Subnuclear Compartments Research Articles

Spatial 3D genome organization controls the activity of bivalent chromatin during human neurogenesis.

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1-5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery - not the presence of H3K27me3 - is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains

The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.

ScGHOST: identifying single-cell 3D genome subcompartments.

Single-cell Hi-C (scHi-C) technologies allow for probing of genome-wide cell-to-cell variability in three-dimensional (3D) genome organization from individual cells. Computational methods have been developed to reveal single-cell 3D genome features based on scHi-C, including A/B compartments, topologically associating domains and chromatin loops. However, no method exists for annotating single-cell subcompartments, which is important for understanding chromosome spatial localization in single cells. Here we present scGHOST, a single-cell subcompartment annotation method using graph embedding with constrained random walk sampling. Applications of scGHOST to scHi-C data and contact maps derived from single-cell 3D genome imaging demonstrate reliable identification of single-cell subcompartments, offering insights into cell-to-cell variability of nuclear subcompartments. Using scHi-C data from complex tissues, scGHOST identifies cell-type-specific or allele-specific subcompartments linked to gene transcription across various cell types and developmental stages, suggesting functional implications of single-cell subcompartments. scGHOST is an effective method for annotating single-cell 3D genome subcompartments in a broad range of biological contexts.

Abstract 1650: Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells

Abstract Nucleoli are large nuclear sub-compartments where vital cellular processes, such as rRNA production and ribosome assembly, take place. Aggressive tumor types, such as Triple Negative Breast Cancer (TNBC), require increased ribosome biogenesis to support their enhanced proliferation. Therefore, it has been suggested that the nucleolus could be a valid anti-neoplastic target. However, we still have a limited understanding of the cancer-specific functions of the nucleolar components, preventing the effective development of therapeutic agents. The characterization of nucleolar proteins is technically challenging. Several nucleolar proteins are essential, and their complete abrogation cannot be achieved through conventional RNAi or CRISPR-KO approaches. Additionally, many nucleolar proteins display peculiar biophysical properties, and their functions are connected to their abundance. Therefore, artificial overexpression systems might not fully recapitulate their endogenous biological functions. To overcome these challenges, we implemented an Auxin Inducible Degron (AID) system to acutely abrogate endogenous nucleolin, one of the most abundant nucleolar proteins, and asses its impact on the proliferation of TNBC cell lines. We used CRISPR/Cas9 editing in TNBC cell lines to modify the endogenous NCL gene, fusing it with an AID tag. Then, we stably transduced the cells with a modified Oryza Sativa TIR1 (OsTIR1-F74G) E3 ubiquitin ligase. In response to the synthetic phytohormone phenyl-auxin, OsTIR1 leads to the proteasomal degradation of AID-containing endogenous nucleolin in less than 6h, sparing any other human protein. RNA sequencing analysis showed that nucleolin degradation significantly alters the expression of genes involved in cell cycle progression. Accordingly, nucleolin degradation reduces cancer cell proliferation in vitro, associated with increased propidium iodide staining, suggestive of a G2/M cell cycle arrest. However, immunofluorescence analyses revealed that nucleolin abrogation leads to the accumulation of bi-nucleated cells with actin patches. These findings are indicative of potential cytokinesis defects due to altered spindle tension during mitosis. Notably, nucleolin abrogation increased the efficacy of chemical inhibitors of the Anaphase Promoting Complex on suppression of cell proliferation. To the best of our knowledge, this study is the first reporting the acute abrogation of an endogenous, highly abundant nucleolar protein, using the AID system. This approach allowed the characterization of nucleolin biological functions with unprecedented temporal resolution, shedding light on new biological functions in the regulation of cell division. Finally, our results suggest that nucleolin inhibition could enhance the therapeutic activities of drugs affecting cell cycle progression in TNBC. Citation Format: Nastaran Samadi Rad, Joseph Mills, Anna Tessari, Damu Sunil Kumar, Vollter Anastas, Saranya Lamba, Ashley B Reers, Ilaria Cosentini, Zirui Zhu, Thomas J Magliery, Emanuele Cocucci, Lara Rizzotto, Dario Palmieri. Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1650.

Topological formation of transcriptional condensates relative to subnuclear compartments

Topological formation of transcriptional condensates relative to subnuclear compartments

Chromatin Remodeling Due to Transient-Link-and-Pass Activity Enhances Subnuclear Dynamics.

Spatiotemporal coordination of chromatin and subnuclear compartments is crucial for cells. Numerous enzymes act inside nucleus-some of those transiently link and pass two chromatin segments. Here, we study how such an active perturbation affects fluctuating dynamics of an inclusion in the chromatic medium. Using numerical simulations and a versatile effective model, we categorize inclusion dynamics into three distinct modes. The transient-link-and-pass activity speeds up inclusion dynamics by affecting a slow mode related to chromatin remodeling, viz., size and shape of the chromatin meshes.

Dose-Dependent Transcriptional Response to Ionizing Radiation Is Orchestrated with DNA Repair within the Nuclear Space.

Radiation therapy is commonly used to treat glioblastoma multiforme (GBM) brain tumors. Ionizing radiation (IR) induces dose-specific variations in transcriptional programs, implicating that they are tightly regulated and critical components in the tumor response and survival. Yet, our understanding of the downstream molecular events triggered by effective vs. non-effective IR doses is limited. Herein, we report that variations in the genetic programs are positively and functionally correlated with the exposure to effective or non-effective IR doses. Genome architecture analysis revealed that gene regulation is spatially and temporally coordinated with DNA repair kinetics. The radiation-activated genes were pre-positioned in active sub-nuclear compartments and were upregulated following the DNA damage response, while the DNA repair activity shifted to the inactive heterochromatic spatial compartments. The IR dose affected the levels of DNA damage repair and transcription modulation, but not the order of the events, which was linked to their spatial nuclear positioning. Thus, the distinct coordinated temporal dynamics of DNA damage repair and transcription reprogramming in the active and inactive sub-nuclear compartments highlight the importance of high-order genome organization in synchronizing the molecular events following IR.

Cell-type differential targeting of SETDB1 prevents aberrant CTCF binding, chromatin looping, and cis-regulatory interactions

SETDB1 is an essential histone methyltransferase that deposits histone H3 lysine 9 trimethylation (H3K9me3) to transcriptionally repress genes and repetitive elements. The function of differential H3K9me3 enrichment between cell-types remains unclear. Here, we demonstrate mutual exclusivity of H3K9me3 and CTCF across mouse tissues from different developmental timepoints. We analyze SETDB1 depleted cells and discover that H3K9me3 prevents aberrant CTCF binding independently of DNA methylation and H3K9me2. Such sites are enriched with SINE B2 retrotransposons. Moreover, analysis of higher-order genome architecture reveals that large chromatin structures including topologically associated domains and subnuclear compartments, remain intact in SETDB1 depleted cells. However, chromatin loops and local 3D interactions are disrupted, leading to transcriptional changes by modifying pre-existing chromatin landscapes. Specific genes with altered expression show differential interactions with dysregulated cis-regulatory elements. Collectively, we find that cell-type specific targets of SETDB1 maintain cellular identities by modulating CTCF binding, which shape nuclear architecture and transcriptomic networks.

NOC1 is a direct MYC target, and its protein interactome dissects its activity in controlling nucleolar function.

The nucleolus is a subnuclear compartment critical in ribosome biogenesis and cellular stress responses. These mechanisms are governed by a complex interplay of proteins, including NOC1, a member of the NOC family of nucleolar proteins responsible for controlling rRNA processing and ribosomal maturation. This study reveals a novel relationship between NOC1 and MYC transcription factor, known for its crucial role in controlling ribosomal biogenesis, cell growth, and proliferation. Here, we demonstrate that NOC1 functions as a direct target of MYC, as it is transcriptionally induced through a functional MYC-binding E-box sequence in the NOC1 promoter region. Furthermore, protein interactome analysis reveals that NOC1-complex includes the nucleolar proteins NOC2 and NOC3 and other nucleolar components such as Nucleostemin1 Ns1 transporters of ribosomal subunits and components involved in rRNA processing and maturation. In response to MYC, NOC1 expression and localization within the nucleolus significantly increase, suggesting a direct functional link between MYC activity and NOC1 function. Notably, NOC1 over-expression leads to the formation of large nuclear granules and enlarged nucleoli, which co-localize with nucleolar fibrillarin and Ns1. Additionally, we demonstrate that NOC1 expression is necessary for Ns1 nucleolar localization, suggesting a role for NOC1 in maintaining nucleolar structure. Finally, the co-expression of NOC1 and MYC enhances nucleolus size and maintains their co-localization, outlining another aspect of the cooperation between NOC1 and MYC in nucleolar dynamics. This study also reveals an enrichment with NOC1 with few proteins involved in RNA processing, modification, and splicing. Moreover, proteins such as Ythdc1, Flacc, and splenito are known to mediate N6-methyladenosine (m6A) methylation of mRNAs in nuclear export, revealing NOC1's potential involvement in coordinating RNA splicing and nuclear mRNA export. In summary, we uncovered novel roles for NOC1 in nucleolar homeostasis and established its direct connection with MYC in the network governing nucleolar structure and function. These findings also highlight NOC1's interaction with proteins relevant to specific RNA functions, suggesting a broader role in addition to its control of nucleolar homeostasis and providing new insight that can be further investigated.

Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1.

The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an 'Escaping' effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for 'Escaping'. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.

T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13.

T cells require rapid proliferation to initiate adaptive immunity to prevent pathogen attacks. The nucleolus, a distinct subnuclear membrane-less compartment for ribosomal biogenesis, is indispensable for cell proliferation. However, specific nucleolar proteins involved in rapid T cell proliferation and their underlying molecular regulatory mechanism remain elusive. Here, we identified an essential nucleolar protein, DCAF13, in T cells and revealed its significant regulation of rapid T cell proliferation. Its depletion drastically impairs T cell proliferation due to severe 18S rRNA maturation failure, consequent abnormal ribosome assembly in nucleoli, and insufficient production of nascent proteins. Mechanistically, we propose that DCAF13 promotes NPM1 phase separation to accelerate pre-RNA enrichment and its endonuclease UTP23 for 18S rRNA maturation during T cell proliferation. Our findings reveal the modulatory effect of nucleolar NPM1/DCAF13 phase separation on ribosomal maturation to ensure rapid T cell proliferation and further pathogen clearance for the first time.

Analysis of Giardia lamblia Nucleolus as Drug Target: A Review.

Giardia lamblia (G. lamblia) is the main causative agent of diarrhea worldwide, affecting children and adults alike; in the former, it can be lethal, and in the latter a strong cause of morbidity. Despite being considered a predominant disease in low-income and developing countries, current migratory flows have caused an increase in giardiasis cases in high-income countries. Currently, there is a wide variety of chemotherapeutic treatments to combat this parasitosis, most of which have potentially serious side effects, such as genotoxic, carcinogenic, and teratogenic. The necessity to create novel treatments and discover new therapeutic targets to fight against this illness is evident. The current review centers around the controversial nucleolus of G. lamblia, providing a historical perspective that traces its apparent absence to the present evidence supporting its existence as a subnuclear compartment in this organism. Additionally, possible examples of ncRNAs and proteins ubiquitous to the nucleolus that can be used as targets of different therapeutic strategies are discussed. Finally, some examples of drugs under research that could be effective against G. lamblia are described.

Increased body mass index is linked to systemic inflammation through altered chromatin co-accessibility in human preadipocytes

Obesity-induced adipose tissue dysfunction can cause low-grade inflammation and downstream obesity comorbidities. Although preadipocytes may contribute to this pro-inflammatory environment, the underlying mechanisms are unclear. We used human primary preadipocytes from body mass index (BMI) -discordant monozygotic (MZ) twin pairs to generate epigenetic (ATAC-sequence) and transcriptomic (RNA-sequence) data for testing whether increased BMI alters the subnuclear compartmentalization of open chromatin in the twins’ preadipocytes, causing downstream inflammation. Here we show that the co-accessibility of open chromatin, i.e. compartmentalization of chromatin activity, is altered in the higher vs lower BMI MZ siblings for a large subset ( ~ 88.5 Mb) of the active subnuclear compartments. Using the UK Biobank we show that variants within these regions contribute to systemic inflammation through interactions with BMI on C-reactive protein. In summary, open chromatin co-accessibility in human preadipocytes is disrupted among the higher BMI siblings, suggesting a mechanism how obesity may lead to inflammation via gene-environment interactions.

Abstract 2815: Analyzing nuclear paraspeckle-dependent protection of unphosphorylated MicroRNA

Abstract MicroRNA-34 (miR-34) is widely studied for its role in tumor suppression, DNA regulation, and cell cycle regulation. During DNA damage, miR-34 is directly upregulated by p53, inhibiting proliferation and promoting DNA repair. Accordingly, radiation therapy induces upregulation of miR-34 expression, coordinating several downstream radiation-induced stress responses. Recent work has demonstrated the existence of a stable, unphosphorylated pool of miR-34, which is rapidly phosphorylated and activated via loading onto Argonaute after ionizing radiation, independent of de novo transcription. Since Argonaute binding to the 5’ phosphate is also important for miRNA stability, we hypothesize that a separate mechanism must exist to stabilize the unphosphorylated pool of miR-34. Mass spectrometry performed on co-precipitates of biotinylated mimics of unphosphorylated miR-34 identified predominantly nuclear proteins, with a notable enrichment for proteins localized in nuclear paraspeckles. Nuclear paraspeckles are a phase-separated nuclear subcompartment comprised of several key structural proteins and a long noncoding RNA NEAT1_2 that prevents mixing of internal contents with the rest of the nucleoplasm and are canonically implicated in regulating gene expression by sequestering inosine-modified mRNA. To further investigate the role of nuclear paraspeckles in the stability or sequestering of unphosphorylated miR-34, stable cell lines with deletion of a a motif required for stability of paraspeckles through disruption of the lncRNA NEAT1_2 were created (A549 NEAT1 dTH). Interestingly, luciferase reporter assays for miR-34 activity in the NEAT1 dTH lines showed a general loss of miR-34 activity after radiation compared to wildtype cells which exhibit radiation induced activation of miR-34. This loss of activity occurred both immediately after radiation, which is attributable to phosphorylation of the unphosphorylated pool, and at later time points after radiation, which has been attributed to de novo miRNA production. Ongoing research will monitor colocalization of proteins known to activate the unphosphorylated pool with paraspeckles to determine if paraspeckles coordinate activation of unphosphorylated miR-34, as well as will investigate the potential impact of disruption of paraspeckles on de novo miR-34 production. A better understanding of the interplay between nuclear paraspeckles and stress-responsive miRNA production may provide a means to selectively modify cancer stress responses and uncover novel mechanisms in miRNA production and coordination. Citation Format: Graham H. Read, David W. Salzman, Julian Whitelegge, Whitaker Cohn, Tiffany Yang, Cynthia Tsang, Ihsan A. Turk, Joanne B. Weidhaas. Analyzing nuclear paraspeckle-dependent protection of unphosphorylated MicroRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2815.

Abstract P6-04-03: Computational pathology based HER2 expression quantification in HER2-low breast cancer

Abstract Background HER2 directed therapies for breast cancer (BC) rely on accurate estimation of HER2 expression by pathologist scoring of immunohistochemically (IHC) stained tissue according to ASCO/CAP guidelines Emerging HER2-targeted antibody drug conjugates (ADCs) like trastuzumab deruxtecan (T-DXd), have demonstrated efficacy in the HER2-low (IHC 1+ or IHC 2+/ISH-) population (Modi, NEJM 2022). A deeper understanding of the spectrum of HER2 expression and its spatial distribution could provide insights about the mode of action of ADCs, including potential bystander activity. Computational pathology-based methods like Quantitative Continuous Scoring (QCS) can help here by objectively quantifying HER2 expression levels on a per cell basis from digitized HER2 IHC slides (Gustavson, SABCS 2020). We applied QCS to a cohort of HER2-negative (HER2-neg) patients (pts) from a retrospective study (NCT04807595) to quantify the prevalence of HER2 expression in this population and investigate the relationship with manual scoring. Methods To analyze the prevalence of HER2 expression in the HER2-neg population, we used available digital images (N=207 pts) from retrospectively rescored HER2 slides from tumors categorized as HER2-low or IHC 0 (IHC 0 or >0< 1+). QCS algorithm was applied to perform an instance segmentation of each tumor cell into the membrane, cytoplasmic and nuclear sub-compartments. HER2 expression levels on the membrane were estimated from a Hue-Saturation-Density model (Van der Laak, JQCS 2000) in terms of optical density (OD). Descriptive statistics and spatial modelling were used to aggregate cell-level information to a slide level score using the membrane OD values and tumor cell locations. A novel Spatial Proximity Score (SPS) was used to mathematically model the proportion of tumor cells that could potentially be targeted either directly or via bystander activity of ADCs. The analysis is ongoing, complete results with additional patient data to be presented. Analytical validation of the QCS algorithm demonstrated high correlation between OD values as measured on the automatically detected membranes from QCS and those measured on consolidated manual membrane annotations (N=2157 cells) from three pathologists (R = 0.993). This is very similar to the correlation observed between individual pathologists (R = 0.995). Results In the analyzed cohort (N=207), median OD of HER2-low tumors was significantly higher compared to IHC 0 tumors (one-sided Wilcoxon p-value < 0.001). A significant increase of OD values was observed for increasing IHC categories from 0 through >0 < 1+ and 1+ to 2+/ISH- (one-sided Jonckheere-Terpstra p-value < 0.001). OD values within each IHC category showed considerable variability, particularly in IHC 1+ and IHC 2+. In 49% of pts (N=101), greater than 88% of tumor cells expressed HER2 at any intensity (OD≥10). Among the remaining 106 pts, the number of potentially ADC-susceptible cells (within 25μm radius of HER2 expressing cells) as estimated by SPS was at least double the amount estimated by single cell-based scores alone in 45 cases (42%) and increased by at least 50% in another 12 cases (11%). Conclusions Computational approaches such as QCS can help us to objectively characterize the spectrum and spatial distribution of HER2 expression. These mathematical models contribute to our understanding of potential mechanisms of action of ADCs. While this study confirmed a general association of QCS-based scores with manual IHC categories, we also saw considerable variation, as some IHC 1+ or 2+ samples had low OD. Building on these and other promising initial results (Gustavson et al, SABCS 2020), we will further explore clinical relevance of QCS-based scoring. Eventually, digital scoring may be able to define data-driven signatures to select HER2-low pts that might benefit from HER2 targeted therapies. Citation Format: Andreas Spitzmüller, Ansh Kapil, Anatoliy Shumilov, Jessica Chan, Lemonia Konstantinidou, Zonera Hassan, Mark Gustavson, Danielle Carroll, Della Varghese, Gareth D. James, Akira Moh, Andrew Livingston, Victoria de Giorgio-Miller. Computational pathology based HER2 expression quantification in HER2-low breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-03.

SUMO conjugation regulates the activity of the Integrator complex.

RNA polymerase II (RNAPII) transcribes small nuclear RNA (snRNA) genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that overexpression of USPL1 as well as of another nuclear SUMO isopeptidase, SENP6, alters snRNA 3'-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different RNAPII transcripts. While several subunits of the complex are SUMO conjugation substrates, we found that the SUMOylation of the INTS11 subunit is regulated by USPL1 and SENP6. We defined Lys381, Lys462 and Lys475 as bona fide SUMO attachment sites on INTS11 and observed that SUMOylation of this protein modulates its subcellular localization and is required for Integrator activity. Moreover, while an INTS11 SUMOylation-deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. These findings point to a regulatory role for SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

A nuclear redox sensor modulates gene activation and var switching in Plasmodium falciparum

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.