Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.
© 2022. The Author(s).

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.
Copyright © 2021 Pronot, Kieffer, Gay, Debayle, Forquet, Poupon, Schorova, Martin and Gwizdek.

The importin superfamily member Importin-13 is a bidirectional nuclear transporter. To delineate its functional roles, we performed transcriptomic analysis on wild-type and Importin-13-knockout mouse embryonic stem cells, revealing enrichment of differentially expressed genes involved in stress responses and apoptosis regulation. De novo promoter motif analysis on 277 Importin-13-dependent genes responsive to oxidative stress revealed an enrichment of motifs aligned to consensus sites for the transcription factors specificity protein 1, SP1, or Kruppel like factor 4, KLF4. Analysis of embryonic stem cells subjected to oxidative stress revealed that Importin-13-knockout cells were more resistant, with knockdown of SP1 or KLF4 helping protect wild-type embryonic stem cells against stress-induced death. Importin-13 was revealed to bind to SP1 and KLF4 in a cellular context, with a key role in oxidative stress-dependent nuclear export of both transcription factors. The results are integral to understanding stress biology, highlighting the importance of Importin-13 in the stress response.
© 2021. The Author(s).

HIV-1 latency generates reservoirs that prevent viral eradication by the current therapies. To find strategies toward an HIV cure, detailed understandings of the molecular mechanisms underlying establishment and persistence of the reservoirs are needed. The cellular transcription factor KAP1 is known as a potent repressor of gene transcription. Here we report that KAP1 represses HIV-1 gene expression in myeloid cells including microglial cells, the major reservoir of the central nervous system. Mechanistically, KAP1 interacts and colocalizes with the viral transactivator Tat to promote its degradation via the proteasome pathway and repress HIV-1 gene expression. In myeloid models of latent HIV-1 infection, the depletion of KAP1 increased viral gene elongation and reactivated HIV-1 expression. Bound to the latent HIV-1 promoter, KAP1 associates and cooperates with CTIP2, a key epigenetic silencer of HIV-1 expression in microglial cells. In addition, Tat and CTIP2 compete for KAP1 binding suggesting a dynamic modulation of the KAP1 cellular partners upon HIV-1 infection. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of HIV-1 latency in myeloid cells.

The Ran-binding protein 2 (Ranbp2/Nup358) is a cytoplasmic and peripheral nucleoporin comprised of 4 Ran-GTP-binding domains (RBDs) that are interspersed among diverse structural domains with multifunctional activities. Our prior studies found that the RBD2 and RBD3 of Ranbp2 control mitochondrial motility independently of Ran-GTP-binding in cultured cells, whereas loss of Ran-GTP-binding to RBD2 and RBD3 are essential to support cone photoreceptor development and the survival of mature retinal pigment epithelium (RPE) in mice. Here, we uncover that loss of Ran-GTP-binding to RBD3 alone promotes the robust age-dependent increase of ubiquitylated substrates and S1 subunit (Pmsd1) of the 19S cap of the proteasome in the retina and RPE and that such loss in RBD3 also compromises the structural integrity of the outer segment compartment of cone photoreceptors only and without affecting the viability of these neurons. We also found that the E2-ligase and partner of Ranbp2, ubc9, is localized prominently in the mitochondrial-rich ellipsoid compartment of photoreceptors, where Ranbp2 is also known to localize with and modulate the activity of mitochondrial proteins. However, the natures of Ranbp2 and ubc9 isoforms to the mitochondria are heretofore elusive. Subcellular fractionation, co-immunolocalization and immunoaffinity purification of Ranbp2 complexes show that novel isoforms of Ranbp2 and ubc9 with molecular masses distinct from the large Ranbp2 and unmodified ubc9 isoforms localize specifically to the mitochondrial fraction or associate with mitochondrial components, whereas unmodified and SUMOylated Ran GTPase are excluded from the mitochondrial fraction. Further, liposome-mediated intracellular delivery of an antibody against a domain shared by the mitochondrial and nuclear pore isoforms of Ranbp2 causes the profound fragmentation of mitochondria and their delocalization from Ranbp2 and without affecting Ranbp2 localization at the nuclear pores. Collectively, the data support that Ran GTPase-dependent and independent and moonlighting roles of Ranbp2 or domains thereof and ubc9 control selectively age-dependent, neural-type and mitochondrial functions.