Nuclear Organization Research Articles

Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics.

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

Nuclear Mechanosensation and Mechanotransduction in Vascular Cells.

Vascular cells are constantly subjected to physical forces associated with the rhythmic activities of the heart, which combined with the individual geometry of vessels further imposes oscillatory, turbulent, or laminar shear stresses on vascular cells. These hemodynamic forces play an important role in regulating the transcriptional program and phenotype of endothelial and smooth muscle cells in different regions of the vascular tree. Within the aorta, the lesser curvature of the arch is characterized by disturbed, oscillatory flow. There, endothelial cells become activated, adopting pro-inflammatory and athero-prone phenotypes. This contrasts the descending aorta where flow is laminar and endothelial cells maintain a quiescent and atheroprotective phenotype. While still unclear, the specific mechanisms involved in mechanosensing flow patterns and their molecular mechanotransduction directly impact the nucleus with consequences to transcriptional and epigenetic states. The linker of nucleoskeleton and cytoskeleton (LINC) protein complex transmits both internal and external forces, including shear stress, through the cytoskeleton to the nucleus. These forces can ultimately lead to changes in nuclear integrity, chromatin organization, and gene expression that significantly impact emergence of pathology such as the high incidence of atherosclerosis in progeria. Therefore, there is strong motivation to understand how endothelial nuclei can sense and respond to physical signals and how abnormal responses to mechanical cues can lead to disease. Here, we review the evidence for a critical role of the nucleus as a mechanosensor and the importance of maintaining nuclear integrity in response to continuous biophysical forces, specifically shear stress, for proper vascular function and stability.

Abstract 1233: A novel 14-gene signature to predict response to neoadjuvant chemotherapy and endocrine treatment in breast cancer patients

Abstract mRNA transcriptomic markers have become a key tool in the classification of breast cancer and deciding treatment regimens for patients. However, limited studies have evaluated markers that might be predictive of response to neoadjuvant treatment (chemotherapy and endocrine treatment). Defining such key markers that are associated with treatment response could offer new insights on the relative molecular mechanisms and provide more tailored treatment regimens to target pathways that might be over-represented. We aimed to identify mRNA expression markers associated with breast cancer patients’ response to neoadjuvant treatment from a pool of studies in which tumor samples were collected at pre-treatment and on-treatment timepoints. We collated 1194 pre- and on-treatment samples (721 unique patients) from 9 publicly available gene expression datasets that met our inclusion criteria. The standardized gene expression values from each study were merged in a global matrix of 1551 genes and 1194 samples. Differential Gene Expression Analysis adjusted for timepoint, pam50 subtype (as predicted by the genefu package in R), treatment and batch effects was conducted. 14 significantly (FDR = 0.05) differentially expressed were identified (CCNA2, RFC3, VRK1, PSMB2, ALDH7A1, RRM2, ADRM1, TSPAN4, ZNF473, RNH1, HDAC1, CDK1, SMC1A and TOR1AIP1) when responders and non-responders were compared, some of which are known drug targets (e.g. PSMB2/carfilzomib). Using the set of identified genes we performed Monte-Carlo consensus clustering on the full set (optimal number of clusters: 6). Clusters were significantly associated with response (p = 2·10-8), timepoint (p < 10-15) and pam50 subtype (p < 10-15), but not treatment (p = 0.35). We then split our data into a training, a validation and a test set (776, 299, 119 samples respectively) and used the 14 genes (alone or in combination with metadata) as predictors to fit three types of machine learning models (lasso-regularized Logistic Regression, Decision Trees and Support Vector Machines). Support Vector Machines demonstrated the best classification performance on the validation set (75% classification accuracy) and achieved accuracy of 80% on the test set. Pathway analysis based on the identified genes revealed enriched nuclear membrane organization, protein deubiquitination, DNA replication and histone modification pathways. Prediction of response to treatment at baseline or mid-treatment can aid in patient stratification in the neoadjuvant setting and separate patients who would benefit from treatment the most and could undergo a less extensive surgical operation from patients who are unlikely to respond and should be scheduled for surgery sooner. This kind of early intervention has the potential to lead to improved patient outcomes and reduced side effects from unnecessary treatment administration. Citation Format: Aristeidis Sionakidis, Jonine D. Figueroa, Timothy I. Cannings. A novel 14-gene signature to predict response to neoadjuvant chemotherapy and endocrine treatment in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1233.

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

Observation of quantum phase synchronization in a nuclear-spin system

We report an experimental study of phase synchronization in a pair of interacting nuclear spins subjected to an external drive in nuclear magnetic resonance architecture. A weak transition-selective radio-frequency field applied on one of the spins is observed to cause phase localization, which is experimentally established by measuring the Husimi distribution function under various drive conditions. To this end, we have developed a general interferometric technique to directly extract values of the Husimi function via the transverse magnetization of the undriven nuclear spin. We further verify the robustness of synchronization to detuning in the system by studying the Arnold tongue behavior.

Lamin A and telomere maintenance in aging: Two to Tango

Lamin proteins which constitute the nuclear lamina in almost all higher eukaryotes, are mainly of two types A & B encoded by LMNA and LMNB1/B2 genes respectively. While lamin A remains the principal product of LMNA gene, variants like lamin C, C2 and A∆10 are also formed as alternate splice products. Role of lamin A in aging has been highlighted in recent times due to its association with progeroid or premature aging syndromes which is classified as a type of laminopathy. Progeria caused by accelerated accumulation of lamin A Δ50 or progerin occurs due to a mutation in this LMNA gene leading to defects in post translational modification of lamin A. One of the most common and severe symptoms of progeroid laminopathy is accelerated cellular senescence or aging along with bone resorption, muscle weakness, lipodystrophy and cardiovascular disorders. On the other hand, progerin accumulation and telomere dysfunction merge as common traits in the process of chronological aging. Two major hallmarks of physiological aging in humans include loss of genomic integrity and telomere attrition which can result from defective laminar organization leading to deformed nuclear architecture and culminates into replicative senescence. This also adversely affects epigenetic landscape, mitochondrial dysfunction and several signalling pathways like DNA repair, mTOR, MAPK, TGFβ. In this review, we discuss the telomere-lamina interplay in the context of physiological aging and progeria.

CENP-A Regulation and Cancer.

In mammals, CENP-A, a histone H3 variant found in the centromeric chromatin, is critical for faithful chromosome segregation and genome integrity maintenance through cell divisions. Specifically, it has dual functions, enabling to define epigenetically the centromere position and providing the foundation for building up the kinetochore. Regulation of its dynamics of synthesis and deposition ensures to propagate proper centromeres on each chromosome across mitosis and meiosis. However, CENP-A overexpression is a feature identified in many cancers. Importantly, high levels of CENP-A lead to its mislocalization outside the centromere. Recent studies in mammals have begun to uncover how CENP-A overexpression can affect genome integrity, reprogram cell fate and impact 3D nuclear organization in cancer. Here, we summarize the mechanisms that orchestrate CENP-A regulation. Then we review how, beyond its centromeric function, CENP-A overexpression is linked to cancer state in mammalian cells, with a focus on the perturbations that ensue at the level of chromatin organization. Finally, we review the clinical interest for CENP-A in cancer treatment.

Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.

Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

Investigation of the Potential Mechanisms Underlying Nuclear F-Actin Organization in Ovarian Cancer Cells by High-Throughput Screening in Combination With Deep Learning.

Increasing evidence supports the notion that filamentous actin (F-actin) and globular actin exist in the nuclei of somatic cells, and are involved in chromatin remodeling, gene transcription regulation and DNA damage repair. However, the underlying mechanisms of how nuclear F-actin are polymerized in cells remain incompletely understood. Here, we identify potential kinase targets that participate in nuclear F-actin polymerization in ovarian cancer cells using small-molecule inhibitor library screening in combination with a deep learning approach. The analysis of the targets of the inhibitors used in this study suggest that the PI3K-AKT pathway are involved in regulating nuclear F-actin organization in ovarian cancer cells. Our work lays the foundation for uncovering the important roles of nuclear F-actin in the context of ovarian cancer, and for understanding how nuclear F-actin structures are organized.

Extrachromosomal DNA in Cancer.

In cancer, complex genome rearrangements and other structural alterations, including the amplification of oncogenes on circular extrachromosomal DNA (ecDNA) elements, drive the formation and progression of tumors. ecDNA is a particularly challenging structural alteration. By untethering oncogenes from chromosomal constraints, it elevates oncogene copy number, drives intratumoral genetic heterogeneity, promotes rapid tumor evolution, and results in treatment resistance. The profound changes in DNA shape and nuclear architecture generated by ecDNA alter the transcriptional landscape of tumors by catalyzing new types of regulatory interactions that do not occur on chromosomes. The current suite of tools for interrogating cancer genomes is well suited for deciphering sequence but has limited ability to resolve the complex changes in DNA structure and dynamics that ecDNA generates. Here, we review the challenges of resolving ecDNA form and function and discuss the emerging tool kit for deciphering ecDNA architecture and spatial organization, including what has been learned to date about how this dramatic change in shape alters tumor development, progression, and drug resistance.

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation.

Over 50% of the world’s population is infected with Human Cytomegalovirus (HCMV). HCMV is responsible for serious complications in the immuno-compromised and is a leading cause of congenital birth defects. The molecular function of many HCMV proteins remains unknown, and a deeper understanding of the viral effectors that modulate virion maturation is required. In this study, we observed that UL34 is a viral protein expressed with leaky late kinetics that localises to the nucleus during infection. Deletion of UL34 from the HCMV genome (ΔUL34) did not abolish the spread of HCMV. Instead, over >100-fold fewer infectious virions were produced, so we report that UL34 is an augmenting gene. We found that ΔUL34 is dispensable for viral DNA replication, and its absence did not alter the expression of IE1, MCP, gB, UL26, UL83, or UL99 proteins. In addition, ΔUL34 infections were able to progress through the replication cycle to form a viral assembly compartment; however, virion maturation in the cytoplasm was abrogated. Further examination of the nucleus in ΔUL34 infections revealed replication compartments with aberrant morphology, containing significantly less assembled capsids, with almost none undergoing subsequent maturation. Therefore, this work lays the foundation for UL34 to be further investigated in the context of nuclear organization and capsid maturation during HCMV infection.

Portable two-filter dual-flow-loop <sup>222</sup>Rn detector: stand-alone monitor and calibration transfer device

Abstract. Little overlap exists in the required capabilities of 222Rn (radon) monitors for public health and atmospheric research. The former requires robust, compact, easily transportable instruments to characterise daily to yearly variability >100 Bq m−3, whereas the latter requires static instruments capable of characterising sub-hourly variability between 0.1 and 100 Bq m−3. Consequently, detector development has evolved independently for the two research communities, and while many radon measurements are being made world-wide, the full potential of this measurement network can't be realised because not all results are comparable. Development of a monitor that satisfies the primary needs of both measurement communities, including a calibration traceable to the International System of Units (SI), would constitute an important step toward (i) increasing the availability of radon measurements to both research communities, and (ii) providing a means to harmonize and compare radon measurements across the existing eclectic global network of radon detectors. To this end, we describe a prototype detector built by the Australian Nuclear Science and Technology Organisation (ANSTO), in collaboration with the EMPIR 19ENV01 traceRadon Project and Physikalisch-Technische Bundesanstalt (PTB). This two-filter dual-flow-loop radon monitor can be transported in a standard vehicle, fits in a 19′′ instrument rack, has a 30 min temporal resolution, and a detection limit of ∼0.14 Bq m−3. It is capable of continuous, long-term, low-maintenance, low-power, indoor or outdoor monitoring with a high sensitivity and an uncertainty of ∼15 % at 1 Bq m−3. Furthermore, we demonstrate the successful transfer of an SI traceable calibration from this portable monitor to a 1500 L two-filter radon monitor under field conditions.

Nde1 is required for heterochromatin compaction and stability in neocortical neurons

SummaryThe NDE1 gene encodes a scaffold protein essential for brain development. Although biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we demonstrate Nde1 controls neurogenesis through facilitating H4K20 trimethylation-mediated heterochromatin compaction. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We demonstrate that NDE1 can undergo dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations and DNA double-strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and protecting neuronal heterochromatin. They suggest that heterochromatin instability predisposes a wide range of brain dysfunction.

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 4D Nucleome Data Portal as a resource for searching and visualizing curated nucleomics data

The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal (https://data.4dnucleome.org/), a repository for datasets generated in the 4DN network and relevant external datasets. Datasets were generated with a wide range of experiments, including chromosome conformation capture assays such as Hi-C and other innovative sequencing and microscopy-based assays probing chromosome architecture. All together, the 4DN data portal hosts more than 1800 experiment sets and 36000 files. Results of sequencing-based assays from different laboratories are uniformly processed and quality-controlled. The portal interface allows easy browsing, filtering, and bulk downloads, and the integrated HiGlass genome browser allows interactive visualization and comparison of multiple datasets. The 4DN data portal represents a primary resource for chromosome contact and other nuclear architecture data for the scientific community.

Coilin Modulates Nuclear Organization by Promoting Protein SUMOylation

Coilin was first identified as the marker protein for Cajal Bodies (CBs). However, CBs are only abundant in neuronal, embryonic, and cancer cells, while primary cells have few CBs. Even in cells with CBs, 70% of coilin is nucleoplasmic, not found in CBs. CBs are subnuclear domains that function in small nuclear ribonucleoprotein (snRNP) biogenesis as well as production of telomerase RNP, expression and 3’end processing of small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and small CB‐associated RNAs (scaRNAs). The function of nucleoplasmic coilin is not well understood. We have previously shown that coilin promotes miRNA biogenesis by promoting phosphorylation of the Microprocessor component, DGCR8, at its S377 residue in both cells with and without CBs. We have since identified 7 additional serine residues of DGCR8 with decreased phosphorylation in coilin knockdown through phosphoproteomic analysis of HeLa cells. In addition to phosphorylation, the addition of a small ubiquitin‐like modifier (SUMO), or SUMOylation, of DGCR8 also increases its stability. Because of coilin’s role in the promotion of one post‐translational modification, here we aimed to test the hypothesis that coilin knockdown alters protein SUMOylation and results in altered nuclear organization. We conducted a series of siRNA knockdowns, Western blots, and immunoprecipitation experiments. Here we show, for the first time, that coilin knockdown results in global decrease of protein SUMOylation in cells with (HeLa) and without CBs (WI‐38), suggesting that regulation of protein SUMOylation may be a function of nucleoplasmic coilin. Specifically, we found that coilin knockdown results in decreased SUMOylation of DGCR8 and Speckled protein 100kDa (Sp100), a major component of the promyelocytic leukemia nuclear body (PML‐NB). Sp100 is vital to PML‐NB formation, and decreased SUMOylation of Sp100 decreases its stability. This information led us to the second part of our hypothesis. We used immunofluorescence and found that coilin knockdown results in decreased numbers of PML‐NBs per cell. To summarize, we have identified coilin as a regulator of protein phosphorylation, SUMOylation, and nuclear organization.

Immunofluorescent and Immunohistochemical (Colorimetric) Characterization of B‐DNA, Z‐DNA and G4‐Quadruplex DNA in Human Tissues Demonstrating the Spatial Genomic Organization of Different DNA Structures: Genomesorganizomics

Spatial genomic organization, relative to DNA and RNA structures, has not been examined. DNA can adopt a variety of different shapes (helical conformations), i.e., canonical right‐handed double‐stranded (ds‐) B‐DNA, alternative left‐handed ds‐Z‐DNA and multi‐stranded G4‐quadruplex DNA. Each of these DNAs has a unique structure and plays a different role in cells. The structure of the nucleus is complex, and has a major role in gene expression. For the first time, our group has demonstrated the spatial genomic distribution of three totally different DNAs concurrently in HeLa cells, paraffin‐embedded tissue sections, and frozen sections. This was achieved by means of anti‐B‐DNA, anti‐Z‐DNA and anti‐G4 quadruplex DNA antibodies, via immunofluorescence (IF), and immunohistochemistry (IHC) (colorimetric). No one is investigating the relationship between spatial genomic organization and structures of different nucleic acids. All three types of DNA exist in the human genome and perform diverse cellular genomic functions. Tissues were processed in 10% NBF, and staining protocols were done both manually and by autostainer. Antigen retrieval enhanced all DNA‐based immunostaining procedures. IF was performed using Alexa Fluor conjugated secondary antibodies [i.e., Alexa Fluor 405 (blue), Alexa Fluor 488 (green) and Alexa Fluor 555 (red)], and wheat germ agglutinin conjugated with Alexa Fluor 647 (white) (counterstain). IHC protocols (i.e., enzyme: HRP or AP) were accomplished by using enzymes conjugated to secondary antibodies, and color developed by chromogens [i.e., B‐DNA (Blue) AP Substrate, Z‐DNA (Red/Magenta) AP Substrate, and G4‐quadruplex DNA (Brown), DAB Peroxidase (HRP) Substrate]. Negative and positive controls, isotype controls, and blocking background were performed in triplicate. The order of primary antibodies (and substrates: IHC) is critical for obtaining accurate data. Images were obtained by confocal and light microscopes (i.e., 200x, 600x). Advanced computer‐based image analysis (e.g., HALO, Image Pro Plus) allowed for qualitative and quantitative characterization of immunostained DNAs. The largest percentage of DNA was in the form of B‐DNA, followed by the other two remaining structures of DNA. The novel method signifies a new subfield of structural and functional genomics, which we have named Spatial Genomic Organization of DNA Structures, i.e., Genomesorganizomics. It will aid spatial transcriptomics in the analysis of gene expression and provide information on transcriptional activity based upon the conformation of DNA in genes and telomeres. An in‐depth comparative analysis between IF and IHC will yield information about specific advantages and disadvantages of these immuno‐based tissue staining systems (e.g., sensitivity, dynamic range, easier multiplexing, photobleaching, better target colocalization). This genomics approach is both sensitive and reliable, and will allow for the characterization of nuclear genomic architecture. Future projects are examining both DNA and RNA structures of four or more nucleic acids.

Tau modulates mRNA transcription, alternative polyadenylation (APA) profiles of hnRNPs, chromatin remodeling and spliceosome complexes

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer ’s disease (AD) and frontotemporal dementia (FTD). It is well‐established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription and RNA processing. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau‐inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing, including several RNA‐Binding Proteins (RBPs). We isolated total mRNA before and after over‐expressing tau and then performed Poly(A)‐ClickSeq (PAC‐Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up‐regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss‐of‐function at the transcriptional level. WT tau up‐regulates genes contributing to cytoskeleton‐dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down‐regulate genes responsible for ubiquitin‐proteasome system. In addition, WT tau significantly down‐regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post‐transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis.

Radial pair correlation of molecular brightness fluctuations maps protein diffusion as a function of oligomeric state within live-cell nuclear architecture

Nuclear proteins can modulate their DNA binding activity and the exploration volume available during DNA target search by self-associating into higher-order oligomers. Directly tracking this process in the nucleoplasm of a living cell is, however, a complex task. Thus, here we present a microscopy method based on radial pair correlation of molecular brightness fluctuations (radial pCOMB) that can extract the mobility of a fluorescently tagged nuclear protein as a function of its oligomeric state and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture. By simply performing a rapid frame scan acquisition, radial pCOMB has the capacity to detect, within each pixel, protein oligomer formation and the size-dependent obstruction nuclear architecture imparts on this complex’s transport across sub-micrometer distances. From application of radial pCOMB to an oligomeric transcription factor and DNA repair protein, we demonstrate that homo-oligomer formation differentially regulates chromatin accessibility and interaction with the DNA template.

Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy.

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.