Nucleus

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Abnormalities in nuclear morphology are an important diagnostic tool to determine malignancy in cancer cells and are characterised by nuclear blebbing and deformations. Nuclear shape is mostly maintained by a dense protein meshwork of lamins, consisting of 4 lamin subtypes, of which the individual contribution to nuclear shape maintenance remains elusive. In this study, we decouple the roles of lamin A, C, and B1 across cancer cell lines with varying malignant potential (HeLa, HT1080, and MDA-MB-231). Using single-cell correlation analysis, we directly link reduced lamin A/C, and not lamin B1, expression levels to nuclear deformability. We found that the nuclear shape of the more malignant MDA-MB-231 cells is approximately 4-fold more sensitive to lamin A/C than HeLa and HT1080 cells. Biochemical analyses reveal cell-type-specific variation in lamin A/C interactions and homodimer formation that correlates with nuclear shape deformations. In contrast to healthy mouse embryonic fibroblast cells, malignant cells exhibit reduced dimerisation, which correlates with nuclear deformability. As such, our study links, for the first time, the lamin A/C dimerisation state to nuclear abnormalities, thereby providing new avenues for investigating cancer progression.

Heterochromatin protein 1 (HP1 is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1 binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a genetic disease characterized by the accumulation of progerin, a mutant form of lamin A, at the nuclear envelope. Progerin disrupts the stability of the nuclear lamina, leading to genome instability and accelerated aging phenotypes. While structural nuclear defects are well-documented, the impact of progerin on real-time chromatin dynamics and the ability of current therapeutics to rescue these dynamics remains poorly understood. In this work, we employ single-particle tracking to quantify telomere dynamics in HGPS patient fibroblasts. We demonstrate that HGPS cells exhibit significantly increased telomere dynamics, characterized by expanded scan areas, increased diffusion coefficients, and larger jump distances compared to healthy controls. We further evaluated the efficacy of two clinically relevant treatments, the farnesyltransferase inhibitor Lonafarnib and the ICMT inhibitor C75, to determine if emerging treatments can restore chromatin dynamics compared to healthy controls. Our results reveal that Lonafarnib partially rescues telomere dynamics, shifting chromatin motion back towards healthy control levels, and that C75 provides a complete rescue of the dynamics for all parameters quantified. These findings provide a quantitative framework for understanding how nuclear lamina mutations induce aberrant genome dynamics and the efficacy of HGPS therapies on restoring those dynamics.

Abstract/SummaryLong-non coding RNAs (lncRNAs) are now well established players in gene expression regulation, but their detailed molecular mechanisms of action and underlying regulating sequences remain poorly understood. An emerging concept supports the idea that repeated sequences, more notably sequences derived from transposable elements (TEs), contribute to functional domains of lncRNAs. Here, we undertake the characterization of the function, interactors and functional domains of Snhg26, a lncRNA involved in reprogramming towards induced pluripotent stem cells (iPSCs) and maintenance of the pluripotent state of embryonic stem cells (ESCs). First, we show that modulation of Snhg26 expression levels affects expression and splicing of genes involved in pluripotency and chromatin remodeling during the early steps of reprogramming and in ESCs. We also find that down-regulation of Snhg26 increases the expression of SINEB2 transposable elements. Moreover, we identify hundreds of transcripts directly interacting with Snhg26 with a significant enrichment of RNAs containing SINEB2 elements. Strikingly, loss of a SINEB2 sequence embedded within Snhg26 abolishes its function in regulating pluripotent states. Our results thus support the idea that TEs constitute a source of functional units for lncRNAs and encourages further efforts to explore this concept.

Splicing of pre-mRNA can result in multiple possible mRNA isoforms per gene due to alternative splicing. The frequency at which individual isoforms occur depends on the intrinsic splicing kinetics of the pre-mRNA as well as intracellular chemical conditions. Computational modeling can potentially provide a platform to rapidly assess how variations in intracellular and environmental conditions, for example differential levels of regulatory splicing proteins, affect kinetics and resulting mRNA isoforms. Overcoming the vast combinatoric possibilities of splicing, however, has remained a significant challenge in modeling its kinetics. Here we report the development of a stochastic kinetic model of splicing that is extensible to most protein-coding genes in the human genome. Our model allows for variations in site-specific reaction rates as well as the ability to introduce additional splicing factors. We experimentally validate the predictive capability of our computational model by exploring the spliced isoform ratio of a target gene (SRSF6) under normoxia and hypoxia. This work provides a resource for quantitative, computational analysis of pre-mRNA splicing, allowing for a rapid computational-experimental approach to assess biological hypotheses. SignificancemRNA splicing is a key step in human gene expression with deep impact on the molecular processes determining cell physiology and affecting development and disease. Computational models of splicing are highly attractive to understand life processes but so far have been limited in directly accounting for the chemistry of splicing and are not extensible to most of the human genome. We report here a model that overcomes both of these challenges, providing a computational benchtop to probe splicing kinetics for most genes in the human genome. This model has the capability to rapidly pose biological hypotheses for experimental validation. As a targeted demonstration, we explore the effects of the pathologically-relevant chemical condition hypoxia on the pre-mRNA of a single gene.

Genomic DNA is wrapped around core histones to form nucleosomes, which are organized in cells from euchromatin to heterochromatin with distinct genome functions. Although transcription is known to shape chromatin behavior in live cells, it remains unclear how different transcription systems shape chromatin classes and nuclear subcompartments. We developed machine learning-assisted Repli-Histo labeling to classify euchromatin and heterochromatin classes (Classes IA, IB, II, and III) and combined it with single-nucleosome imaging in live cells. Nucleosome motion was progressively constrained from euchromatin to heterochromatin. RNA polymerase II inhibition by THZ1, DRB, or -amanitin increased nucleosome motion in euchromatic Classes IA and IB and in heterochromatin around nucleoli, but not at the nuclear periphery. In contrast, RNA polymerase I inhibition by CX-5461 selectively increased nucleosome motion in Class III heterochromatin around nucleoli. Our study reveals that Pol II and Pol I transcription shape chromatin behavior in distinct chromatin classes and nuclear subcompartments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/736477v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@127137dorg.highwire.dtl.DTLVardef@709a16org.highwire.dtl.DTLVardef@94550corg.highwire.dtl.DTLVardef@5ba6ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

Biomolecular structure prediction tools such as AlphaFold have achieved remarkable success in predicting structures of single proteins and multiprotein complexes. AlphaFold3 now incorporates the capability to model complexes containing nucleic acids and chemically modified side chains. Investigators can now predict structures of proteins bound to chromatin, where interactions with nucleosomal DNA and histone post-translational modifications converge to control genome function. To evaluate its robustness in modeling chromatin complexes, we benchmarked AlphaFold3 on 115 structures containing nucleosomes whose coordinates were released by the Protein Data Bank after the training set cutoff date. We find that AlphaFold3 excels at predicting histone-driven interactions and accurately models complexes that deposit and recognize post-translational modifications. By contrast, AlphaFold3 struggles to predict structures of chromatin factors that primarily engage nucleosomal DNA, notably transcription factors and chromatin remodelers. Finally, we show that AlphaFold3 can faithfully recapitulate known post-translational modification recognition patterns, matching experimentally determined specificity profiles. This assessment of the capabilities and limitations of AF3 in chromatin structural biology provides a roadmap for its effective application to studies of chromatin regulation and PTM readout, while identifying key areas for future algorithmic refinement. SignificanceStructure prediction with AlphaFold has become an invaluable tool in experimental biology, and the accuracy of many of its predictions has been verified in structural and biochemical studies. With the recent incorporation into AlphaFold3 of nucleic acids and post-translational modifications, this prediction tool can now be applied to chromatin structural biology. Our benchmarking of AlphaFold3 reveals its strengths and weaknesses in predicting structures of proteins bound to nucleosomes, thereby providing a framework for using these models in mechanistic studies of chromatin regulation. We introduce metrics for evaluating structures of nucleosome complexes that highlight AlphaFold3s strengths in predicting protein-nucleosome interactions and post-translational modification specificity.

Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation [&le;] acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A N-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A in nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we systematically tested the contribution of each N-terminal histone region to maintaining H3 histones by utilizing a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of H3/CENP-A chimera histone nucleosomes containing targeted swaps of the tail, latch, and N-helix regions. Removal or substitution of individual H3 with CENP-A N-terminal regions increased DNA accessibility and nucleosome unwrapping. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the wrapped DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and N-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins. SIGNIFICANCEThe centromere is marked by nucleosomes containing CENP-A, which adopt a more open and accessible DNA conformation than canonical nucleosomes. However, the molecular determinants underlying this difference remain unclear. Previous structural investigations of the centromeric nucleosome have placed less emphasis on the intrinsically disordered N-terminal regions of CENP-A. Here, we systematically dissect the contributions of the N-terminal tail, latch, and N-helix via MNase digestion assays and molecular dynamics simulations on nucleosomes containing H3/CENP-A chimeras. We demonstrate that no individual H3-derived region is sufficient to impart a closed conformation to the nucleosomal DNA. Instead, multiple regions act together to stabilize DNA, revealing that nucleosome conformation is controlled by concerted histone-DNA interactions.

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

BackgroundEukaryotic gene regulation depends on transcription factors (TFs) recognizing short DNA motifs within chromatin. Many of these motifs lie within nucleosomes, where DNA is sharply bent, rotationally phased, and constrained by histone-DNA contacts. Yet only a subset is occupied in any cellular context. Motif identity alone, therefore, cannot fully explain selective TF engagement with nucleosomal DNA. We asked whether sequence-derived DNA conformational flexibility provides an interpretable representation of sequence context relevant to TF recognition on nucleosomes. ResultsWe compiled five DNA flexibility descriptors in the Python package DNAflexpy, representing bendability, torsional deformation, backbone conformational variability, and stiffness. We built quantitative models of TF binding affinity across 226 datasets from a high-throughput in vitro TF-nucleosome binding assay. Flexibility-augmented models improved prediction over mononucleotide baselines in most datasets, with smaller but reproducible gains over trinucleotide baselines. The gains were not uniform: they varied across TF families and were concordant with DNA shape-fluctuation features, suggesting that DNAflexpy descriptors capture a sequence-encoded structural signal. In PIONEAR-seq data, model performance generalized across nucleosomal templates in a TF- and sequence-dependent manner. Beyond prediction, position-resolved flexibility footprints revealed deformation signatures at cognate motifs and flanking regions across diverse TF families. For SOX11, model-derived footprints aligned with DNA shape fluctuations from nanosecond-to-microsecond molecular dynamics trajectories of SOX11-bound nucleosomes, consistent with independently observed DNA conformational dynamics and bound-state stabilization. The in vivo data showed a similar but more context-dependent pattern. OCT4 occupancy tended to correlate with local flexibility, whereas GATA3-pioneered regions showed flexibility coupled with altered rotational positioning of cognate motifs. Flexibility-augmented classifiers further improved discrimination of occupied nucleosomal motifs across ENCODE datasets. Torsional flexibility features, particularly twist dispersion and trx, were most informative for classification. ConclusionsSequence-derived DNA conformational flexibility provides a quantitative and interpretable representation of sequence context in TF recognition on nucleosomes. By augmenting sequence with structural information, these models help quantify and interpret an indirect-readout contribution in which DNA deformation tendencies may complement motif sequence and DNA shape. This framework may help explain why only selected motif instances are engaged in chromatin, without treating flexibility as independent of primary sequence.

The nuclear lamina provides mechanical integrity to the eukaryotic nucleus and organizes lamina-associated chromatin domains that are important for chromatin architecture and gene-expression regulation. Lamin A/C, a major component of the nuclear lamina, is disrupted in hereditary laminopathies and has also been implicated in aging-associated nuclear dysfunction. Although the mechanical role of Lamin A/C has been extensively studied in vitro, particularly in monolayer cell culture and isolated nuclei, its role in maintaining nuclear mechanics and chromatin organization in intact tissues remains incompletely understood. Here, we investigated how partial and complete Lamin A/C disruption affects nuclear shape, chromatin architecture, intranuclear mechanics, and gene expression in vivo. Across multiple murine tissues, Lamin A/C deficiency did not cause a generalized collapse of nuclear shape or gross tissue architecture. Instead, Lamin A/C disruption preferentially altered chromatin architecture in mechanically stiff tissues, including skeletal muscle and heart. Using live in vivo deformation microscopy during controlled hindlimb muscle stimulation, we quantified real-time multiscale deformation of skeletal muscle tissue and nuclei. These measurements revealed reduced effective nuclear stiffness and altered load sharing between euchromatin-rich and heterochromatin-rich domains after Lamin A/C loss. Super-resolution imaging further showed that partial and complete Lamin A/C disruption uncoupled H3K9me3 from DAPI-dense heterochromatin, indicating a spatial disruption of repressive chromatin organization. Exploratory ATAC-seq suggested increased chromatin accessibility in heterozygous muscle, whereas RNA-seq showed that complete Lamin A/C loss caused broad myopathic transcriptional dysregulation while partial loss preserved a near-wild-type transcriptomic state. Integrated analysis identified HDAC2 as a candidate mechanosensitive compensatory node that may help buffer gene expression after partial Lamin A/C disruption. Together, these results establish Lamin A/C as an in vivo coordinator of nuclear mechanics, heterochromatin organization, and transcriptional homeostasis in skeletal muscle.

MYC oncoprotein binding at promoters and enhancers influences RNA polymerase II (RNAPII)-driven gene expression. Numerous genes also bind MYC near their transcriptional end sites (TESs). This often allows direct promoter-TES contact via looping and further regulates total and 'read-through' transcription that extends beyond standard termination sites. We aimed here to better clarify the rules governing TES associated MYC and/or RNAPII binding cross-talk in human and murine cells. Using ChIPseq and RNAseq datasets from the ENCODE portal and elsewhere, MYC and RNAPII binding profiles were found to differ around TESs and transcriptional start sites (TSSs). Variations in E box flanking sequences likely accounted for the somewhat lower affinities of MYC for TES-associated sites. Motifs for numerous other transcription factors were also observed to cluster non-randomly and in close proximity to MYC and RNAPII binding site peak summits. On average, genes with TES-proximal MYC or RNAPII sites were more highly expressed than those without, although co-binding tended to be suppressive. Both normal and neoplastic proliferative stimuli altered the MYC and RNAPII binding patterns of many genes, indicating that 'category switching' was common, subject to disparate external signals and often reversible. Functionally related gene sets with high levels of read-through transcription were uniformly marked by significant amounts of TES-associated MYC and/or RNAPII binding. These findings indicate that, both independently and together, MYC and RNAPII binding near TESs dynamically impact total and read-through transcription while also coordinating the expression of many common purpose gene sets.

Transcription factors form clusters often described as condensates that exhibit emergent biophysical properties. Here we present a software package to simulate transcription factor spatial distributions from molecular diffusion and binding kinetics alone. The software simulates microscopy data and FRAP experiments and recapitulates the clustering behavior of experimentally characterized transcription factors. Our results demonstrate that condensate-like structures can emerge from molecular kinetics principles without invoking higher-order processes like phase separation.

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proper connection between the sperm head and tail is critical for fertility and is mediated by the head-tail coupling apparatus (HTCA). Recent evidence suggests that the nuclear pore complex (NPC) may be important in male fertility, though a specific role at the HTCA has not been described. To investigate this, we performed a testis-specific RNAi screen targeting nucleoporins of the NPC. We identified Nup133 and Nup107 as regulators of HTCA development. We found that Nup133 and Nup107 were required to form the initial connection between the nucleus and centriole during HTCA establishment. We determined that failure to build the HTCA following Nup133 and Nup107 depletion was due to loss of nuclear envelope dynein/dynactin. Finally, we showed that loss of the NPC cytoplasmic filament component Nup358 results in the most severe centriole detachment phenotype, thus potentially functioning as the dynein anchor. Together, our data indicate that NPCs are critical regulators of early HTCA establishment and are required to recruit dynein to the nuclear envelope to bring the nucleus and centriole together during spermiogenesis.