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Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents., It has been proposed that resistance to targeted therapies in non-small cell lung carcinoma (NSCLC) is due to a nonhomogeneous cell population. Here the authors analyse preclinical NSCLC models using single-cell RNA-seq and identify drug tolerant cell states and subpopulations, as well as associated genes.

Background Due to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen. Results Two targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against β-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains. Conclusions The findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.

Background Although it is becoming evident that individual’s immune system has a decisive influence on SARS-CoV-2 disease progression, pathogenesis is largely unknown. In this study, we aimed to profile the host transcriptome of COVID-19 patients from nasopharyngeal samples along with virus genomic features isolated from respective host, and a comparative analyses of differential host responses in various SARS-CoV-2 infection systems. Results Unique and rare missense mutations in 3C-like protease observed in all of our reported isolates. Functional enrichment analyses exhibited that the host induced responses are mediated by innate immunity, interferon, and cytokine stimulation. Surprisingly, induction of apoptosis, phagosome, antigen presentation, hypoxia response was lacking within these patients. Upregulation of immune and cytokine signaling genes such as CCL4, TNFA, IL6, IL1A, CCL2, CXCL2, IFN, and CCR1 were observed in lungs. Lungs lacked the overexpression of ACE2 as suspected, however, high ACE2 but low DPP4 expression was observed in nasopharyngeal cells. Interestingly, directly or indirectly, viral proteins specially non-structural protein mediated overexpression of integrins such as ITGAV, ITGA6, ITGB7, ITGB3, ITGA2B, ITGA5, ITGA6, ITGA9, ITGA4, ITGAE, and ITGA8 in lungs compared to nasopharyngeal samples suggesting the possible way of enhanced invasion. Furthermore, we found comparatively highly expressed transcription factors such as CBP, CEBP, NFAT, ATF3, GATA6, HDAC2, TCF12 which have pivotal roles in lung injury. Conclusions Even though this study incorporates a limited number of cases, our data will provide valuable insights in developing potential studies to elucidate the differential host responses on the viral pathogenesis in COVID-19, and incorporation of further data will enrich the search of an effective therapeutics.

The constant rise of the death toll and cases of COVID-19 has made this pandemic a serious threat to human civilization. Understanding of host-SARS-CoV-2 interaction in viral pathogenesis is still in its infancy. In this study, we utilized a blend of computational and knowledgebase approaches to model the putative virus-host interplay in host signaling pathways by integrating the experimentally validated host interactome proteins and differentially expressed host genes in SARS-CoV-2 infection. While searching for the pathways in which viral proteins interact with host proteins, we discovered various antiviral immune response pathways such as hypoxia-inducible factor 1 (HIF-1) signaling, autophagy, retinoic acid-inducible gene I (RIG-I) signaling, Toll-like receptor signaling, fatty acid oxidation/degradation, and IL-17 signaling. All these pathways can be either hijacked or suppressed by the viral proteins, leading to improved viral survival and life cycle. Aberration in pathways such as HIF-1 signaling and relaxin signaling in the lungs suggests the pathogenic lung pathophysiology in COVID-19. From enrichment analysis, it was evident that the deregulated genes in SARS-CoV-2 infection might also be involved in heart development, kidney development, and AGE-RAGE signaling pathway in diabetic complications. Anomalies in these pathways might suggest the increased vulnerability of COVID-19 patients with comorbidities. Moreover, we noticed several presumed infection-induced differentially expressed transcription factors and epigenetic factors, such as miRNAs and several histone modifiers, which can modulate different immune signaling pathways, helping both host and virus. Our modeling suggests that SARS-CoV-2 integrates its proteins in different immune signaling pathways and other cellular signaling pathways for developing efficient immune evasion mechanisms while leading the host to a more complicated disease condition. Our findings would help in designing more targeted therapeutic interventions against SARS-CoV-2.

Background Cervical cancer is a gynecologic cancer type that develops in the cervix, accounting for 8% mortality of all female cancer patients. Infection with specific human papillomavirus (HPV) types is considered the most severe risk factor for cervical cancer. In the context of our socioeconomic conditions, an increasing burden of this disease and high mortality rate prevail in Bangladesh. Although several researches related to the epidemiology, HPV vaccination, and treatment modalities were conducted, researches on the mutation profiles of marker genes in cervical cancer in Bangladesh remain unexplored. Methods In this study, five different genomic regions within the top three most frequently mutated genes (EGFR, KRAS and PIK3CA) in COSMIC database with a key role in the development of cervical cancers were selected to study the mutation frequency in Bangladeshi patients. In silico analysis was done in two steps: nucleotide sequence analysis and its corresponding amino acid analysis. Results DNA from 46 cervical cancer tissue samples were extracted and amplified by PCR, using 1 set of primers designed for EGFR and 2 sets of primers designed for two different regions of both PIK3CA and KRAS gene. In total, 39 mutations were found in 26 patient samples. Eleven different mutations (23.91%), twenty-four different mutations (52.17%) and four mutations (8.7%) were found in amplified EGFR, PIK3CA and KRAS gene fragments, respectively; among which 1 (EGFR) was common in seven patient samples and 2 (PIKCA) were found in more than 1 patient. Our study shows that except for KRAS, the frequency of observed mutations in our patients is higher than those reported earlier in other parts of the world. Most of the exonic mutations were found only in the PIK3CA and EGFR genes. Conclusions The study can be used as a basis to build a mutation database for cervical cancer in Bangladesh with the possibility of targetable oncogenic mutations. Further explorations are needed to establish future diagnostics, personalized medicine decisions, and other pharmaceutical applications for specific cancer subtypes.

Background MicroRNAs are ~ 22-nucleotide-long biological modifiers that act as the post-transcriptional modulator of gene expression. Some of them are identified to be embedded within the introns of protein-coding genes, these miRNAs are called the intronic miRNAs. Previous findings state that these intronic miRNAs are co-expressed with their host genes. This co-expression is necessary to maintain the robustness of the biological system. Till to date, only a few experiments are performed discretely to elucidate the functional relationship between few co-expressed intronic miRNAs and their associated host genes. Results In this study, we have interpreted the underlying modulatory mechanisms of intronic miRNA hsa-miR-933 on its target host gene ATF2 and found that aberration can lead to several disease conditions. A protein-protein interaction network-based approach was adopted, and functional enrichment analysis was performed to elucidate the significantly over-represented biological functions and pathways of the common targets. Our approach delineated that hsa-miR-933 might control the hyperglycemic condition and hyperinsulinism by regulating ATF2 target genes MAP4K4, PRKCE, PEA15, BDNF, PRKACB, and GNAS which can otherwise lead to the development of type II diabetes mellitus. Moreover, we showed that hsa-miR-933 can regulate a target of ATF2, brain-derived neurotrophic factor (BDNF), to modulate the optimal expression of ATF2 in neuron cells to render neuroprotection for the inhibition of neurodegenerative diseases. Conclusions Our in silico model provides interesting resources for experimentations in a model organism or cell line for further validation. These findings may extend the common perception of gene expression analysis with new regulatory functionality.

Studies in three mouse models of breast cancer identified profound discrepancies between cell autonomous and systemic Akt1 or Akt2 deletion on breast cancer tumorigenesis and metastasis. First, unlike systemic Akt1 deletion, which inhibits metastasis, cell autonomous Akt1 deletion does not. Second, systemic Akt2 deletion does not inhibit mammary tumorigenesis and metastasis, but cell autonomous Akt2 deletion eliminates ErbB2 expressing cells in the mammary gland and prevents tumorigenesis. However, the elevation in insulin by Akt2 systemic deletion hyperactivates tumor Akt, enabling ErbB2 expression, and exacerbates mammary tumorigenesis. Decreasing insulin level inhibits accelerated tumorigenesis by systemic Akt2 deletion. Single cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing the survival and mobilization of tumor-associated neutrophils. Importantly, neutrophil-specific deletion of Akt1 is sufficient to exert resistance to metastasis. The results underscore the importance of determining systemic effects rather than cell autonomous effects as a proof of concept for cancer therapy.

A detailed understanding of the molecular mechanism of SARS-CoV-2 pathogenesis is still elusive, and there is a need to address its deadly nature and to design effective therapeutics. Here, we present a study that elucidates the interplay between the SARS-CoV and SARS-CoV-2 viruses' and host's miRNAs, an epigenetic regulator, as a mode of pathogenesis; and we explored how the SARS-CoV and SARS-CoV-2 infections differ in terms of their miRNA-mediated interactions with the host and the implications this has in terms of disease complexity. We have utilized computational approaches to predict potential host and viral miRNAs and their possible roles in different important functional pathways. We have identified several putative host antiviral miRNAs that can target the SARS viruses and also predicted SARS viruses-encoded miRNAs targeting host genes. In silico predicted targets were also integrated with SARS-infected human cell microarray and RNA-seq gene expression data. A comparison between the host miRNA binding profiles on 67 different SARS-CoV-2 genomes from 24 different countries with respective country's normalized death count surprisingly uncovered some miRNA clusters, which are associated with increased death rates. We have found that induced cellular miRNAs can be both a boon and a bane to the host immunity, as they have possible roles in neutralizing the viral threat; conversely, they can also function as proviral factors. On the other hand, from over representation analysis, our study revealed that although both SARS-CoV and SARS-CoV-2 viral miRNAs could target broad immune-signaling pathways; only some of the SARS-CoV-2 miRNAs are found to uniquely target some immune-signaling pathways, such as autophagy, IFN-I signaling, etc., which might suggest their immune-escape mechanisms for prolonged latency inside some hosts without any symptoms of COVID-19. Furthermore, SARS-CoV-2 can modulate several important cellular pathways that might lead to the increased anomalies in patients with comorbidities like cardiovascular diseases, diabetes, breathing complications, etc. This might suggest that miRNAs can be a key epigenetic modulator behind the overcomplications amongst the COVID-19 patients. Our results support that miRNAs of host and SARS-CoV-2 can indeed play a role in the pathogenesis which can be further concluded with more experiments. These results will also be useful in designing RNA therapeutics to alleviate the complications from COVID-19.

BackgroundSince December 2019, the world is experiencing an unprecedented crisis due to a novel coronavirus, SARS-CoV-2. Owing to poor understanding of pathogenicity, the virus is eluding treatment and complicating recovery. Regulatory roles of long non-coding RNAs (lncRNAs) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. To elucidate possible functions of lncRNAs in the COVID-19 pathobiology, we have utilized RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells.ResultsOur analyses uncover 21 differentially expressed lncRNAs whose functions are broadly involved in cell survival and regulation of gene expression. By network enrichment analysis we find that these lncRNAs can directly interact with differentially expressed protein-coding genes ADAR, EDN1, KYNU, MALL, TLR2 and YWHAG; and also AKAP8L, EXOSC5, GDF15, HECTD1, LARP4B, LARP7, MIPOL1, UPF1, MOV10 and PRKAR2A, host genes that interact with SARS-CoV-2 proteins. These genes are involved in cellular signaling, metabolism, immune response and RNA homeostasis. Since lncRNAs have been known to sponge microRNAs and protect expression of upregulated genes, we also identified 9 microRNAs that are induced in viral infections; however, some lncRNAs are able to block their usual suppressive effect on overexpressed genes and consequently contribute to host defense and cell survival.ConclusionsOur investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel RNA therapeutics as a treatment option.

Amongst the anti-TNF-α therapy for rheumatoid arthritis and other autoimmune diseases, Adalimumab mAb is one of the best candidates. However, several risk factors are found to be associated with higher doses. Improvement of the binding properties will therefore significantly increase its therapeutic efficacy, reduce the dosage requirements, and ultimately the associated toxicity and treatment cost. Here, we proposed a systematic in silico approach of finding newer mAb variants with improved binding properties. Using various bioinformatics tools, we have identified the significant amino acid residues on Adalimumab mAb. Next, we searched for the suitability of the other residues for mutating the significant residues and from the combinations of suitable mutations, variants were designed. To find the most significant ones, binding properties of the variants were compared with the wild type Adalimumab mAb using molecular docking scrutiny and molecular dynamics simulation. Finally, structural properties between the variant and wild type were analyzed. We have identified the six most significant residues on Adalimumab mAb involved in the antigen-antibody interactions. Using the suitable mutations replacing each of these residues, we have modeled 143 variants. From several docking analyses, we have found five significant variants and after molecular dynamics simulation, one most significant variant with improved binding affinity was identified whose structural properties are similar to the wild type Adalimumab mAb. Designed variant from this study, may provide newer insights on the structure-based affinity improvements of monoclonal antibodies and likewise modifications of the Fc region will also improve the therapeutic effector functions of antibodies too.

The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single cell RNA-sequencing and identify Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved.

Since the first recorded case of the SARS-CoV-2, it has acquired several mutations in its genome while spreading throughout the globe. In this study, we investigated the significance of these mutations by analyzing the host miRNA binding and virus's internal ribosome entry site (IRES). Strikingly, we observed that due to the acquired mutations, five host miRNAs which lost their affinity for targeting the viral genome, and another five can target the mutated viral genome. Moreover, functional enrichment analysis suggests that targets of both of these miRNAs might be involved in various host immune signaling pathways. Remarkably, we detected that three particular mutations in the IRES can disrupt its secondary structure which can further make the virus less functional. These results could be valuable in exploring the functional importance of the mutations of SARS-CoV-2 and could provide novel insights into the differences observed different parts of the world.

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

In Drosophila, the wing disc‐associated muscle precursor cells give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single‐cell RNA‐sequencing experiments and built a cell atlas of myoblasts associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM myoblasts that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of myoblasts, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM myoblasts at various states of differentiation by immunofluorescence and genetic cell‐tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Amalgam which is functionally important in muscle development. Our work provides a framework for leveraging scRNA‐seq for gene discovery and details a strategy that can be applied to other scRNA‐seq datasets., A single‐cell atlas of the Drosophila wing disc identifies the transcriptional profiles and markers for diverse cell types. The myoblasts exhibit distinct Notch‐dependent states of differentiation, and diversification into fiber fate specification.

Long intergenic noncoding RNAs (lincRNAs) are more than 200 bases long, transcribed from intergenic genomic regions and do not undergo translation. They have regulatory roles in differentiation and development. However, how their transcription is activated and how their expression is differentially modulated in differentiation is quite unclear. In this study, we explored and analyzed data at the transcriptomic and epigenetic level to address these questions. Here, we identified novel lincRNAs that are differentially expressed in neuronal and hematopoietic differentiation and showed that such differential modulations are achieved under epigenetic regulations. lincRNAs that are upregulated in mature cells than in progenitor are activated from a bivalent poised state where activating H3K4me3/H3K9ac/H3K27ac and suppressive H3K9me3/H3K27me3 marks are colocalized. And, lincRNAs that are downregulated in mature cells after differentiation are suppressed by the addition of H3K9me3/H3K27me3 marks. Moreover, here we show a tissue-specific expression pattern of lincRNAs in various cell lines and normal tissues. The study reveals bidirectional histone marks as an epigenetic means of directing the differential expression of lincRNAs which are found to be involved in the process of cellular differentiation.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). Accurate and reliable diagnosis of IEMs by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-offs of the analytes. This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. A total of 570 enrolled healthy participants were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days–7 years, and (3) 8–17 years, to establish the age-specific cut-offs for AAs and ACs. Also, 273 suspected patients with IEMs were enrolled to evaluate the reliability of the established cut-off values. Quantitation of AAs and ACs was performed on an automated LC-MS/MS system using dried blood spot (DBS) cards. Then the specimens of the enrolled clinically suspected patients were analyzed by the established method. Nine patients came out as screening positive for different IEMs, including two borderline positive cases of medium-chain acyl-CoA dehydrogenase deficiency (MCAD). A second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography- mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis. In conclusion, along with establishment of a validated LC-MS/MS method for quantitation of AAs and ACs from the DBS cards, the study also demonstrates the presence of predominantly available IEMs in Bangladesh.

Aim: In silico approach is used to identify most potent epitope and drug against pathogenic Hantavirus against which no approved therapeutics exist. Methods: Nucleocapsid protein sequences were retrieved, aligned and conserved regions were analyzed for the presence of B- and T-cell epitopes, and pockets for potential drugs. Results: T-cell epitope SYLRRTQSM and B-cell epitopes SYLRRTQ and YLRRTQSM appeared to be highly conserved, antigenic, nonallergenic. The T-cell epitope bound to maximum alleles. Thus, SYLRRTQSM would likely elicit both T- and B-cell immunity. High-throughput screening of Traditional Chinese Medicine database by docking technique revealed a potential drug, compound 46547 (1R,11S,15S,18S,20S,21R,22S)-12-oxa-8,17-diazaheptacyclo[15.5.2.0^{1,18}.0^{2,7}.0^{8,22}.0^{11,21}.0^{15,20}]tetracosa-2,4,6-trien-9-one. Conclusion: Our results predict potential therapeutics against multiple strains of pathogenic Hantavirus, but requires validation by in vivo experimentation.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life. We thank Drs. Richard Harvey (University of Sydney, Australia) and Michael Schneider (Imperial College, London, UK) for providing Nkx2.5cre/+ and α-MHCcre/+ transgenic lines, respectively, Dr. S. Bartlett for English editing, and Gemma Benito and Ana Guío for technical assistance. J.M.R. is supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad; SAF 2012 34296 and SAF 2015 63633-R), the Spanish Ministry of Health (Ministerio de Sanidad y Consumo) Red de Investigación Cardiovascular (RIC; grant RD06/0042/0022), and the Fundación La Marató TV3 (264/C/2012). The RIC also supports J.L.P. (RD12/0042/0005) and L.J.J.-B. (RD12/0042/0056). RIC grants are partially funded by FEDER funds. P.M.-C., J.A.E., and J.L.P. are supported by SAF2012-38547, SAF2015-67369-R, SAF2012-1207, and SAF2013-45543-R, respectively. E.P. and P.M.-C. were also supported by SAF2015-67369-R. NGS experiments were performed at the CNIC Genomics Unit. IF of adult hearts was performed at the Advanced Fluorescence Microscopy Unit of (IBMB-CSIC) Barcelona. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). R.A.-P. was supported by a Ramón y Cajal grant and is a holder of a Marie Curie grant (RA-P:UEO/MCA1108). M.Z. was a holder of a Marie Curie Outgoing International Fellowship (MOIF-CT-2006-039327), and P.G.A. was supported by a Ramón y Cajal grant from the Spanish Ministry of Education, Science and Sport.

The constitutively-expressed cyclooxygenase 1 (COX-1) and the inducible COX-2 are both involved in the conversion of arachidonic acid (AA) to prostaglandins (PGs). However, the functional roles of COX-1 at the cellular level remain unclear. We hypothesized that by comparing differential gene expression and eicosanoid metabolism in lung fibroblasts from wild-type (WT) mice and COX-2(-/-) or COX-1(-/-) mice may help address the functional roles of COX-1 in inflammation and other cellular functions. Compared to WT, the number of specifically-induced transcripts were altered descendingly as follows: COX-2(-/-)COX-1(-/-)WT+IL-1β. COX-1(-/-) or COX-2(-/-) cells shared about 50% of the induced transcripts with WT cells treated with IL-1β, respectively. An interactive "anti-inflammatory, proinflammatory, and redox-activated" signature in the protein-protein interactome map was observed in COX-2(-/-) cells. The augmented COX-1 mRNA (in COX-2(-/-) cells) was associated with the upregulation of mRNAs for glutathione S-transferase (GST), superoxide dismutase (SOD), NAD(P)H dehydrogenase quinone 1 (NQO1), aryl hydrocarbon receptor (AhR), peroxiredoxin, phospholipase, prostacyclin synthase, and prostaglandin E synthase, resulting in a significant increase in the levels of PGE2, PGD2, leukotriene B4 (LTB4), PGF1α, thromboxane B2 (TXB2), and PGF2α. The COX-1 plays a dominant role in shifting AA toward the LTB4 pathway and anti-inflammatory activities. Compared to WT, the upregulated COX-1 mRNA in COX-2(-/-) cells generated an "eicosanoid storm". The genomic characteristics of COX-2(-/-) is similar to that of proinflammatory cells as observed in IL-1β induced WT cells. COX-1(-/-) and COX-2(-/-) cells exhibited compensation of various eicosanoids at the genomic and metabolic levels.

The histone deacetylase HDAC7 interacts with and represses myeloid and T cell genes in pro–B cells. HDAC7 deletion blocks early B cell development and results in severe lymphopenia., Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro–B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML. This work was supported by the European Research Council (CoG-2014-646903 to P.M), the Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065, RTC-2016-4603-1 to P.M), the Asociación Española Contra el Cáncer (AECC-CI-2015), FERO Foundation, and the ISCIII/FEDER (PI14-01191) to C.B and the ‘‘Fundación Hay Esperanza’’ to E.A. P.M also acknowledges financial support from The Obra Social La Caixa-Fundaciò Josep Carreras, The Inocente Inocente Foundation and The Generalitat de Catalunya (SGR330). P.M an investigator of the Spanish Cell Therapy cooperative network (TERCEL). We thank Celgene Corporation (San Diego, CA) for providing IMiDs. We thank Judit Sopena and Mariano Graupera (IDIBELL, Barcelona) for their technical assistance with immunohistochamical analysis

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

The Scientific Foundation ‘Asociación Española Contra el Cáncer’ (AECC, Stable Coordinated Group, Hereditary Cancer); the BBVA Foundation; the Eugenio Rodríguez Pascual Foundation grant 2012; Generalitat de Catalunya AGAUR SGR 2012 grants 283, 290 and 312, and SGR 2014 grants 364, 530, and 535; Spanish Ministry of Health ISCIII FIS grants PI10/00057, PI10/00222, PI10/01422, PI12/01528, PI13/00132, and PI14/00336. ISCIII RTICC grants RD06/0020/1051, RD12/0036/0007, RD12/0036/0008 and RD12/0036/0063; Spanish Ministry of Science and Innovation, ‘Fondo Europeo de Desarrollo Regional (FEDER), una manera de hacer Europa’, MINECO grants SAF2010-20203 and SAF2013-46196; and the Telemaraton 2014 ‘Todos Somos Raros, Todos Somos Únicos’ grant P35 (...), Mateo, F.; Arenas, E. J.; Aguilar, H.; Serra-Musach, J.; de Garibay, G. Ruiz; Boni, J.; Maicas, M.; Du, S.; Iorio, F.; Herranz-Ors, C.; Islam, A.; Prado, X.; Llorente, A.; Petit, A.; Vidal, A.; Catala, I.; Soler, T.; Venturas, G.; Rojo-Sebastian, A.; Serra, H.; Cuadras, D.; Blanco, I.; Lozano, J.; Canals, F.; Sieuwerts, A. M.; de Weerd, V.; Look, M. P.; Puertas, S.; Garcia, N.; Perkins, A. S.; Bonifaci, N.; Skowron, M.; Gomez-Baldo, L.; Hernandez, V.; Martinez-Aranda, A.; Martinez-Iniesta, M.; Serrat, X.; Ceron, J.; Brunet, J.; Barretina, M. P.; Gil, M.; Falo, C.; Fernandez, A.; Morilla, I.; Pernas, S.; Pla, M. J.; Andreu, X.; Segui, M. A.; Ballester, R.; Castella, E.; Nellist, M.; Morales, S.; Valls, J.; Velasco, A.; Matias-Guiu, X.; Figueras, A.; Sanchez-Mut, J. V.; Sanchez-Cespedes, M.; Cordero, A.; Gomez-Miragaya, J.; Palomero, L.; Gomez, A.; Gajewski, T. F.; Cohen, E. E. W.; Jesiotr, M.; Bodnar, L.; Quintela-Fandino, M.; Lopez-Bigas, N.; Valdes-Mas, R.; Puente, X. S.; Vinals, F.; Casanovas, O.; Graupera, M.; Hernandez-Losa, J.; Ramon y Cajal, S.; Garcia-Alonso, L.; Saez-Rodriguez, J.; Esteller, M.; Sierra, A.; Martin-Martin, N.; Matheu, A.; Carracedo, A.; Gonzalez-Suarez, E.; Nanjundan, M.; Cortes, J.; Lazaro, C.; Odero, M. D.; Martens, J. W. M.; Moreno-Bueno, G.; Barcellos-Hoff, M. H.; Villanueva, A.; Gomis, R. R.; Pujana, M. A.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF., Ministerio de Economía y Competitividad (MINECO) [SAF2012-38078 to O.B. and BFU2015-65311-R to R.A.]; Instituto de Salud Carlos III [RD12/0036/0054 to O.B. and CB16/12/00244 (CIBERONC) to A.B.]; ‘La Caixa’ Foundation 2016-052407 (to R.A.). Funding for open access charge: MInisterio the Economia y Competitividad, Spain La Caixa Foundation. Spain.

SUMMARY The importance of the retinoblastoma tumor suppressor protein pRB in cell cycle control is well established. However, less is known about its role in differentiation during animal development. Here, we investigated the role of Rbf, the Drosophila pRB homolog, in adult skeletal muscles. We found that the depletion of Rbf severely reduced muscle growth and altered myofibrillogenesis but only minimally affected myoblast proliferation. We identified an Rbf-dependent transcriptional program in late muscle development that is distinct from the canonical role of Rbf in cell cycle control. Unexpectedly, Rbf acts as a transcriptional activator of the myogenic and metabolic genes in the growing muscles. The genomic regions bound by Rbf contained the binding sites of several factors that genetically interacted with Rbf by modulating Rbf-dependent phenotype. Thus, our results reveal a distinctive role for Rbf as a direct activator of the myogenic transcriptional program that drives late muscle differentiation., Graphical Abstract, In Brief Inactivation of the tumor suppressor RB, an obligatory step in most cancers, results in unrestrained cell cycle progression. Zappia et al. show that Rbf, the RB Drosophila ortholog, directly activates the metabolic program that accompanies muscle development. This work expands the understanding of the plethora of Rbf functions.

SummaryE2F/DP transcription factors regulate cell proliferation and apoptosis. Here, we investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because we show that this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. We attribute this defect to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, our results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation.

Nature Communications 6: Article number: 8510 10.1038/ncomms9510 (2015); Published: October142015; Updated: March042016 The original version of this Article contained a typographical error in the spelling of the author Zhenyi Liu, which was incorrectly given as Zenhy Liu. This has now been corrected in both the PDF and HTML versions of the Article.

Background We examined whether differences in tumor DNA methylation were associated with more aggressive hormone receptor-negative breast cancer in an ethnically diverse group of patients in the Breast Cancer Care in Chicago (BCCC) study and using data from The Cancer Genome Atlas (TCGA). Results DNA was extracted from formalin-fixed, paraffin-embedded samples on 75 patients (21 White, 31 African-American, and 23 Hispanic) (training dataset) enrolled in the BCCC. Hormone receptor status was defined as negative if tumors were negative for both estrogen and progesterone (ER/PR) receptors (N = 22/75). DNA methylation was analyzed at 1505 CpG sites within 807 gene promoters using the Illumina GoldenGate assay. Differential DNA methylation as a predictor of hormone receptor status was tested while controlling for false discovery rate and assigned to the gene closest to the respective CpG site. Next, those genes that predicted ER/PR status were validated using TCGA data with respect to DNA methylation (validation dataset), and correlations between CpG methylation and gene expression were examined. In the training dataset, 5.7 % of promoter mean methylation values (46/807) were associated with receptor status at P

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., It is unclear how Notch1 signals regulate both the maintenance of the endothelial fate and the endothelial-to-hematopoietic transition in the embryonic aorta. Here the authors show that those cells in which Notch1 ligand Jag1 is out-competed by Dll4 remain endothelial, while higher Jag1 activity leads to generation of hematopoietic stem cells.

Background: Extracellular stimuli induce gene expression responses through intracellular signaling mediators. The p38 signaling pathway is a paradigm of the mitogen-activated protein kinase (MAPK) family that, although originally identified as stress-response mediator, contributes to establishing stem cell differentiation fates. p38α is central for induction of the differentiation fate of the skeletal muscle stem cells (satellite cells) through not fully characterized mechanisms. Methods: To investigate the global gene transcription program regulated by p38α during satellite cell differentiation (myogenesis), and to specifically address whether this regulation occurs through direct action of p38α on gene promoters, we performed a combination of microarray gene expression and genome-wide binding analyses. For experimental robustness, two myogenic cellular systems with genetic and chemical loss of p38α function were used: (1) satellite cells derived from mice with muscle-specific deletion of p38α, and (2) the C2C12 murine myoblast cell line cultured in the absence or presence of the p38α/β inhibitor SB203580. Analyses were performed at cell proliferation and early differentiation stages. Results: We show that p38α binds to a large set of active promoters during the transition of myoblasts from proliferation to differentiation stages. p38α-bound promoters are enriched with binding motifs for several transcription factors, with Sp1, Tcf3/E47, Lef1, FoxO4, MyoD, and NFATc standing out in all experimental conditions. p38α association with chromatin correlates very well with high levels of transcription, in agreement with its classical function as an activator of myogenic differentiation. Interestingly, p38α also associates with genes repressed at the onset of differentiation, thus highlighting the relevance of p38-dependent chromatin regulation for transcriptional activation and repression during myogenesis. Conclusions: These results uncover p38α association and function on chromatin at novel classes of target genes during skeletal muscle cell differentiation. This is consistent with this MAPK isoform being a transcriptional regulator. The authors acknowledge funding from MINECO, Spain (SAF2012-38547, PLE2009-0124, SAF2015-67369-R, "María de Maeztu" Programme for Units of Excellence in R&D MDM-2014-0370), AFM, MDA, DDP-Netherlands, E-RARE, Fundació Marató TV3 and EU-FP7 (Myoage, Optistem and Endostem). JS is recipient of a Juan de la Cierva postdoctoral fellowship.

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Introduction]: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. [Methods]: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. [Results]: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. [Conclusions]: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer., This work was supported by grants from the Eugenio Rodríguez Pascual Foundation (2012, to MAP), the Government of Catalonia (2009-SGR283, to AV and MAP), the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R01 DK015556, to JAK), the Red Cooperative Research Thematic Network on Cancer (RTICC) (12/0036/0002 to XRB and 12/0036/0008 to XRB and MAP) and the Spanish Ministry of Health, Fund for Health Research–Institute of Health Carlos III (11/00951 to AU and 12/01528 to MAP).

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., Author Summary Through the hematopoietic system, all the distinct mature blood cell types are generated, thereby constituting one of the best-studied paradigms for cell lineage commitment and differentiation in biology. B lymphocytes are generated through several cell-commitment, lineage-choice, and differentiation processes. To date, the central role of lineage-specific transcription factors in positively regulating these distinct developmental steps is well established. However, in the absence of proper transcriptional repression, an “adolescent cell” will never be able to reach its “adulthood identity,” having a potential impact in the development of hematological malignancies. In this article, we examined the molecular mechanism responsible for the gene silencing of lineage undesirable genes in B cell precursors and uncovered the role played in this process by the histone deacetylase HDAC7. We show that HDAC7 is expressed in B cell precursors where it interacts with the transcription factor MEF2C and is recruited to the promoters of non-B cell genes. While HDAC7 is down-regulated during the lineage conversion of pre-B cells into macrophages, re-expression of HDAC7 interferes with both the acquisition of the myeloid gene transcriptional program and macrophage-specific cell functions. We therefore have identified a novel lineage-specific transcriptional repressor in the hematopoietic system.

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., M.M. is an investigator of the Sara Borrell program (CD09/00421). E.L.A. is a recipient of a predoctoral fellowship from the Department of Education, Universities and Research of the Basque Government (BFI-2011), and V.R. was a recipient of FIMIM predoctoral fellowship. L.E. is an investigator at the Carlos III program. This work was supported by a grant from the Instituto de Salud Carlos III (PI041890), Fundación Mutua Madrileña, AGAUR (SGR23), and RTICCS/FEDER (RD06/0020/0098 and RD12/0036/0054). M.D. was funded by the Ministerio de Ciencia e Innovación (BFU2009-09074 and MEC-CONSOLIDER CSD2007-00023), Generalitat Valenciana (PROMETEO 2008/134), and a grant from European Union Research (UE-HEALH-F2-2008-201666). J.V-F. was funded by a MICINN grant (CTQ2008-00755) and the EC VPH (no. FP7-2007-IST-223920).

Results Surprisingly, gene expression profiling revealed that the normal transcription program induced in response to irradiation in wild-type animals was also properly induced in dDP mutant animals. However, we found that the loss of dDP resulted in down-regulation of expression of a large panel of mitochondria-related genes. ChIP-seq and ChIP confirmed that many of these genes are indeed direct dDP targets. Consistently, mitochondrial function was severely compromised in dDP mutant eye discs. Down-regulation of several of mitochondria-related dDP target genes mimicked the dDP mutant phenotype and was sufficient to protect the eye discs from irradiation-induced apoptosis.

Cancer is characterized by aberrant patterns of expression of multiple genes. These major shifts in gene expression are believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including histone modifications. However, it is unclear whether the binding of histone-modifying proteins to genomic regions and the placing of histone modifications efficiently discriminates corresponding genes from the rest of the genes in the human genome. We performed gene expression analysis of histone demethylases (HDMs) and histone methyltransferases (HMTs), their target genes and genes with relevant histone modifications in normal and tumor tissues. Surprisingly, this analysis revealed the existence of correlations in the expression levels of different HDMs and HMTs. The observed HDM/HMT gene expression signature was specific to particular normal and cancer cell types and highly correlated with target gene expression and the expression of genes with histone modifications. Notably, we observed that trimethylation at lysine 4 and lysine 27 separated preferentially expressed and underexpressed genes, which was strikingly different in cancer cells compared to normal cells. We conclude that changes in coordinated regulation of enzymes executing histone modifications may underlie global epigenetic changes occurring in cancer This project was funded by R01CA138631 (E.V.B.) and grant number 115347-RSG-08-271-01-GMC from the American Cancer Society (E.V.B.). N.L.-B. acknowledges funding from the Spanish Ministry of Science and Education, grant number SAF2009-06954. A.B.M.M.K.I. is supported by a fellowship from AGAUR of the Catalonian Government, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3). The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me3. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome. This work was supported by 115347-RSG-08-271-01-GMC from ACS, and by CA138631 grant from NIH.

Background: DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved. Results: Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation. Conclusions: The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process. This work was supported by grant SAF2011-29635 from the Spanish Ministry of Science and Innovation, grant CIVP16A1834 from Fundación Ramón Areces and grant 2009SGR184 from AGAUR (Catalan Government). LR is supported by a PFIS predoctoral fellowship

Background MERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV. Results Multiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein’s antigenic sites are found to be conserved with those in HKU4 and HKU5. Conclusion This sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity. Electronic supplementary material The online version of this article (doi:10.1186/s13029-016-0049-7) contains supplementary material, which is available to authorized users.