Down-regulation of LATS kinases alters p53 to promote cell migration.

p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.

RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation.

Embryonic stem cells (ESCs) maintain high genomic plasticity, which is essential for their capacity to enter diverse differentiation pathways. Posttranscriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner.

Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues.

BACKGROUND: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. RESULTS: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. CONCLUSIONS: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

PML, YAP, and p73 are components of a proapoptotic autoregulatory feedback loop.

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.

Pathway-based personalized analysis of cancer.

We introduce Pathifier, an algorithm that infers pathway deregulation scores for each tumor sample on the basis of expression data. This score is determined, in a context-specific manner, for every particular dataset and type of cancer that is being investigated. The algorithm transforms gene-level information into pathway-level information, generating a compact and biologically relevant representation of each sample. We demonstrate the algorithm's performance on three colorectal cancer datasets and two glioblastoma multiforme datasets and show that our multipathway-based representation is reproducible, preserves much of the original information, and allows inference of complex biologically significant information. We discovered several pathways that were significantly associated with survival of glioblastoma patients and two whose scores are predictive of survival in colorectal cancer: CXCR3-mediated signaling and oxidative phosphorylation. We also identified a subclass of proneural and neural glioblastoma with significantly better survival, and an EGF receptor-deregulated subclass of colon cancers.

A module of negative feedback regulators defines growth factor signaling.

Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

Guanine polynucleotides are self-antigens for human natural autoantibodies and are significantly reduced in the human genome.

In the course of investigating anti-DNA autoantibodies, we examined IgM and IgG antibodies to poly-G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20-mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo-nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti-G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170,000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain-associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti-G20 antibodies; so natural anti-G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti-G20 antibodies in human health and disease and of runs of G20 in the human genome.

Pemphigus vulgaris is characterized by low IgG reactivities to specific self-antigens along with high IgG reactivity to desmoglein 3.

Pemphigus vulgaris (PV) is an autoimmune skin disease, which has been characterized by IgG autoantibodies to desmoglein 3. Here we studied the antibody signatures of PV patients compared with healthy subjects and with patients with two other autoimmune diseases with skin manifestations (systemic lupus erythematosus and scleroderma), using an antigen microarray and informatics analysis. We now report a previously unobserved phenomenon--patients with PV, compared with the healthy subjects and the two other diseases, show a significant decrease in IgG autoantibodies to a specific set of self-antigens. This novel finding demonstrates that an autoimmune disease may be associated with a loss of specific, healthy IgG autoantibodies and not only with a gain of specific, pathogenic IgG autoantibodies.

Epstein-Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti-DNA.

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities - anti-dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two-thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two-thirds of SLE patients reacted to a large spectrum of self-molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein-Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.

An accessible database for mouse and human whole transcriptome qPCR primers.

MOTIVATION: Real time quantitative polymerase chain reaction (qPCR) is an important tool in quantitative studies of DNA and RNA molecules; especially in transcriptome studies, where different primer combinations allow identification of specific transcripts such as splice variants or precursor messenger RNA. Several softwares that implement various rules for optimal primer design are available. Nevertheless, as designing qPCR primers needs to be done manually, the repeated task is tedious, time consuming and prone to errors. RESULTS: We used a set of rules to automatically design all possible exon-exon and intron-exon junctions in the human and mouse transcriptomes. The resulting database is included as a track in the UCSC genome browser, making it widely accessible and easy to use. AVAILABILITY: The database is available from the UCSC genome browser (http://genome.ucsc.edu/), track name 'Whole Transcriptome qPCR Primers' for the hg19 (Human) and mm10 (Mouse) genome versions. Batch query is available in the following: http://www.weizmann.ac.il/complex/compphys/software/Amit/primers/batch_query_qpcr_primers.htm CONTACT: amit.zeisel@weizmann.ac.il or eytan.domany@weizmann.ac.il SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Blastocyst implantation failure relates to impaired translational machinery gene expression.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

Context-specific microRNA analysis: identification of functional microRNAs and their mRNA targets.

MicroRNAs (miRs) function primarily as post-transcriptional negative regulators of gene expression through binding to their mRNA targets. Reliable prediction of a miR's targets is a considerable bioinformatic challenge of great importance for inferring the miR's function. Sequence-based prediction algorithms have high false-positive rates, are not in agreement, and are not biological context specific. Here we introduce CoSMic (Context-Specific MicroRNA analysis), an algorithm that combines sequence-based prediction with miR and mRNA expression data. CoSMic differs from existing methods--it identifies miRs that play active roles in the specific biological system of interest and predicts with less false positives their functional targets. We applied CoSMic to search for miRs that regulate the migratory response of human mammary cells to epidermal growth factor (EGF) stimulation. Several such miRs, whose putative targets were significantly enriched by migration processes were identified. We tested three of these miRs experimentally, and showed that they indeed affected the migratory phenotype; we also tested three negative controls. In comparison to other algorithms CoSMic indeed filters out false positives and allows improved identification of context-specific targets. CoSMic can greatly facilitate miR research in general and, in particular, advance our understanding of individual miRs' function in a specific context.

BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors.

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

EGR1 and the ERK-ERF axis drive mammary cell migration in response to EGF.

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.

Coupled pre-mRNA and mRNA dynamics unveil operational strategies underlying transcriptional responses to stimuli.

Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude.

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Association of survival and disease progression with chromosomal instability: a genomic exploration of colorectal cancer.

During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1-4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA, MYC, MET, FGF6, FGF23, LYN, MMP9, MYBL2, AURKA, UBE2C, and PTEN. Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68, CSMD1, POLR1D, and PMEPA1. From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.

Integrative analysis correlates donor transcripts to recipient autoantibodies in primary graft dysfunction after lung transplantation.

Up to one in four lung-transplanted patients develop pulmonary infiltrates and impaired oxygenation within the first days after lung transplantation. Known as primary graft dysfunction (PGD), this condition increases mortality significantly. Complex interactions between donor lung and recipient immune system are the suspected cause. We took an integrative, systems-level approach by first exploring whether the recipient's immune response to PGD includes the development of long-lasting autoreactivity. We next explored whether proteins displaying such differential autoreactivity also display differential gene expression in donor lungs that later develop PGD compared with those that did not. We evaluated 39 patients from whom autoantibody profiles were already available for PGD based on chest radiographs and oxygenation data. An additional nine patients were evaluated for PGD based on their medical records and set aside for validation. From two recent donor lung gene expression studies, we reanalysed and paired gene profiles with autoantibody profiles. Primary graft dysfunction can be distinguished by a profile of differentially reactive autoantibodies binding to 17 proteins. Functional analysis showed that 12 of these proteins are part of a protein-protein interaction network (P=3 x 10⁻6) involved in proliferative processes. A nearest centroid classifier assigned correct PGD grades to eight out of the nine patients in the validation cohort (P=0·048). We observed significant positive correlation (r=0·63, P=0·011) between differences in IgM reactivity and differences in gene expression levels. This connection between donor lung gene expression and long-lasting recipient IgM autoantibodies towards a specific set of proteins suggests a mechanism for the development of autoimmunity in PGD.

Gene expression following exposure to celecoxib in humans: pathways of inflammation and carcinogenesis are activated in tumors but not normal tissues.

BACKGROUND: The Cox-2 inhibitor, celecoxib (Pfizer Inc., N.Y., USA), is a promising chemopreventive agent [Arber et al.: N Engl J Med 2006;355:885-895; Bertagnolli et al.: N Engl J Med 2006;355:873-884]. This study aims to explore its mechanism by defining changes in gene expression between neoplastic and normal tissue samples before and after treatment. METHODS: Patients with documented colorectal neoplasia in screening colonoscopy, destined to undergo surgical colectomy, were randomized for treatment with celecoxib (n = 11; 400 mg/day) or placebo (n = 3) for 30 days. Tissue samples were taken from the tumor and from normal adjacent mucosa during both colonoscopy and surgery. RNA was extracted and analyzed using Affymetrix Genechip®. RESULTS: 687 genes differentiated tumor samples before and after treatment, among which 310 genes did not show the same differential expression in the placebo group or normal samples. These genes were significantly related to pathways of cell cycle regulation and inflammation, and of note was the TGF-ß pathway, which held a strong association with the list of genes formerly found to be associated with the colorectal cancer expression profile in microarray analyses, as summarized in a meta-analysis by Cardoso et al. [Biochim Biophys Acta 2007;1775:103-137]. CONCLUSIONS: Celecoxib selectively affects genes and pathways involved in inflammation and malignant transformation in tumor but not normal tissues, this may assist in the development of safer and more effective chemopreventive agents.