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Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Summary: Advances in high-throughput DNA sequencing technologies and decreasing costs have fueled the identification of small genetic variants (such as single nucleotide variants and indels) across tumors. Despite efforts to standardize variant formats and vocabularies, many sources of variability persist across databases and computational tools that annotate variants, hindering their integration within cancer genomic analyses. In this context, we present OpenVariant, an easily extendable Python package that facilitates seamless reading, parsing and refinement of diverse input file formats in a customizable structure, all within a single process., Availability and Implementation: OpenVariant is an open-source package available at https://github.com/bbglab/openvariant. Documentation may be found at https://openvariant.readthedocs.io., (© The Author(s) 2024. Published by Oxford University Press.)

Clonal hematopoiesis (CH) is a phenomenon of clonal expansion of hematopoietic stem cells driven by somatic mutations affecting certain genes. Recently, CH has been linked to the development of hematologic malignancies, cardiovascular diseases, and other conditions. Although the most frequently mutated CH driver genes have been identified, a systematic landscape of the mutations capable of initiating this phenomenon is still lacking. In this study, we trained machine learning models for 12 of the most recurrent CH genes to identify their driver mutations. These models outperform expert-curated rules based on prior knowledge of the function of these genes. Moreover, their application to identify CH driver mutations across almost half a million donors of the UK Biobank reproduces known associations between CH driver mutations and age, and the prevalence of several diseases and conditions. We thus propose that these models support the accurate identification of CH across healthy individuals. Significance: We developed and validated gene-specific machine learning models to identify CH driver mutations, showing their advantage with respect to expert-curated rules. These models can support the identification and clinical interpretation of CH mutations in newly sequenced individuals. See related commentary by Arends and Jaiswal, p. 1581., (©2024 The Authors; Published by the American Association for Cancer Research.)

Insulinomas are rare neuroendocrine tumors arising from pancreatic β cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in β cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of β cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pediatric cancers are rare diseases, and children without known germline predisposing conditions who develop a second malignancy during developmental ages are extremely rare. We present four such clinical cases and, through whole-genome and error-correcting ultra-deep duplex sequencing of tumor and normal samples, we explored the origin of the second malignancy in four children, uncovering different routes of development. The exposure to cytotoxic therapies was linked to the emergence of a secondary acute myeloid leukemia. A common somatic mutation acquired early during embryonic development was the driver of two solid malignancies in another child. In two cases, the two tumors developed from completely independent clones diverging during embryogenesis. Importantly, we demonstrate that platinum-based therapies contributed at least one order of magnitude more mutations per day of exposure than aging to normal tissues in these children., Significance: Using whole-genome and error-correcting ultra-deep duplex sequencing, we uncover different origins for second neoplasms in four children. We also uncover the presence of platinum-related mutations across 10 normal tissues of exposed individuals, highlighting the impact that the use of cytotoxic therapies may have on cancer survivors. See related commentary by Pacyna and Nangalia, p. 900. This article is featured in Selected Articles from This Issue, p. 897., (©2024 The Authors; Published by the American Association for Cancer Research.)

DNA base damage is a major source of oncogenic mutations 1 . Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation 2 . Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication 3,4 , we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts 5 . The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution., (© 2024. The Author(s).)

Resistance to MAPK inhibitors (MAPKi), the main cause of relapse in BRAF-mutant melanoma, is associated with the production of alternative BRAF mRNA isoforms (altBRAFs) in up to 30% of patients receiving BRAF inhibitor monotherapy. These altBRAFs have been described as being generated by alternative pre-mRNA splicing, and splicing modulation has been proposed as a therapeutic strategy to overcome resistance. In contrast, we report that altBRAFs are generated through genomic deletions. Using different in vitro models of altBRAF-mediated melanoma resistance, we demonstrate the production of altBRAFs exclusively from the BRAF V600E allele, correlating with corresponding genomic deletions. Genomic deletions are also detected in tumor samples from melanoma and breast cancer patients expressing altBRAFs. Along with the identification of altBRAFs in BRAF wild-type and in MAPKi-naive melanoma samples, our results represent a major shift in our understanding of mechanisms leading to the generation of BRAF transcripts variants associated with resistance in melanoma., Competing Interests: Declaration of interests F.A. received honoraria from MSD and Novartis. J.V. is a member of the scientific advisory boards of Remix Therapeutics, Stoke Therapeutics and IntronX., (Copyright © 2024 Center for Genomic Regulation. Published by Elsevier Inc. All rights reserved.)

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines., (© 2024. The Author(s).)

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

Clonal hematopoiesis (CH) is a phenomenon of clonal expansion of hematopoietic stem cells driven by somatic mutations affecting certain genes. Recently, CH has been linked to the development of a number of hematologic malignancies, cardiovascular diseases and other conditions. Although the most frequently mutated CH driver genes have been identified, a systematic landscape of the mutations capable of initiating this phenomenon is still lacking. Here, we train high-quality machine-learning models for 12 of the most recurrent CH driver genes to identify their driver mutations. These models outperform an experimental base-editing approach and expert-curated rules based on prior knowledge of the function of these genes. Moreover, their application to identify CH driver mutations across almost half a million donors of the UK Biobank reproduces known associations between CH driver mutations and age, and the prevalence of several diseases and conditions. We thus propose that these models support the accurate identification of CH across healthy individuals., Competing Interests: The authors declare no potential conflicts of interest.

Genetic information has been crucial to understand the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) at diagnosis and at relapse, but still nowadays has a limited value in a clinical context. Few genetic markers are associated with the outcome of T-ALL patients, independently of measurable residual disease (MRD) status after therapy. In addition, the prognostic relevance of genetic features may be modulated by the specific treatment used. We analyzed the genetic profile of 145 T-ALL patients by targeted deep sequencing. Genomic information was integrated with the clinicalbiological and survival data of a subset of 116 adult patients enrolled in two consecutive MRD-oriented trials of the Spanish PETHEMA (Programa Español de Tratamientos en Hematología) group. Genetic analysis revealed a mutational profile defined by DNMT3A/ N/KRAS/ MSH2/ U2AF1 gene mutations that identified refractory/resistant patients. Mutations in the DMNT3A gene were also found in the non-leukemic cell fraction of patients with T-ALL, revealing a possible mutational-driven clonal hematopoiesis event to prime T-ALL in elderly. The prognostic impact of this adverse genetic profile was independent of MRD status on day +35 of induction therapy. The combined worse-outcome genetic signature and MRD on day +35 allowed risk stratification of T-ALL into standard or high-risk groups with significantly different 5- year overall survival (OS) of 52% (95% confidence interval: 37-67) and 17% (95% confidence interval: 1-33), respectively. These results confirm the relevance of the tumor genetic profile in predicting patient outcome in adult T-ALL and highlight the need for novel gene-targeted chemotherapeutic schedules to improve the OS of poor-prognosis T-ALL patients.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS) high -B cell receptor (BCR) low -signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT., (© 2022. The Author(s).)

In celebration of the 20th anniversary of Nature Reviews Genetics, we asked 12 leading researchers to reflect on the key challenges and opportunities faced by the field of genetics and genomics. Keeping their particular research area in mind, they take stock of the current state of play and emphasize the work that remains to be done over the next few years so that, ultimately, the benefits of genetic and genomic research can be felt by everyone.

DYRK (dual-specificity tyrosine-regulated kinases) are an evolutionary conserved family of protein kinases with members from yeast to humans. In humans, DYRKs are pleiotropic factors that phosphorylate a broad set of proteins involved in many different cellular processes. These include factors that have been associated with all the hallmarks of cancer, from genomic instability to increased proliferation and resistance, programmed cell death, or signaling pathways whose dysfunction is relevant to tumor onset and progression. In accordance with an involvement of DYRK kinases in the regulation of tumorigenic processes, an increasing number of research studies have been published in recent years showing either alterations of DYRK gene expression in tumor samples and/or providing evidence of DYRK-dependent mechanisms that contribute to tumor initiation and/or progression. In the present article, we will review the current understanding of the role of DYRK family members in cancer initiation and progression, providing an overview of the small molecules that act as DYRK inhibitors and discussing the clinical implications and therapeutic opportunities currently available.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion 1,2 . Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

The discovery of drivers of cancer has traditionally focused on protein-coding genes 1-4 . Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers 6,7 , raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

In the version of this article initially published, the x axis on the fourth plot in Fig. 2e was incorrectly labeled "H3K36me3 exon-to-intron ratio (lower to higher)." The x axis on this plot should read "Genic H3K36me3 coverage bins (higher to lower)".

Purpose: Diffuse gliomas are the most common primary tumor of the brain and include different subtypes with diverse prognosis. The genomic characterization of diffuse gliomas facilitates their molecular diagnosis. The anatomical localization of diffuse gliomas complicates access to tumor specimens for diagnosis, in some cases incurring high-risk surgical procedures and stereotactic biopsies. Recently, cell-free circulating tumor DNA (ctDNA) has been identified in the cerebrospinal fluid (CSF) of patients with brain malignancies. Experimental Design: We performed an analysis of IDH1, IDH2, TP53, TERT , ATRX, H3F3A , and HIST1H3B gene mutations in two tumor cohorts from The Cancer Genome Atlas (TCGA) including 648 diffuse gliomas. We also performed targeted exome sequencing and droplet digital PCR (ddPCR) analysis of these seven genes in 20 clinical tumor specimens and CSF from glioma patients and performed a histopathologic characterization of the tumors. Results: Analysis of the mutational status of the IDH1, IDH2, TP53, TERT, ATRX, H3F3A , and HIST1H3B genes allowed the classification of 79% of the 648 diffuse gliomas analyzed, into IDH-wild-type glioblastoma, IDH-mutant glioblastoma/diffuse astrocytoma and oligodendroglioma, each subtype exhibiting diverse median overall survival (1.1, 6.7, and 11.2 years, respectively). We developed a sequencing platform to simultaneously and rapidly genotype these seven genes in CSF ctDNA allowing the subclassification of diffuse gliomas. Conclusions: The genomic analysis of IDH1, IDH2, TP53, ATRX, TERT, H3F3A , and HIST1H3B gene mutations in CSF ctDNA facilitates the diagnosis of diffuse gliomas in a timely manner to support the surgical and clinical management of these patients. Clin Cancer Res; 24(12); 2812-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

While recent studies have identified higher than anticipated heterogeneity of mutation rate across genomic regions, mutations in exons and introns are assumed to be generated at the same rate. Here we find fewer somatic mutations in exons than expected from their sequence content and demonstrate that this is not due to purifying selection. Instead, we show that it is caused by higher mismatch-repair activity in exonic than in intronic regions. Our findings have important implications for understanding of mutational and DNA repair processes and knowledge of the evolution of eukaryotic genes, and they have practical ramifications for the study of evolution of both tumors and species.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Distinguishing the driver mutations from somatic mutations in a tumor genome is one of the major challenges of cancer research. This challenge is more acute and far from solved for non-coding mutations. Here we present OncodriveFML, a method designed to analyze the pattern of somatic mutations across tumors in both coding and non-coding genomic regions to identify signals of positive selection, and therefore, their involvement in tumorigenesis. We describe the method and illustrate its usefulness to identify protein-coding genes, promoters, untranslated regions, intronic splice regions, and lncRNAs-containing driver mutations in several malignancies.

Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Acquisition of the arterial and haemogenic endothelium fates concurrently occur in the aorta-gonad-mesonephros (AGM) region prior to haematopoietic stem cell (HSC) generation. The arterial programme depends on Dll4 and the haemogenic endothelium/HSC on Jag1-mediated Notch1 signalling. How Notch1 distinguishes and executes these different programmes in response to particular ligands is poorly understood. By using two Notch1 activation trap mouse models with different sensitivity, here we show that arterial endothelial cells and HSCs originate from distinct precursors, characterized by different Notch1 signal strengths. Microarray analysis on AGM subpopulations demonstrates that the Jag1 ligand stimulates low Notch strength, inhibits the endothelial programme and is permissive for HSC specification. In the absence of Jag1, endothelial cells experience high Dll4-induced Notch activity and select the endothelial programme, thus precluding HSC formation. Interference with the Dll4 signal by ligand-specific blocking antibodies is sufficient to inhibit the endothelial programme and favour specification of the haematopoietic lineage.

The generation of B cells is a complex process requiring several cellular transitions, including cell commitment and differentiation. Proper transcriptional control to establish the genetic programs characteristic of each cellular stage is essential for the correct development of B lymphocytes. Deregulation of these particular transcriptional programs may result in a block in B-cell maturation, contributing to the development of hematological malignancies such as leukemia and lymphoma. However, very little is currently known about the role of transcriptional repressors in normal and aberrant B lymphopoiesis. Here we report that histone deacetylase 7 (HDAC7) is underexpressed in pro-B acute lymphoblastic leukemia (pro-B-ALL) and Burkitt lymphoma. Ectopic expression of HDAC7 induces apoptosis, leads to the downregulation of c-Myc and inhibits the oncogenic potential of cells in vivo, in a xenograft model. Most significantly, we have observed low levels of HDAC7 expression in B-ALL patient samples, which is correlated with the increased levels of c-Myc. From a mechanistic angle, we show that ectopically expressed HDAC7 localizes to the nucleus and interacts with the transcription factor myocyte enhancer factor C (MEF2C) and the corepressors HDAC3 and SMRT. Accordingly, both the HDAC7-MEF2C interaction domain as well as its catalytic domain are involved in the reduced cell viability induced by HDAC7. We conclude that HDAC7 has a potent anti-oncogenic effect on specific B-cell malignancies, indicating that its deregulation may contribute to the pathogenesis of the disease.

DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Extracting relevant information from large-scale data offers unprecedented opportunities in cancerology. We applied independent component analysis (ICA) to bladder cancer transcriptome data sets and interpreted the components using gene enrichment analysis and tumor-associated molecular, clinicopathological, and processing information. We identified components associated with biological processes of tumor cells or the tumor microenvironment, and other components revealed technical biases. Applying ICA to nine cancer types identified cancer-shared and bladder-cancer-specific components. We characterized the luminal and basal-like subtypes of muscle-invasive bladder cancers according to the components identified. The study of the urothelial differentiation component, specific to the luminal subtypes, showed that a molecular urothelial differentiation program was maintained even in those luminal tumors that had lost morphological differentiation. Study of the genomic alterations associated with this component coupled with functional studies revealed a protumorigenic role for PPARG in luminal tumors. Our results support the inclusion of ICA in the exploitation of multiscale data sets., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence., (© 2014 Guiu et al.)

The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.

The quiescent center (QC) maintains the activity of the surrounding stem cells within the root stem cell niche, yet specific molecular players sustaining the low rate of QC cell division remain poorly understood. Here, we identified a R2R3-MYB transcription factor, BRAVO (BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER), acting as a cell-specific repressor of QC divisions in the primary root of Arabidopsis. Ectopic BRAVO expression restricts overall root growth and ceases root regeneration upon damage of the stem cells, demonstrating the role of BRAVO in counteracting Brassinosteroid (BR)-mediated cell division in the QC cells. Interestingly, BR-regulated transcription factor BES1 (BRI1-EMS SUPRESSOR 1) directly represses and physically interacts with BRAVO in vivo, creating a switch that modulates QC divisions at the root stem cell niche. Together, our results define a mechanism for BR-mediated regulation of stem cell quiescence in plants., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression.

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.

MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes., Competing Interests: The authors have declared that no competing interests exist.

Autoimmune rheumatic diseases are complex disorders, whose etiopathology is attributed to a crosstalk between genetic predisposition and environmental factors. Both variants of autoimmune susceptibility genes and environment are involved in the generation of aberrant epigenetic profiles in a cell-specific manner, which ultimately result in dysregulation of expression. Furthermore, changes in miRNA expression profiles also cause gene dysregulation associated with aberrant phenotypes. In rheumatoid arthritis, several cell types are involved in the destruction of the joints, synovial fibroblasts being among the most important. In this study we performed DNA methylation and miRNA expression screening of a set of rheumatoid arthritis synovial fibroblasts and compared the results with those obtained from osteoarthritis patients with a normal phenotype. DNA methylation screening allowed us to identify changes in novel key target genes like IL6R, CAPN8 and DPP4, as well as several HOX genes. A significant proportion of genes undergoing DNA methylation changes were inversely correlated with expression. miRNA screening revealed the existence of subsets of miRNAs that underwent changes in expression. Integrated analysis highlighted sets of miRNAs that are controlled by DNA methylation, and genes that are regulated by DNA methylation and are targeted by miRNAs with a potential use as clinical markers. Our study enabled the identification of novel dysregulated targets in rheumatoid arthritis synovial fibroblasts and generated a new workflow for the integrated analysis of miRNA and epigenetic control., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth., Results: We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation., Conclusions: Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27.

14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting.

The E2F family of transcription factors regulates the expression of both genes associated with cell proliferation and genes that regulate cell death. The net outcome is dependent on cellular context and tissue environment. The mir-11 gene is located in the last intron of the Drosophila E2F1 homolog gene dE2f1, and its expression parallels that of dE2f1. Here, we investigated the role of miR-11 and found that miR-11 specifically modulated the proapoptotic function of its host gene, dE2f1. A mir-11 mutant was highly sensitive to dE2F1-dependent, DNA damage-induced apoptosis. Consistently, coexpression of miR-11 in transgenic animals suppressed dE2F1-induced apoptosis in multiple tissues, while exerting no effect on dE2F1-driven cell proliferation. Importantly, miR-11 repressed the expression of the proapoptotic genes reaper (rpr) and head involution defective (hid), which are directly regulated by dE2F1 upon DNA damage. In addition to rpr and hid, we identified a novel set of cell death genes that was also directly regulated by dE2F1 and miR-11. Thus, our data support a model in which the coexpression of miR-11 limits the proapoptotic function of its host gene, dE2f1, upon DNA damage by directly modulating a dE2F1-dependent apoptotic transcriptional program.

Several large ongoing initiatives that profit from next-generation sequencing technologies have driven--and in coming years will continue to drive--the emergence of long catalogs of missense single-nucleotide variants (SNVs) in the human genome. As a consequence, researchers have developed various methods and their related computational tools to classify these missense SNVs as probably deleterious or probably neutral polymorphisms. The outputs produced by each of these computational tools are of different natures and thus difficult to compare and integrate. Taking advantage of the possible complementarity between different tools might allow more accurate classifications. Here we propose an effective approach to integrating the output of some of these tools into a unified classification; this approach is based on a weighted average of the normalized scores of the individual methods (WAS). (In this paper, the approach is illustrated for the integration of five tools.) We show that this WAS outperforms each individual method in the task of classifying missense SNVs as deleterious or neutral. Furthermore, we demonstrate that this WAS can be used not only for classification purposes (deleterious versus neutral mutation) but also as an indicator of the impact of the mutation on the functionality of the mutant protein. In other words, it may be used as a deleteriousness score of missense SNVs. Therefore, we recommend the use of this WAS as a consensus deleteriousness score of missense mutations (Condel)., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Endocrine therapies targeting the proliferative effect of 17β-estradiol through estrogen receptor α (ERα) are the most effective systemic treatment of ERα-positive breast cancer. However, most breast tumors initially responsive to these therapies develop resistance through molecular mechanisms that are not yet fully understood. The long-term estrogen-deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibitors in postmenopausal women. To elucidate this resistance, genomic, transcriptomic and molecular data were integrated into the time course of MCF7-LTED adaptation. Dynamic and widespread genomic changes were observed, including amplification of the ESR1 locus consequently linked to an increase in ERα. Dynamic transcriptomic profiles were also observed that correlated significantly with genomic changes and were predicted to be influenced by transcription factors known to be involved in acquired resistance or cell proliferation (for example, interferon regulatory transcription factor 1 and E2F1, respectively) but, notably, not by canonical ERα transcriptional function. Consistently, at the molecular level, activation of growth factor signaling pathways by EGFR/ERBB/AKT and a switch from phospho-Ser118 (pS118)- to pS167-ERα were observed during MCF7-LTED adaptation. Evaluation of relevant clinical settings identified significant associations between MCF7-LTED and breast tumor transcriptome profiles that characterize ERα-negative status, early response to letrozole and tamoxifen, and recurrence after tamoxifen treatment. In accordance with these profiles, MCF7-LTED cells showed increased sensitivity to inhibition of FGFR-mediated signaling with PD173074. This study provides mechanistic insight into acquired resistance to endocrine therapies of breast cancer and highlights a potential therapeutic strategy.

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Background: Melon (Cucumis melo) is a horticultural specie of significant nutritional value, which belongs to the Cucurbitaceae family, whose economic importance is second only to the Solanaceae. Its small genome of approx. 450 Mb coupled to the high genetic diversity has prompted the development of genetic tools in the last decade. However, the unprecedented existence of a transcriptomic approaches in melon, highlight the importance of designing new tools for high-throughput analysis of gene expression., Results: We report the construction of an oligo-based microarray using a total of 17,510 unigenes derived from 33,418 high-quality melon ESTs. This chip is particularly enriched with genes that are expressed in fruit and during interaction with pathogens. Hybridizations for three independent experiments allowed the characterization of global gene expression profiles during fruit ripening, as well as in response to viral and fungal infections in plant cotyledons and roots, respectively. Microarray construction, statistical analyses and validation together with functional-enrichment analysis are presented in this study., Conclusion: The platform validation and enrichment analyses shown in our study indicate that this oligo-based microarray is amenable for future genetic and functional genomic studies of a wide range of experimental conditions in melon.