Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation.

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (fdark) in the ensemble. To date, fdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine fdark values between 30 and 60% for the different VFPs. The high values for fdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.

IL-1ß primed mesenchymal stromal cells moderate hemorrhagic shock-induced organ injuries.

BACKGROUND: Organ damages following hemorrhagic shock (HS) have been partly attributed to an immunological dysfunction. The current challenge in the management of HS patients is to prevent organ injury-induced morbidity and mortality which currently has not etiological treatment available. Mesenchymal stromal cells (MSC) are used in clinical cell therapy for immunomodulation and tissue repair. In vitro priming is often used to improve the immunomodulation efficiency of MSC before administration. OBJECTIVE: Assess the effect of naive MSC (MSCn) or interleukin (IL)-1ß primed (MSCp) treatment in a context of HS-induced organ injury. METHODS: Rats underwent fixed pressure HS and were treated with allogenic MSCn or MSCp. Liver and kidney injuries were evaluated 6h later by histological and biochemical analysis. Whole blood was collected to measure leukocytes phenotypes. Then, in vitro characterization of MSCn or MSCp was carried out. RESULTS: Plasma creatinine, blood urea nitrogen, and cystatin C were decrease by MSCp infusion as well as kidney injury molecule (KIM)-1 on histological kidney sections. Transaminases, GGT, and liver histology were normalized by MSCp. Systemic cytokines (IL-1α, IL-6, and IL-10) as well as CD80, 86, and PD-1/PDL-1 axis were decreased by MSCp on monocytes and granulocytes. In vitro, MSCp showed higher level of secreted immunomodulatory molecules than MSCn. CONCLUSION: An early administration of MSCp moderates HS-induced kidney and liver injury. IL-1ß priming improves MSC efficiency by promoting their immunomodulatory activity. These data provide proof of concept that MSCp could be a therapeutic tool to prevent the appearance of organs injury following HS.

Bromodomain factors of BET family are new essential actors of pericentric heterochromatin transcriptional activation in response to heat shock.

The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).

Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains.

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in-frame fusion of the double bromodomain protein, BRD4, with a testis-specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4-NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient-derived cell line, we show that p300 sequestration into the BRD4-NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4-NUT released p300 and restored p53-dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off-context activity of a testis-specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation.

Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes.

Astroglial reactivity associated with increased production of NFkappaB-dependent proinflammatory molecules is an important component of the pathophysiology of chronic neurological disorders such as multiple sclerosis (MS). The use of estrogens as potential anti-inflammatory and neuroprotective drugs is a matter of debate. Using mouse experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we report that implants reproducing pregnancy levels of 17beta-estradiol (E2) alleviate ongoing disease and decrease astrocytic production of CCL2, a proinflammatory chemokine that drives the local recruitment of inflammatory myeloid cells. Immunohistochemistry and confocal imaging reveal that, in spinal cord white matter EAE lesions, reactive astrocytes express estrogen receptor (ER)alpha (and to a lesser extent ERbeta) with a preferential nuclear localization, whereas other cells including infiltrated leukocytes express ERs only in their membranes or cytosol. In cultured rodent astrocytes, E2 or an ERalpha agonist, but not an ERbeta agonist, inhibits TNFalpha-induced CCL2 expression at nanomolar concentrations, and the ER antagonist ICI 182,170 blocks this effect. We show that this anti-inflammatory action is not associated with inhibition of NFkappaB nuclear translocation but rather involves direct repression of NFkappaB-dependent transcription. Chromatin immunoprecipitation assays further indicate that estrogen suppresses TNFalpha-induced NFkappaB recruitment to the CCL2 enhancer. These data uncover reactive astrocytes as an important target for nuclear ERalpha inhibitory action on chemokine expression and suggest that targeting astrocytic nuclear NFkappaB activation with estrogen receptor alpha modulators may improve therapies of chronic neurodegenerative disorders involving astroglial neuroinflammation.

HMGB4, a novel member of the HMGB family, is preferentially expressed in the mouse testis and localizes to the basal pole of elongating spermatids.

We identified HMGB4, a novel member of the HMGB family lacking the acidic tail typically found in this family. HMGB4 is strongly and preferentially expressed in the adult mouse testis and weakly in the brain, but not in many other tissues. HMGB4 associates with chromatin, and in transfection assays, in contrast to HMGB1, it acts as a potent transcriptional repressor. During spermatogenesis, HMGB4 is present in the euchromatin of late pachytene spermatocytes and haploid round spermatids, whereas stronger expression is observed during the elongation phase, where it localizes to the basal pole of the nucleus in a manner mutually exclusive with H1FNT (H1T2) localized at the apical pole. HMGB4 basal localization is lost in H1FNT-mutant spermatids, showing that H1FNT provides a positional cue for organizing chromatin domains within the nucleus. These results show that HMGB4 and H1FNT specify distinct chromatin domains at the apical and basal poles of the elongating spermatid nucleus.

Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization.

Before fertilization, the genome packaging of male and female gametes is very different. Indeed, whereas the female haploid genome is associated with histones in a somatic-like chromatin structure, most of the male genome is tightly bound to protamines. However, it has recently been demonstrated that the pericentric heterochromatin regions of the male genome are associated with specific H2A-like histone variants, named H2AL1 and H2AL2, suggesting a heterogeneous organization. The fate and role of the sex-specific genome packaging transmitted by germinal cells to the embryo are not well understood. The aim of the present study was to follow reprogramming of the parental genomes in early embryos after in vivo fertilization. We show here that two typical epigenetic markers, trimethylated lysine 9 of histone H3 (TriMethylH3K9) and acetylated H4, are asymmetrically distributed between the parental genomes in one-cell mouse embryos, confirming data from embryos obtained after intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Indeed, whereas the maternal genome is highly enriched with trimethylH3K9, this mark is not detected in the paternal genome. On the contrary, histone H4 incorporated in the paternal genome is highly acetylated at an early stage, while in the maternal pronucleus, the level of acetylated H4 remains low in early one-cell embryos and becomes enriched at a later stage. Moreover, our results suggest a very quick disappearance of histone H2A variants H2AL1 and H2Al2 from the paternal pericentric heterochromatin regions after sperm-egg fusion.

HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates.

A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation.

Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis.

During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.

Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis.

HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.

Structural characterization of the histone variant macroH2A.

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses.

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Testis-specific histone H3 expression in somatic cells.

Histone variants functionally differentiate individual nucleosomes and, hence, act as key regulators of chromatin structure and function. Large-scale proteomic projects are now valuable sources of histone-variant discovery, showing, in particular, that somatic mammalian cells express a larger panel of histone H3 variants than previously thought, including testis-specific variants and as yet uncharacterized species. These data also suggest a tight relationship between the complexity of histone-variant expression and physiopathological states of the cells.

How to pack the genome for a safe trip.

The transformation of the somatic chromatin into a unique and highly compact structure occurring during the post-meiotic phase of spermatogenesis is one of the most dramatic known processes of chromatin remodeling. Paradoxically, no information is available on the mechanisms controlling this specific reorganization of the haploid cell genome. The only existing hints suggest a role for histone variants, as well as for stage-specific post-translational histone modifications,before and during the incorporation of testis-specific basic nuclear proteins. Moreover, the exact functions of the latter remain obscure. This chapter summarizes the major chromatin-associated events taking place during the post-meiotic differentiation of male haploid cells in mammals and discusses some of the basic issues that remain to be solved to finally understand chromatin remodeling during spermatogenesis.

Cellular uptake of the EBV transcription factor EB1/Zta.

A number of viral proteins have the property to penetrate into the cells when present in the extra-cellular compartment. Here, we report that the Epstein-Barr virus (EBV) transcriptional activator EB1/Zta, which is responsible for the activation of the EBV lytic replication, binds to lymphoid cells surface, is efficiently translocated and accumulates in the nucleus. The internalization of EB1/Zta is energy-dependent and shares common features with endocytosis. As the EB1/Zta was not degraded in the cells and reached the nucleus, the potential effect of its internalisation on viral reactivation was assessed.

Regulatory cross-talk between lysine acetylation and ubiquitination: role in the control of protein stability.

It is now becoming apparent that cross-talk between two protein lysine modifications, acetylation and ubiquitination, is a critical regulatory mechanism controlling vital cellular functions. The most apparent effect is the inhibition of proteasome-mediated protein degradation by lysine acetylation. Analysis of the underlying mechanisms, however, shows that, besides a direct competition between the two lysine modifications, more complex and indirect processes also connect these two signalling pathways. These findings point to protein lysine acetylation as a potential regulator of various cellular functions involving protein ubiquitination.

Establishment of male-specific epigenetic information.

The setting of male-specific epigenetic information is a complex process, which involves a major global re-organisation, as well as localized changes of the nucleus structure during the pre-meiotic, meiotic and post-meiotic stages of the male germ cell differentiation. Although it has long been known that DNA methylation in targeted regions of the genome is associated with male-specific genomic imprinting, or that most core histones are hyperacetylated and then replaced by sperm-specific proteins during the post-meiotic condensation of the nucleus, many questions remain unanswered. How these changes interact, how they affect the epigenetic information and how the paternal epigenetic marks contribute to the future genome are indeed major issues remaining to be explored.

Role of the histone acetyl transferase Tip60 in the p53 pathway.

The histone acetyl transferase Tip60 (HTATIP) shares many properties with the tumor suppressor p53 (TP53). Both proteins are involved in the cellular response to DNA damage, are subjected to proteasomal digestion following Mdm2-mediated ubiquitination, and accumulate after UV irradiation. We found here that knock-down of Tip60 affects the p53-dependent response following actinomycin D treatment, most likely because it inhibits p21 (CDKN1A) accumulation. Moreover, Tip60 is required for p53 to activate the endogenous p21 promoter, suggesting that it functions as a p53 co-activator. However, we also found that knock-down of Tip60 increases the turnover rate of p53 under normal growth conditions. Tip60 interferes with Mdm2-mediated degradation of p53, probably because it affects its subcellular localization. Taken together, our results suggest that Tip60 plays a double role in the p53 pathway: under normal growth conditions, Tip60 contributes to maintain a basal pool of p53 by interfering with its degradation; following DNA damage, Tip60 functions as p53 co-activator. That these two distinct roles are linked during the p53-dependent response is an attractive hypothesis.

The role of histones in chromatin remodelling during mammalian spermiogenesis.

One of the most dramatic chromatin remodelling processes takes place during mammalian spermatogenesis. Indeed, during the postmeiotic maturation of male haploid germ cells, or spermiogenesis, histones are replaced by small basic proteins, which in mammals are transition proteins and protamines. However, nothing is known of the mechanisms controlling the process of histone replacement. Two hints from the literature could help to shed light on the underlying molecular events: one is the massive synthesis of histone variants, including testis-specific members, and the second is a stage specific post-translational modification of histones. A new testis-specific 'histone code' can therefore be generated combining both histone variants and histone post-translational modifications. This review will detail these two phenomena and discuss possible functional significance of the global chromatin alterations occurring prior to histone replacement during spermiogenesis.

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.