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1. Exploring chromosomal structural heterogeneity across multiple cell lines

   https://doi.org/10.7554/eLife.60312  

   Cheng, Ryan R; Contessoto, Vinicius G; Lieberman Aiden, Erez; Wolynes, Peter G; Di Pierro, Michele; Onuchic, Jose N (October 2020, eLife)

   null (Ed.)

   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. 

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2. Three-color single-molecule localization microscopy in chromatin

   https://doi.org/10.1038/s41377-025-01786-1  

   Acosta, Nicolas; Gong, Ruyi; Su, Yuanzhe; Frederick, Jane; Medina, Karla_I; Li, Wing_Shun; Mohammadian, Kiana; Almassalha, Luay; Wang, Geng; Backman, Vadim (March 2025, Light: Science & Applications)

   Abstract Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy. 

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3. MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface

   https://doi.org/10.7554/eLife.73348  

   Houlard, Martin; Cutts, Erin E; Shamim, Muhammad S; Godwin, Jonathan; Weisz, David; Presser Aiden, Aviva; Lieberman Aiden, Erez; Schermelleh, Lothar; Vannini, Alessandro; Nasmyth, Kim (December 2021, eLife)

   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. 

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4. Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes

   https://doi.org/10.3390/cells9020339  

   George, Phillip; Kinney, Nicholas A.; Liang, Jiangtao; Onufriev, Alexey V.; Sharakhov, Igor V. (February 2020, Cells)

   Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism’s development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome–nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome–chromosome interactions, which is a key feature of the cell type-specific nuclear architecture. 

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5. Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

   https://doi.org/10.1083/jcb.201902117  

   Bertero, Alessandro; Fields, Paul A.; Smith, Alec S. T.; Leonard, Andrea; Beussman, Kevin; Sniadecki, Nathan J.; Kim, Deok-Ho; Tse, Hung-Fat; Pabon, Lil; Shendure, Jay; et al (August 2019, The Journal of Cell Biology)

   Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency. 

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