation by facilitating BRD4 recruitment to active enhancers. Zhu et al. report that NUP93 is largely dispensable for direct control of the higher-order 3D genome architecture. [ABSTRACT FROM AUTHOR]

In eukaryotic organisms, genetic information is usually carried on multiple chromosomes. Whether and how the number and configuration of chromosomes affect organismal fitness and speciation remain unclear. Here, we have successfully established several single-chromosome fission yeast Schizosaccharomyces pombe strains, in which the three natural chromosomes have been fused into one giant chromosome in different orders. Chromosome fusions accompanied by the deletions of telomeres and centromeres result in the loss of chromosomal interactions and a drastic change of global chromosome organization, but alter gene expression marginally. The single-chromosome strains display little defects in cell morphology, mitosis, genotoxin sensitivity, and meiosis. Crosses between a wild-type strain and a single-chromosome strain or between two single-chromosome strains with different fusion orders suffer defective meiosis and poor spore viability. We conclude that eukaryotic genomes are robust against dramatic chromosomal reconfiguration, and stochastic changes in chromosome number and genome organization during evolution underlie reproductive isolation and speciation. [Display omitted] • S. pombe can tolerate the fusion of its chromosomes into one chromosome • The 3D structure change of the genome has no significant effect on gene expression • Chromosome fusions cause few defects in cell morphology and growth • Chromosome number and genome organization affect meiosis and natural speciation Gu et al. fuse three native chromosomes of the fission yeast Schizosaccharomyces pombe into one giant chromosome in different orders. They characterize these single-chromosome strains in terms of genome 3D structure, transcriptome, mitosis, and meiosis. [ABSTRACT FROM AUTHOR]

The mammalian genome has a complex, functional 3D organization. However, it remains largely unknown how DNA contacts are orchestrated by chromatin organizers. Here, we infer from only Hi-C the cell-type-specific arrangement of DNA binding sites sufficient to recapitulate, through polymer physics, contact patterns genome wide. Our model is validated by its predictions in a set of duplications at Sox9 against available independent data. The binding site types fall in classes that well match chromatin states from segmentation studies, yet they have an overlapping, combinatorial organization along chromosomes necessary to accurately explain contact specificity. The chromatin signatures of the binding site types return a code linking chromatin states to 3D architecture. The code is validated by extensive de novo predictions of Hi-C maps in an independent set of chromosomes. Overall, our results shed light on how 3D information is encrypted in 1D chromatin via the specific combinatorial arrangement of binding sites. [Display omitted] • Polymer physics identifies a code linking 3D chromatin structure to 1D chromatin states • The code is shown to be sufficient to explain Hi-C data genome wide • DNA-DNA contact binding sites have a genomic overlapping, combinatorial organization • The code is validated by de novo predictions of contact maps in wild-types and mutants Esposito et al. identify a code linking 3D chromatin structure to 1D chromatin states by combining polymer physics and machine learning. The code is sufficient to produce 3D conformations consistent with Hi-C genome wide. That sheds light on how the multitude of specific regulatory DNA contacts is orchestrated by chromatin organizing factors. [ABSTRACT FROM AUTHOR]

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation. [Display omitted] • DNA hypomethylation is associated with single-cell replication timing heterogeneity • Hypomethylation induces loss of allelic replication at cancer-related gene loci • Hypomethylation alters higher-order genome architecture of PMD boundaries • Non-canonical H3K4me3-H3K9me3 domains form to protect silent late replication Du et al. demonstrate the importance of DNA methylation in the maintenance of 3D genome regulation. They show that hypomethylation, a hallmark of cancer, is associated with the disruption of 3D genome compartmentalization, gain in replication timing heterogeneity, and loss of allelic replication, potentially through the deregulation of DNA replication and genome organization pathways. [ABSTRACT FROM AUTHOR]