Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

PML nuclear bodies and chromatin dynamics: catch me if you can!

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment.

Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely "multiple-acute"). Viral genomes in the "multiple-acute" pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the "multiple-latency" pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.

The Tudor protein survival motor neuron (SMN) is a chromatin-binding protein that interacts with methylated lysine 79 of histone H3.

Spinal muscular atrophy (SMA) is a muscular disease characterized by the death of motoneurons, and is a major genetic cause of infant mortality. Mutations in the SMN1 gene, which encodes the protein survival motor neuron (SMN), are responsible for the disease. SMN belongs to the Tudor domain protein family, whose members are known to interact with methylated arginine (R) or lysine (K) residues. SMN has well-defined roles in the metabolism of small non-coding ribonucleoproteins (snRNPs) and spliceosome activity. We previously showed that SMN relocated to damaged interphase centromeres, together with the Cajal-body-associated proteins coilin and fibrillarin, during the so-called interphase centromere damage response (iCDR). Here we reveal that SMN is a chromatin-binding protein that specifically interacts with methylated histone H3K79, a gene expression- and splicing-associated histone modification. SMN relocation to damaged centromeres requires its functional Tudor domain and activity of the H3K79 methyltransferase DOT1L. In vitro pulldown assays showed that SMN interacts with H3K79me1,2 at its functional Tudor domain. Chromatin immunoprecipitation confirmed that SMN binds to H3K79me1,2-containing chromatin in iCDR-induced cells. These data reveal a novel SMN property in the detection of specific chromatin modifications, and shed new light on the involvement of a putative epigenetic dimension to the occurrence of SMA.

A novel cell response triggered by interphase centromere structural instability.

Interphase centromeres are crucial domains for the proper assembly of kinetochores at the onset of mitosis. However, it is not known whether the centromere structure is under tight control during interphase. This study uses the peculiar property of the infected cell protein 0 of herpes simplex virus type 1 to induce centromeric structural damage, revealing a novel cell response triggered by centromere destabilization. It involves centromeric accumulation of the Cajal body-associated coilin and fibrillarin as well as the survival motor neuron proteins. The response, which we have termed interphase centromere damage response (iCDR), was observed in all tested human and mouse cells, indicative of a conserved mechanism. Knockdown cells for several constitutive centromere proteins have shown that the loss of centromeric protein B provokes the centromeric accumulation of coilin. We propose that the iCDR is part of a novel safeguard mechanism that is dedicated to maintaining interphase centromeres compatible with the correct assembly of kinetochores, microtubule binding, and completion of mitosis.

Functional interaction between class II histone deacetylases and ICP0 of herpes simplex virus type 1.

This study describes the physical and functional interactions between ICP0 of herpes simplex virus type 1 and class II histone deacetylases (HDACs) 4, 5, and 7. Class II HDACs are mainly known for their participation in the control of cell differentiation through the regulation of the activity of the transcription factor MEF2 (myocyte enhancer factor 2), implicated in muscle development and neuronal survival. Immunofluorescence experiments performed on transfected cells showed that ICP0 colocalizes with and reorganizes the nuclear distribution of ectopically expressed class I and II HDACs. In addition, endogenous HDAC4 and at least one of its binding partners, the corepressor protein SMRT (for silencing mediator of retinoid and thyroid receptor), undergo changes in their nuclear distribution in ICP0-transfected cells. As a result, during infection endogenous HDAC4 colocalizes with ICP0. Coimmunoprecipitation and glutathione S-transferase pull-down assays confirmed that class II but not class I HDACs specifically interacted with ICP0 through their amino-terminal regions. This region, which is not conserved in class I HDACs but homologous to the MITR (MEF2-interacting transcription repressor) protein, is responsible for the repression, in a deacetylase-independent manner, of MEF2 by sequestering it under an inactive form in the nucleus. Consequently, we show that ICP0 is able to overcome the HDAC5 amino-terminal- and MITR-induced MEF2A repression in gene reporter assays. This is the first report of a viral protein interacting with and controlling the repressor activity of class II HDACs. We discuss the putative consequences of such an interaction for the biology of the virus both during lytic infection and reactivation from latency.