Changes In Nuclear Architecture Research Articles

Foraminifera as a model of eukaryotic genome dynamism.

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Seeing is believing: the impact of RB on nuclear organization

ABSTRACT The retinoblastoma tumor suppressor (RB) prevents G1 to S cell cycle transition by inhibiting E2F activity. This function requires that RB remains un- or underphosphorylated (the so-called active forms of RB). Recently, we showed that active forms of RB cause widespread changes in nuclear architecture that are visible under a microscope. These phenotypes did not correlate with cell cycle arrest or repression of the E2F transcriptional program, but appeared later, and were associated with the appearance of autophagy or in IMR-90 cells with senescence markers. In this perspective, we describe the relative timing of these RB-induced events and discuss the mechanisms that may underlie RB-induced chromatin dispersion. We consider the relationship between RB-induced dispersion, autophagy, and senescence and the potential connection between dispersion and cell cycle exit.

Dynamics of nuclear architecture during early embryonic development and lessons from liveimaging.

Nuclear organization has emerged as a potential key regulator of genome function. During development, the deployment of transcriptional programs must be tightly coordinated with cell division and is often accompanied by major changes in the repertoire of expressed genes. These transcriptional and developmental events are paralleled by changes in the chromatin landscape. Numerous studies have revealed the dynamics of nuclear organization underlying them. In addition, advances in live-imaging-based methodologies enable the study of nuclear organization with high spatial and temporal resolution. In this Review, we summarize the current knowledge of the changes in nuclear architecture in the early embryogenesis of various model systems. Furthermore, to highlight the importance of integrating fixed-cell and live approaches, we discuss how different live-imaging techniques can be applied to examine nuclear processes and their contribution to our understanding of transcription and chromatin dynamics in early development. Finally, we provide future avenues for outstanding questions in this field.

Telomere-Associated Changes in Nuclear Architecture of Cancer-Associated Macrophage-like Cells in Liquid Biopsies from Melanoma Patients.

During phagocytosis, tumor-associated macrophages (TAMs) can incorporate genetic material from tumor cells. The incorporation of extra genetic material may be responsible for advanced malignant behavior observed in some TAMs, making TAMs potentially important players in cancer progression. More recently, similar cells were described in the blood as cancer-associated macrophage-like cells (CAMLs). CAMLs may be equivalent to TAMs cells in the blood, and they express macrophage markers. However, their origin is still unclear. In a previous study, we showed for the first time the distinct telomere 3D structure of circulating tumor cells (CTCs) in melanoma and other cancers. In the present pilot study, we investigated, comparatively, the 3D telomere structure of CAMLs, CTCs and leucocytes from nine melanoma patients with metastatic cutaneous melanoma stage IV. CTC capture was performed by size-based filtration followed by cytological and immunocytological evaluation. Three-dimensional Quantitative Fluorescent in situ Hybridization was performed to measure differences in five 3D telomere parameters. Telomere parameters, such as number, length, telomere aggregates, nuclear volume, and a/c ratio, were compared among different cellular types (CTCs, CAMLs, and normal leucocytes). Three telomere parameters were significantly different between CAMLs and leucocytes. The combination of two telomere parameters (telomere length against the number of telomeres) resulted in the identification of two CAMLs subpopulations with different levels of genomic instability. Those populations were classified as profile 1 and 2. Profile 2, characterized by a high number of short telomeres, was observed in four of the nine melanoma patients. To our knowledge, this is the first pilot study to investigate 3D telomere parameters as hallmarks of nuclear architecture in CAMLs’ population in comparison to leucocytes from the same patient. Further studies involving a larger patient sample size are necessary to validate these findings and explore their potential prognostic value.

Aging is accompanied by T-cell stiffening and reduced interstitial migration through dysfunctional nuclear organization.

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.

Chromatin compaction precedes apoptosis in developing neurons

While major changes in cellular morphology during apoptosis have been well described, the subcellular changes in nuclear architecture involved in this process remain poorly understood. Imaging of nucleosomes in cortical neurons in vitro before and during apoptosis revealed that chromatin compaction precedes the activation of caspase-3 and nucleus shrinkage. While this early chromatin compaction remained unaffected by pharmacological blockade of the final execution of apoptosis through caspase-3 inhibition, interfering with the chromatin dynamics by modulation of actomyosin activity prevented apoptosis, but resulted in necrotic-like cell death instead. With super-resolution imaging at different phases of apoptosis in vitro and in vivo, we demonstrate that chromatin compaction occurs progressively and can be classified into five stages. In conclusion, we show that compaction of chromatin in the neuronal nucleus precedes apoptosis execution. These early changes in chromatin structure critically affect apoptotic cell death and are not part of the final execution of the apoptotic process in developing cortical neurons.

Mitotic genome bookmarking by nuclear receptor VDR advocates transmission of cellular transcriptional memory to progeny cells

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as ‘genome bookmarking factors' and the phenomenon is termed ‘genome bookmarking’. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full ‘DNA binding domain (DBD)’ present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1(Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells.

The Singular Evolution of Olea Genome Structure.

The current view of plant genome evolution proposes that genome size has mainly been determined by polyploidisation and amplification/loss of transposons, with a minor role played by other repeated sequences, such as tandem repeats. In cultivated olive (Olea europaea subsp. europaea var. europaea), available data suggest a singular model of genome evolution, in which a massive expansion of tandem-repeated sequences accompanied changes in nuclear architecture. This peculiar scenario highlights the importance of focusing on Olea genus evolution, to shed light on mechanisms that led to its present genomic structure. Next-generation sequencing technologies, bioinformatics and in situ hybridisation were applied to study the genomic structure of five related Olea taxa, which originated at different times from their last common ancestor. On average, repetitive DNA in the Olea taxa ranged from ~59% to ~73% of the total genome, showing remarkable differences in terms of composition. Among repeats, we identified 11 major families of tandem repeats, with different abundances in the analysed taxa, five of which were novel discoveries. Interestingly, overall tandem repeat abundance was inversely correlated to that of retrotransposons. This trend might imply a competition in the proliferation of these repeat classes. Indeed, O. paniculata, the species closest to the Olea common ancestor, showed very few tandem-repeated sequences, while it was rich in long terminal repeat retrotransposons, suggesting that the amplification of tandem repeats occurred after its divergence from the Olea ancestor. Furthermore, some tandem repeats were physically localised in closely related O. europaea subspecies (i.e., cultivated olive and O. europaea subsp. cuspidata), which showed a significant difference in tandem repeats abundance. For 4 tandem repeats families, a similar number of hybridisation signals were observed in both subspecies, apparently indicating that, after their dissemination throughout the olive genome, these tandem repeats families differentially amplified maintaining the same positions in each genome. Overall, our research identified the temporal dynamics shaping genome structure during Olea speciation, which represented a singular model of genome evolution in higher plants.

Macronuclear development in ciliates, with a focus on nuclear architecture.

Ciliates are defined by the presence of dimorphic nuclei as they have both a somatic macronucleus and germline micronucleus within each individual cell. The size and structure of both germline micronuclei and somatic macronuclei vary tremendously among ciliates. Except just after conjugation (i.e. the nuclear exchange in their life cycle), the germline micronucleus is transcriptionally inactive and contains canonical chromosomes that will be inherited between generations. In contrast, the transcriptionally active macronucleus contains chromosomes that vary in size in different classes of ciliates, with some lineages having extensively fragmented gene-sized somatic chromosomes while others contain longer multigene chromosomes. Here, we describe the variation in somatic macronuclear architecture in lineages sampled across the ciliate tree of life, specifically focusing on lineages with extensively fragmented chromosomes (e.g. the classes Phyllopharyngea and Spirotrichea). Further, we synthesize information from the literature on the development of ciliate macronuclei, focusing on changes in nuclear architecture throughout life cycles. These data highlight the tremendous diversity among ciliate nuclear cycles, extend our understanding of patterns of genome evolution, and provide insight into different germline and somatic nuclear features (e.g. nuclear structure and development) among eukaryotes.

Down-syndrome-induced senescence disrupts the nuclear architecture of neural progenitors.

Down syndrome (DS) is a genetic disorder driven by the triplication of chromosome 21 (T21) and characterized by a wide range of neurodevelopmental and physical disabilities. Transcriptomic analysis of tissue samples from individuals with DS has revealed that T21 induces a genome-wide transcriptional disruption. However, the consequences of T21 on the nuclear architecture and its interplay with the transcriptome remain unknown. In this study, we find that unlike human induced pluripotent stem cells (iPSCs), iPSC-derived neural progenitor cells (NPCs) exhibit genome-wide "chromosomal introversion," disruption of lamina-associated domains, and global chromatin accessibility changes in response to T21, consistent with the transcriptional and nuclear architecture changes characteristic of senescent cells. Treatment of T21-harboring NPCs with senolytic drugs alleviates the transcriptional, molecular, and cellular dysfunctions associated with DS. Our findings provide a mechanistic link between T21 and global transcriptional disruption and indicate that senescence-associated phenotypes may play a key role in the neurodevelopmental pathogenesis of DS.

Inner Nuclear Protein Matrin-3 Coordinates Hematopoietic Cell Transcription and Differentiation By Stabilizing Chromatin Architecture

The nucleus is spatially organized by chromosome and interchromatin functional components. Global reorganization of chromatin interactions and compartmentalization occurring during differentiation requires proper chromosome positioning, but the involvement of nuclear components in this process remains largely underexplored. In particular, blood cell development exemplifies a coordinated process accompanied by dramatic chromatin reorganization, thereby providing a model in which to interrogate chromatin dynamics during differentiation. Here, we show that an abundant inner nuclear protein Matrin-3 (Matr3) plays a critical role in the maintenance of chromatin structure and has a broad effect on erythroid cell differentiation by coordinating gene expression.First, we deleted the entire gene body by CRISPR/Cas9 in mouse erythroleukemia (MEL) cells. The Matr3 knockout (KO) cells proliferate normally and exhibit morphological changes on differentiation suggestive of accelerated maturation. Consistently, erythroid-specific genes were expressed at a higher level in MEL Matr3 KO cells than in parental cells. The consequences of Matr3 deletion were also determined in G1ER cells, in which differentiation is conditional on activation of GATA-1. To assess the global impact of Matr3 loss on erythroid cell maturation, we measured global RNA expression changes. Erythroid-specific genes were expressed at a much higher level upon differentiation of Matr3 KO cells. Differentiation is typically accompanied by specific changes in nuclear architecture. Using super-resolution microscopy, we observed that heterochromatin protein 1α (HP1α) was more dispersed and irregular in appearance in Matr3 KO cells, suggesting that Matr3 loss alters morphological boundaries of heterochromatin.Analysis of the interactions between different regions of chromatin identifies topologically associating domains and classifies the genome into two compartments (A and B). The A and B compartments correspond to the structures and characteristics of known euchromatin and heterochromatin, respectively. We next explored global chromatin structure using a high-throughput chromosome conformation capture (Hi-C) assay. In Matr3 KO cells, insulation at the domain boundaries was reduced, and the compartment strengths between the B compartments became stronger, while those between A-type domains were reduced. Remarkably, we found that these changes in cells lacking Matr3 were similar to changes in chromatin contact during differentiation. To access the genomic features at a higher resolution, we performed the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq). Notably, the newly opened regions in Matr3 KO, as compared to parental, cells were enriched for GATA motifs, which are generally more accessible in differentiated erythroid cells.Architectural proteins function cooperatively to organize chromatin. Using affinity purification followed by mass spectrometry and immunoblotting, we found that Matr3 interacts with proteins involved in chromatin remodeling, such as CTCF and cohesin. To identify whether Matr3 loss alters chromatin occupancy of its interacting partners, we performed ChIP-seq for CTCF and the core cohesin component Rad21. In the absence of Matr3, occupancy of CTCF and Rad21 was perturbed in a subset of genomic regions. Moreover, destabilization of CTCF and cohesin binding correlated with altered transcription and accelerated erythroid differentiation. Most sites with disrupted CTCF and Rad21 binding during differentiation were also sensitive to the absence of the scaffold protein Matr3.Our data demonstrate that the nucleoplasmic protein Matr3 stabilizes the binding of the architectural proteins (CTCF and cohesin) to chromatin and serves to maintain chromatin structure. We speculate that Matr3 negatively regulates cell fate transitions by maintaining cellular state through fine-tuning the binding of CTCF/cohesin to chromatin and associated 3D interactions. Our work reveals a previously unrecognized role of Matr3 in chromatin organization and responses to developmental cues. DisclosuresNo relevant conflicts of interest to declare.

Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells

Trypanosoma cruzi—the causative agent of Chagas disease—like other kinetoplastids, relies mostly on post-transcriptional mechanisms for regulation of gene expression. However, trypanosomatids undergo drastic changes in nuclear architecture and chromatin structure along their complex life cycle which, combined with a remarkable set of reversible histone post-translational modifications, indicate that chromatin is also a target for control of gene expression and differentiation signals in these organisms. Chromatin-modifying enzymes have a direct impact on gene expression programs and DNA metabolism. In this work, we have investigated the function of T. cruzi histone deacetylase 4 (TcHDAC4). We show that, although TcHDAC4 is not essential for viability, metacyclic trypomastigote TcHDAC4 null mutants show a thin cell body and a round and less condensed nucleus located very close to the kinetoplast. Sixty-four acetylation sites were quantitatively evaluated, which revealed H2AT85ac, H4K10ac and H4K78ac as potential target sites of TcHDAC4. Gene expression analyses identified three chromosomes with overrepresented regions of differentially expressed genes in the TcHDAC4 knockout mutant compared with the wild type, showing clusters of either up or downregulated genes. The adjacent chromosomal location of some of these genes indicates that TcHDAC4 participates in gene expression regulation during T. cruzi differentiation.

Piwi–piRNA complexes induce stepwise changes in nuclear architecture at target loci

PIWI‐interacting RNAs (piRNAs) are germline‐specific small RNAs that form effector complexes with PIWI proteins (Piwi–piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co‐factor. Using ChEP and DamID‐seq, we now uncover a Piwi/Nxf2‐dependent target association with nuclear lamins. Hi‐C analysis of Piwi or Nxf2‐depleted cells reveals decreased intra‐TAD and increased inter‐TAD interactions in regions harboring Piwi–piRNA target TEs. Using a forced tethering system, we analyze the functional effects of Piwi–piRNA/Nxf2‐mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. Our data show that the Piwi–piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing.

Altered nuclear architecture in blood cells from Huntington's disease patients.

Cell nuclear architecture has been explored in cancer and laminopathies but not in neurodegenerative disorders. Huntington's disease (HD) is a neurodegenerative disorder that leads to neuronal death. Chromosome-wide changes in gene expression have been reported in HD, not only in the brain but also in peripheral blood cells, but whether this translates into nuclear and chromosome architecture alterations has not yet been studied. We investigate nuclear structure and chromosome organization in HD blood cells using fluorescence in situ hybridization in ultrathin cryosections (cryoFISH), coupled with machine learning image analysis to evaluate size, distribution, and morphology of nuclei and chromosomes. Four chromosomes were analyzed based on up- or downregulation of gene expression in HD. We show that blood cells from HD patients display increased nuclear size and filamentary shape, increased size of gene-rich chromosome 19, decreased filamentary shape of gene-rich chromosome 22, and a more radially centralized position for chromosome 19, whereas chromosomes 4 and 5 do not show detectable differences. We identify gross changes in nuclear architecture and chromosome organization associated with HD in blood. This adds a new layer of information onto disrupting mechanisms in HD and increases the potential of using blood to survey HD.

Precision Surface Microtopography Regulates Cell Fate via Changes to Actomyosin Contractility and Nuclear Architecture.

Cells are able to perceive complex mechanical cues from their microenvironment, which in turn influences their development. Although the understanding of these intricate mechanotransductive signals is evolving, the precise roles of substrate microtopography in directing cell fate is still poorly understood. Here, UV nanoimprint lithography is used to generate micropillar arrays ranging from 1 to 10 µm in height, width, and spacing to investigate the impact of microtopography on mechanotransduction. Using mesenchymal stem cells (MSCs) as a model, stark pattern‐specific changes in nuclear architecture, lamin A/C accumulation, chromatin positioning, and DNA methyltransferase expression, are demonstrated. MSC osteogenesis is also enhanced specifically on micropillars with 5 µm width/spacing and 5 µm height. Intriguingly, the highest degree of osteogenesis correlates with patterns that stimulated maximal nuclear deformation which is shown to be dependent on myosin‐II‐generated tension. The outcomes determine new insights into nuclear mechanotransduction by demonstrating that force transmission across the nuclear envelope can be modulated by substrate topography, and that this can alter chromatin organisation and impact upon cell fate. These findings have potential to inform the development of microstructured cell culture substrates that can direct cell mechanotransduction and fate for therapeutic applications in both research and clinical sectors.

Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma celldevelopment is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.

Characterization of RNA-Binding Motif 3 (RBM3) Protein Levels and Nuclear Architecture Changes in Aggressive and Recurrent Prostate Cancer.

The RNA-binding motif protein 3 (RBM3) has been shown to be up-regulated in several types of cancer, including prostate cancer (PCa), compared to normal tissues. Increased RBM3 nuclear expression has been linked to improved clinical outcomes. Given that RBM3 has been hypothesized to play a role in critical nuclear functions such as chromatin remodeling, DNA damage response, and other post-transcriptional processes, we sought to: (1) quantify RBM3 protein levels in archival PCa samples; (2) develop a nuclear morphometric model to determine if measures of RBM3 protein levels and nuclear features could be used to predict disease aggressiveness and biochemical recurrence. This study utilized two tissue microarrays (TMAs) stained for RBM3 that included 80 total cases of PCa stratified by Gleason score. A software-mediated image processing algorithm identified RBM3-positive cancerous nuclei in the TMA samples and calculated twenty-two features quantifying RBM3 expression and nuclear architecture. Multivariate logistic regression (MLR) modeling was performed to determine if RBM3 levels and nuclear structural changes could predict PCa aggressiveness and biochemical recurrence (BCR). Leave-one-out cross validation (LOOCV) was used to provide insight on how the predictive capabilities of the feature set might behave with respect to an independent patient cohort to address issues such as model overfitting. RBM3 expression was found to be significantly downregulated in highly aggressive GS ≥ 8 PCa samples compared to other Gleason scores (P < 0.0001) and significantly down-regulated in recurrent PCa samples compared to non-recurrent samples (P = 0.0377). An eleven-feature nuclear morphometric MLR model accurately identified aggressive PCa, yielding a receiver operating characteristic area under the curve (ROC-AUC) of 0.90 (P < 0.0001) in the raw data set and 0.77 (95% CI: 0.83-0.97) for LOOCV testing. The same eleven-feature model was then used to predict recurrence, yielding a ROC-AUC of 0.92 (P = 0.0004) in the raw data set and 0.76 (95% CI: 0.64-0.87) for LOOCV testing. The RBM3 biomarker alone is a strong prognostic marker for the prediction of aggressive PCa and biochemical recurrence. Further, RBM3 appears to be down-regulated in aggressive and recurrent tumors.

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.

Single Cell Imaging of Nuclear Architecture Changes.

The dynamic architecture of chromatin, the macromolecular complex comprised primarily of DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to chromatin are important markers of functional and developmental processes in cells. However, chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches to assessing chromatin changes at the single-cell level are required. Here we report the use of FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology and coupling it with cell segmentation analysis approach, we have identified key spectral changes corresponding to changes in DNA levels and chromatin conformation at the single cell level. By further manipulating live single cells using pressure-driven microfluidics, we found that chromatin decondensation – either during general transcriptional activation or during specific immune cell maturation – can ultimately lead to nuclear auxeticity which is a new biological phenomenon recently identified. Taken together our findings demonstrate the tight and, potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear architecture.

Nuclear degradation dynamics in a nonapoptotic programmed cell death.

Nuclear degradation is a major event during programmed cell death (PCD). The breakdown of nuclear components has been well characterized during apoptosis, one form of PCD. Many nonapoptotic forms of PCD have been identified, but our understanding of nuclear degradation during those events is limited. Here, we take advantage of Drosophila oogenesis to investigate nuclear degeneration during stress-induced apoptotic and developmental nonapoptotic cell death in the same cell type in vivo. We find that nuclear Lamin, a caspase substrate, dissociates from the nucleus as an early event during apoptosis, but remains associated with nuclei during nonapoptotic cell death. Lamin reveals a series of changes in nuclear architecture during nonapoptotic death, including nuclear crenellations and involutions. Stretch follicle cells contribute to these architecture changes, and phagocytic and lysosome-associated machinery in stretch follicle cells promote Lamin degradation. More specifically, we find that the lysosomal cathepsin CP1 facilitates Lamin degradation.