Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope.

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML.

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, whether and how SUMO contributes to ALT beyond APB formation remains elusive. Here, we report that SUMO promotes collaboration among DNA repair proteins to achieve APB-independent telomere maintenance. By using ALT cancer cells with PML protein knocked out and thus devoid of APBs, we show that sumoylation is required for manifesting ALT features, including telomere clustering and telomeric DNA synthesis, independent of PML and APBs. Further, small molecule-induced telomere targeting of SUMO produces signatures of phase separation and ALT features in PML null cells in a manner depending on both sumoylation and SUMO interaction with SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are linked to the enrichment of DNA repair proteins, including Rad52, Rad51AP1, and BLM, to the SUMO-containing telomere foci. Finally, we find that Rad52 can undergo phase separation, enrich SUMO on telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that, in addition to forming APBs, SUMO also promotes collaboration among DNA repair proteins to support telomere maintenance in ALT cells. Given the promising effects of sumoylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

Running 'LAPS' Around nLD: Nuclear Lipid Droplet Form and Function.

The nucleus harbours numerous protein subdomains and condensates that regulate chromatin organization, gene expression and genomic stress. A novel nuclear subdomain that is formed following exposure of cells to excess fatty acids is the nuclear lipid droplet (nLD), which is composed of a neutral lipid core surrounded by a phospholipid monolayer and associated regulatory and lipid biosynthetic enzymes. While structurally resembling cytoplasmic LDs, nLDs are formed by distinct but poorly understood mechanisms that involve the emergence of lipid droplets from the lumen of the nucleoplasmic reticulum and de novo lipid synthesis. Luminal lipid droplets that emerge into the nucleoplasm do so at regions of the inner nuclear membrane that become enriched in promyelocytic leukemia (PML) protein. The resulting nLDs that retain PML on their surface are termed lipid-associated PML structures (LAPS), and are distinct from canonical PML nuclear bodies (NB) as they lack key proteins and modifications associated with these NBs. PML is a key regulator of nuclear signaling events and PML NBs are sites of gene regulation and post-translational modification of transcription factors. Therefore, the subfraction of nLDs that form LAPS could regulate lipid stress responses through their recruitment and retention of the PML protein. Both nLDs and LAPS have lipid biosynthetic enzymes on their surface suggesting they are active sites for nuclear phospholipid and triacylglycerol synthesis as well as global lipid regulation. In this review we have summarized the current understanding of nLD and LAPS biogenesis in different cell types, their structure and composition relative to other PML-associated cellular structures, and their role in coordinating a nuclear response to cellular overload of fatty acids.

Super-Resolution Radial Fluctuations (SRRF) Microscopy.

Super-resolution Radial Fluctuations (SRRF) imaging is a computational approach to fixed and live-cell super-resolution microscopy that is highly accessible to life science researchers since it uses common microscopes and open-source software plugins for ImageJ. This allows users to generate super-resolution images using the same equipment, fluorophores, fluorescent proteins and methods they routinely employ for their studies without specialized sample preparations or reagents. Here, we discuss a step-by-step workflow for acquiring and analyzing images using the NanoJ-SRRF software developed by the Ricardo Henriques group, with a focus on imaging chromatin. Increased accessibility of affordable super-resolution imaging techniques is an important step in extending the reach of this revolution in cellular imaging to a greater number of laboratories.

Assessing kinetics and recruitment of DNA repair factors using high content screens.

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

Haploinsufficient tumor suppressor PRP4K is negatively regulated during epithelial-to-mesenchymal transition.

The pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro, and cancer metastasis in mice. These results are consistent with PRP4K being a haploinsufficient tumor suppressor. Increased cell migration and invasion is associated with epithelial-to-mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non-transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple-negative breast cancer cells (MDA-MB-231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA-MB-231 and MCF10A cells and without changes in E-cadherin. Induction of EMT in MCF10A cells, by treatment with WNT-5a and TGF-ß1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT-5a/TGF-ß1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumor suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1.

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40-50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination.

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis.

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis. Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.

An emerging role for calcium signalling in innate and autoimmunity via the cGAS-STING axis.

Type I interferons are effector cytokines essential for the regulation of the innate immunity. A key effector of the type I interferon response that is dysregulated in autoimmunity and cancer is the cGAS-STING signalling axis. Recent work suggests that calcium and associated signalling proteins can regulate both cGAS-STING and autoimmunity. How calcium regulates STING activation is complex and involves both stimulatory and inhibitory mechanisms. One of these is calmodulin-mediated signalling that is necessary for STING activation. The alterations in calcium flux that occur during STING activation can also regulate autophagy, which in turn plays a role in innate immunity through the clearance of intracellular pathogens. Also connected to calcium signalling pathways is the cGAS inhibitor TREX1, a cytoplasmic exonuclease linked to several autoimmune diseases including systemic lupus erythematosus (SLE). In this review, we summarize these and other findings that indicate a regulatory role for calcium signalling in innate and autoimmunity through the cGAS-STING pathway.

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival.

Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

CRISPR/Cas9 Gene Editing: From Basic Mechanisms to Improved Strategies for Enhanced Genome Engineering In Vivo.

INTRODUCTION: Targeted genome editing using the CRISPR/Cas9 technology is becoming a major area of research due to its high potential for the treatment of genetic diseases. Our understanding of this approach has expanded in recent years yet several new challenges have presented themselves as we explore the boundaries of this exciting new technology. Chief among these is improving the efficiency but also the preciseness of genome editing. The efficacy of CRISPR/Cas9 technology relies in part on the use of one of the major DNA repair pathways, Homologous recombination (HR), which is primarily active in S and G2 phases of the cell cycle. Problematically, the HR potential is highly variable from cell type to cell type and most of the cells of interest to be targeted in vivo for precise genome editing are in a quiescent state. CONCLUSION: In this review, we discuss the recent advancements in improving targeted CRISPR/Cas9 based genome editing and the promising ways of delivering this technology in vivo to the cells of interest.

Marker-free coselection for CRISPR-driven genome editing in human cells.

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

PML nuclear bodies contribute to the basal expression of the mTOR inhibitor DDIT4.

The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl2) or genotoxic stress. Analysis of publicly available cancer data also revealed a significant correlation between PML and DDIT4 expression in several cancer types (e.g. lung, breast, prostate). Thus, these findings uncover a novel mechanism by which PML loss may contribute to mTOR activation and cancer progression via dysregulation of basal DDIT4 gene expression.

Myogenic differentiation triggers PML nuclear body loss and DAXX relocalization to chromocentres.

The promyelocytic leukemia protein (PML) is expressed in most normal human tissues and forms nuclear bodies (NBs) that have roles in gene regulation and cellular processes such as DNA repair, cell cycle control, and cell fate decisions. Using murine C2C12 myoblasts, we demonstrate that activation of skeletal muscle differentiation results in loss of PML and PML NBs prior to myotube fusion. Myotube formation was associated with marked chromatin reorganization and the relocalization of DAXX from PML NBs to chromocentres. MyoD expression was sufficient to cause PML NB loss, and silencing of PML induced DAXX relocalization. Fusion of C2C12 cells using the reptilian reovirus p14 fusogenic protein failed to disrupt PML NBs yet still promoted DAXX redistribution and loss; whereas ectopic expression of PML in differentiated cells only partially restored PML NB formation and DAXX localization at NBs. Finally, we determined that the C-terminal SUMO-interacting motif of DAXX is required for its colocalization with ATRX in heterochromatin domains during myotube formation. These data support a model in which activation of myogenic differentiation results in PML NB loss, chromatin reorganization and DAXX relocalization, and provides a paradigm for understanding the consequence of PML loss in other cellular contexts, such as during cancer development and progression.

Precision genome editing in the CRISPR era.

With the introduction of precision genome editing using CRISPR-Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR-Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR-Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

A mechanism for the suppression of homologous recombination in G1 cells.

DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)-RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR-Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells.

Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing.

CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR.

Shigella infection interferes with SUMOylation and increases PML-NB number.

Shigellosis is a severe diarrheal disease that affects hundreds of thousands of individuals resulting in significant morbidity and mortality worldwide. Shigellosis is caused by Shigella spp., a gram-negative bacterium that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into the cytosol of infected human cells. Shigella infection triggers multiple signaling programs that result in a robust host transcriptional response that includes the induction of multiple proinflammatory cytokines. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures that coordinate immune signaling programs and have a demonstrated role in controlling viral infection. We show that PML-NB number increases upon Shigella infection. We examined the effects of Shigella infection on SUMOylation and found that upon Shigella infection the localization of SUMOylated proteins is altered and the level of SUMOylated proteins decreases. Although Shigella infection does not alter the abundance of SUMO activating enzymes SAE1 or SAE2, it dramatically decreases the level of the SUMO conjugating enzyme Ubc9. All Shigella-induced alterations to the SUMOylation system are dependent upon a T3SS. Thus, we demonstrate that Shigella uses one or more T3SS effectors to influence both PML-NB number and the SUMOylation machinery in human cells.