Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters.

Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.

Aberrant activation of five embryonic stem cell-specific genes robustly predicts a high risk of relapse in breast cancers.

BACKGROUND: In breast cancer, as in all cancers, genetic and epigenetic deregulations can result in out-of-context expressions of a set of normally silent tissue-specific genes. The activation of some of these genes in various cancers empowers tumours cells with new properties and drives enhanced proliferation and metastatic activity, leading to a poor survival prognosis. RESULTS: In this work, we undertook an unprecedented systematic and unbiased analysis of out-of-context activations of a specific set of tissue-specific genes from testis, placenta and embryonic stem cells, not expressed in normal breast tissue as a source of novel prognostic biomarkers. To this end, we combined a strict machine learning framework of transcriptomic data analysis, and successfully created a new robust tool, validated in several independent datasets, which is able to identify patients with a high risk of relapse. This unbiased approach allowed us to identify a panel of five biomarkers, DNMT3B, EXO1, MCM10, CENPF and CENPE, that are robustly and significantly associated with disease-free survival prognosis in breast cancer. Based on these findings, we created a new Gene Expression Classifier (GEC) that stratifies patients. Additionally, thanks to the identified GEC, we were able to paint the specific molecular portraits of the particularly aggressive tumours, which show characteristics of male germ cells, with a particular metabolic gene signature, associated with an enrichment in pro-metastatic and pro-proliferation gene expression. CONCLUSIONS: The GEC classifier is able to reliably identify patients with a high risk of relapse at early stages of the disease. We especially recommend to use the GEC tool for patients with the luminal-A molecular subtype of breast cancer, generally considered of a favourable disease-free survival prognosis, to detect the fraction of patients undergoing a high risk of relapse.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

AKR1B10, One of the Triggers of Cytokine Storm in SARS-CoV2 Severe Acute Respiratory Syndrome.

Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1ß, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.

The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of oral squamous cell carcinoma.

BACKGROUND: Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programmes. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test. METHODS: A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts. Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in an independent cohort using immunohistochemistry. RESULTS: A specific gene expression signature, including a combination of three genes, AREG, CCNA1 and DDX20, was found associated with high-risk OSCC in univariate and multivariate analyses. It was translated into an immunohistochemistry-based test, which successfully stratified patients of our own independent cohort. DISCUSSION: The exploration of the whole gene expression profile characterising aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting that CCNA1 could be a good target for therapeutic approaches.

Bromodomain-dependent stage-specific male genome programming by Brdt.

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a 'progenitor cells' gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.

A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.

HDAC6 controls the kinetics of platelet activation.

HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells.

Although there is now evidence that the expression of centromeric (CT) and pericentric (PCT) sequences are key players in major genomic functions, their transcriptional status in human cells is still poorly known. The main reason for this lack of data is the complexity and high level of polymorphism of these repeated sequences, which hampers straightforward analyses by available transcriptomic approaches. Here a transcriptomic macro-array dedicated to the analysis of CT and PCT expression is developed and validated in heat-shocked (HS) HeLa cells. For the first time, the expression status of CT and PCT sequences is analyzed in a series of normal and cancer human cells and tissues demonstrating that they are repressed in all normal tissues except in the testis, where PCT transcripts are found. Moreover, PCT sequences are specifically expressed in HS cells in a Heat-Shock Factor 1 (HSF1)-dependent fashion, and we show here that another independent pathway, involving DNA hypo-methylation, can also trigger their expression. Interestingly, CT and PCT were found illegitimately expressed in somatic cancer samples, whereas PCT were repressed in testis cancer, suggesting that the expression of CT and PCT sequences may represent a good indicator of epigenetic deregulations occurring in response to environmental changes or in cell transformation.

Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains.

BACKGROUND: We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form. RESULTS: Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway. CONCLUSION: Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.

Purification and Analysis of Male Germ Cells from Adult Mouse Testis.

Isolation of pools of spermatogenic cells at specific developmental stages is essential for the investigations of molecular events controlling critical transitions during spermatogenesis. Large-scale cell purification techniques allow for combined proteomics, genomics, and transcriptomics studies. Herein, we describe a procedure for the purification of meiotic and post-meiotic male germ cells from adult mouse testes. We also describe how the fractionated cell populations could be used for further studies. In our laboratory, these protocols are routinely used to specifically investigate the molecular basis of histone acetylation/acylation-driven epigenetic programming.

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching.

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells.

Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

Receptor-Independent Ectopic Activity of Prolactin Predicts Aggressive Lung Tumors and Indicates HDACi-Based Therapeutic Strategies.

AIMS: Ectopic activation of tissue-specific genes accompanies malignant transformation in many cancers. Prolactin (PRL) aberrant activation in lung cancer was investigated here to highlight its value as a biomarker. RESULTS: PRL is ectopically activated in a subset of very aggressive lung tumors, associated with a rapid fatal outcome, in our cohort of 293 lung tumor patients and in an external independent series of patients. Surprisingly PRL receptor expression was not detected in the vast majority of PRL-expressing lung tumors. Additionally, the analysis of the PRL transcripts in lung tumors and cell lines revealed systematic truncations of their 5' regions, including the signal peptide-encoding portions. PRL expression was found to sustain cancer-specific gene expression circuits encompassing genes that are normally responsive to hypoxia. Interestingly, this analysis also indicated that histone deacetylase (HDAC) inhibitors could counteract the PRL-associated transcriptional activity. INNOVATION AND CONCLUSION: Altogether, this work not only unravels a yet unknown oncogenic mechanism but also indicates that the specific category of PRL-expressing aggressive lung cancers could be particularly responsive to an HDAC inhibitor-based treatment.

A six gene expression signature defines aggressive subtypes and predicts outcome in childhood and adult acute lymphoblastic leukemia.

Abnormal gene expression in cancer represents an under-explored source of cancer markers and therapeutic targets. In order to identify gene expression signatures associated with survival in acute lymphoblastic leukemia (ALL), a strategy was designed to search for aberrant gene activity, which consists of applying several filters to transcriptomic datasets from two pediatric ALL studies. Six genes whose expression in leukemic blasts was associated with prognosis were identified:three genes predicting poor prognosis (AK022211, FASTKD1 and STARD4) and three genes associated with a favorable outcome (CAMSAP1, PCGF6 and SH3RF3). Combining the expression of these 6 genes could successfully predict prognosis not only in the two discovery pediatric ALL studies, but also in two independent validation cohorts of adult patients, one from a publicly available study and one consisting of 62 newly recruited Chinese patients. Moreover, our data demonstrate that our six gene based test is particularly efficient in stratifying MLL or BCR.ABL negative patients. Finally, common biological traits characterizing aggressive forms of ALL in both children and adults were found, including features of dormant hematopoietic stem cells, suggesting new therapeutic strategies.

Ectopic activation of germline and placental genes identifies aggressive metastasis-prone lung cancers.

Activation of normally silent tissue-specific genes and the resulting cell "identity crisis" are the unexplored consequences of malignant epigenetic reprogramming. We designed a strategy for investigating this reprogramming, which consisted of identifying a large number of tissue-restricted genes that are epigenetically silenced in normal somatic cells and then detecting their expression in cancer. This approach led to the demonstration that large-scale "off-context" gene activations systematically occur in a variety of cancer types. In our series of 293 lung tumors, we identified an ectopic gene expression signature associated with a subset of highly aggressive tumors, which predicted poor prognosis independently of the TNM (tumor size, node positivity, and metastasis) stage or histological subtype. The ability to isolate these tumors allowed us to reveal their common molecular features characterized by the acquisition of embryonic stem cell/germ cell gene expression profiles and the down-regulation of immune response genes. The methodical recognition of ectopic gene activations in cancer cells could serve as a basis for gene signature-guided tumor stratification, as well as for the discovery of oncogenic mechanisms, and expand the understanding of the biology of very aggressive tumors.

Molecular models for post-meiotic male genome reprogramming.

The molecular basis of post-meiotic male genome reorganization and compaction constitutes one of the last black boxes in modern biology. Although the successive transitions in DNA packaging have been well described, the molecular factors driving these near genome-wide reorganizations remain obscure. We have used a combination of different approaches aiming at the discovery of critical factors capable of directing the post-meiotic male genome reprogramming, which is now shedding new light on the nature of the fundamental mechanisms controlling post-meiotic histone replacement and genome compaction. Here we present a summary of these findings. The identification of the first factor capable of reading a precise combination of histone acetylation marks, BRDT, allowed highlighting a critical role for the genome-wide histone hyperacetylation that occurs before generalized histone replacement. In this context, the recent identification of a group of new histone variants capable of forming novel DNA packaging structures on specific regions during late spermatogenesis, when hyperacetylated histones are massively replaced in spermatids, also revealed the occurrence of a post-meiotic region-specific genome reprogramming. Additionally, the functional characterization of other molecular actors and chaperones in action in post-meiotic cells now allows one to describe the first general traits of the mechanisms underlying the structural transitions taking place during the post-meiotic reorganization and epigenetic reprogramming of the male genome.

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev.

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.