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Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

DNA methylation (5mC) is a promising biomarker for detecting circulating tumor DNA (ctDNA), providing information on a cell9s genomic regulation, developmental lineage, and molecular age. Sequencing assays for detecting ctDNA methylation involve pre-processing steps such as immunoprecipitation, enzymatic treatment, or the most common method, sodium bisulfite treatment. These steps add complexity and time that pose a challenge for clinical labs, and bisulfite treatment in particular degrades input DNA and can result in loss of informative ctDNA fragmentation patterns. In this feasibility study, we demonstrate that whole genome sequencing of circulating cell-free DNA using conventional Oxford Nanopore Technologies (ONT) sequencing can accurately detect cell-of-origin and cancer-specific 5mC changes while preserving important fragmentomic information. The simplicity of this approach makes it attractive as a liquid biopsy assay for cancer as well as non-cancer applications in emergency medicine.

ERBB2 amplification is a prognostic marker for aggressive tumors and a predictive marker for prolonged survival following treatment with HER2 inhibitors. We attempt to sub-group HER2+ tumors based on amplicon structures and co-amplified genes. We examined five HER2+ cell lines, three HER2+ xenographs and 57 HER2+ tumor tissues. ERBB2 amplification was analyzed using digital droplet PCR and low coverage whole genome sequencing. In some HER2+ tumors PPM1D, that encodes WIP1, is co-amplified. Cell lines were treated with HER2 and WIP1 inhibitors. We find that inverted duplication is the amplicon structure in the majority of HER2+ tumors. In patients suffering from an early stage disease the ERBB2 amplicon is composed of a single segment while in patients suffering from advanced cancer the amplicon is composed of several different segments. We find robust WIP1 inhibition in some HER2+ PPM1D amplified cell lines. Sub-grouping HER2+ tumors using low coverage whole genome sequencing identifies inverted duplications as the main amplicon structure and based on the number of segments, differentiates between local and advanced tumors. In addition, we found that we could determine if a tumor is a recurrent tumor or second primary tumor and identify co-amplified oncogenes that may serve as targets for therapy.

ERBB2 amplification is a prognostic marker for aggressive tumors and a predictive marker for prolonged survival following treatment with HER2 inhibitors. We attempt to sub-group HER2+ tumors based on amplicon structures and co-amplified genes. We examined five HER2+ cell lines, three HER2+ xenographs and 57 HER2+ tumor tissues. ERBB2 amplification was analyzed using digital droplet PCR and low coverage whole genome sequencing. In some HER2+ tumors PPM1D, that encodes WIP1, is co-amplified. Cell lines were treated with HER2 and WIP1 inhibitors. We find that inverted duplication is the amplicon structure in the majority of HER2+ tumors. In patients suffering from an early stage disease the ERBB2 amplicon is composed of a single segment while in patients suffering from advanced cancer the amplicon is composed of several different segments. We find robust WIP1 inhibition in some HER2+ PPM1D amplified cell lines. Sub-grouping HER2+ tumors using low coverage whole genome sequencing identifies inverted duplications as the main amplicon structure and based on the number of segments, differentiates between local and advanced tumors. In addition, we found that we could determine if a tumor is a recurrent tumor or second primary tumor and identify co-amplified oncogenes that may serve as targets for therapy.

The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are “left behind” and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.

SMARCB1 (Snf5/Ini1/Baf47) is a potent tumor suppressor, the loss of which serves as the diagnostic feature in malignant rhabdoid tumors (MRT) and atypical teratoid/rhabdoid tumors (AT/RT), two highly aggressive forms of pediatric neoplasms. SMARCB1 is a core subunit of Swi/Snf chromatin remodeling complexes, and loss of SMARCB1 or other subunits of these complexes has been observed in a variety of tumor types. Here, we restore Smarcb1 expression in cells derived from Smarcb1-deficient tumors, which developed in Smarcb1 heterozygous p53(-/-) mice. We find that while re-introduction of Smarcb1 does not induce growth arrest, it restores sensitivity to programmed cell death and completely abolishes the ability of the tumor cells to grow as xenografts. We describe persistent activation of AKT signaling in Smarcb1-deficient cells, which stems from PI3K (phosphatidylinositol 3'-kinase)-mediated signaling and which contributes to the survival and proliferation of the tumor cells. We further demonstrate that inhibition of AKT is effective in preventing proliferation of Smarcb1-deficient cells in vitro and inhibits the development of xenografted tumors in vivo. Profiling Smarcb1-dependent gene expression, we find genes that require Smarcb1 and Swi/Snf for their expression to be enriched for extracellular matrix and cell adhesion functions. We find that Smarcb1 is required for transcriptional activation of Igfbp7, a member of the insulin-like growth factor-binding proteins family and a tumor suppressor in itself, and show that re-introduction of Igfbp7 alone can hinder tumor development. Our results define a novel mechanism for Smarcb1-mediated tumorigenesis and highlight potential therapeutic targets.

SummaryMost adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.Video Abstract

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.

Background The SWI/SNF ATP dependent chromatin remodeling complex is a multi-subunit complex, conserved in eukaryotic evolution that facilitates nucleosomal re-positioning relative to the DNA sequence. In recent years the SWI/SNF complex has emerged to play a role in cancer development as various sub-units of the complex are found to be mutated in a variety of tumors. One core-subunit of the complex, which has been well established as a tumor suppressor gene is SMARCB1 (SNF5/INI1/BAF47). Mutation and inactivation of SMARCB1 have been identified as the underlying mechanism leading to Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid/Rhabdoid Tumors (AT/RT), two highly aggressive forms of pediatric neoplasms. Methods We present a phosphoproteomic study of Smarcb1 dependent changes in signaling networks. The SILAC (Stable Isotopic Labeling of Amino Acids in Cell Culture) protocol was used to quantify in an unbiased manner any changes in the phosphoproteomic profile of Smarcb1 deficient murine rhabdoid tumor cell lines following Smarcb1 stable re-expression and under different serum conditions. Results This study illustrates broad changes in the regulation of multiple biological networks including cell cycle progression, chromatin remodeling, cytoskeletal regulation and focal adhesion. Specifically, we identify Smarcb1 dependent changes in phosphorylation and expression of the EGF receptor, demonstrate downstream signaling and show that inhibition of EGFR signaling specifically hinders the proliferation of Smarcb1 deficient cells. Conclusions These results support recent findings regarding the effectivity of EGFR inhibitors in hindering the proliferation of human MRT cells and demonstrate that activation of EGFR signaling in Rhabdoid tumors is SMARCB1 dependent. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0439-5) contains supplementary material, which is available to authorized users.

Genome-wide DNA hypomethylation occurs in many human cancers, but whether this epigenetic change is a cause or consequence of tumorigenesis has been unclear. To explore this phenomenon, we generated mice carrying a hypomorphic DNA methyltransferase 1 ( Dnmt1 ) allele, which reduces Dnmt1 expression to 10% of wild-type levels and results in substantial genome-wide hypomethylation in all tissues. The mutant mice were runted at birth, and at 4 to 8 months of age they developed aggressive T cell lymphomas that displayed a high frequency of chromosome 15 trisomy. These results indicate that DNA hypomethylation plays a causal role in tumor formation, possibly by promoting chromosomal instability.

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

Organoleptic compounds produced by yeast during the fermentation of wort have a great impact on beer smell and taste. Among them, fusel alcohols are the major abundant volatile compounds. The availability of Saccharomyces cerevisiae mutants in which the genes coding for the two branched-chain amino acid aminotransferases have been deleted offers the possibility of further defining the role of these enzymes in the formation of higher alcohols. Comparing the production profiles of different strains, it is clear that they are not all influenced in the same way by branched-chain amino acid aminotransferase mutations. First of all, as propanol is synthesised from alpha-ketobutyrate, the first metabolic intermediate in the anabolic pathway of isoleucine, neither the eca39 nor eca40 mutations have any effect on the production of this higher alcohol. On the other hand, it can be concluded that the eca40 mutation has a drastic effect on the production of isobutanol. To a certain extent, the same conclusion can be made for the production of active amyl alcohol and isoamyl alcohol, although the results suggest that another route could lead to the formation of these two higher alcohols.

Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature. Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, ζ-globin (mesoderm), neurofilament 68Kd (ectoderm), and α-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker α-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain. Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine.

Histone variant macroH2A1 (macroH2A1) contains an NH2-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins γ-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.

Direct reprogramming procedures reset the epigenetic memory of cells and convert differentiated somatic cells into pluripotent stem cells. In addition to epigenetic memory of cell identity, which is established during development, somatic cells can accumulate abnormal epigenetic changes that can contribute to pathological conditions. Aberrant promoter hypermethylation and epigenetic silencing of tumor suppressor genes (TSGs) are now recognized as an important mechanism in tumor initiation and progression. Here, we have studied the fate of the silenced TSGs p16(CDKN2A) during direct reprogramming. We find that following reprogramming, p16 expression is restored and is stably maintained even when cells are induced to differentiate. Large-scale methylation profiling of donor cells identified aberrant methylation at hundreds of additional sites. Methylation at many, but not all these sites was reversed following reprogramming. Our results suggest that reprogramming approaches may be applied to repair the epigenetic lesions associated with cancer.

The cytosine analogues 5-azacytidine and 5-aza-2'-deoxycytidine are currently the most advanced drugs for epigenetic cancer therapy. Both drugs function as DNA methyltransferase (DNMT) inhibitors and lead to the reactivation of epigenetically silenced tumour suppressor genes. However, not much is known about their target sequence specificity and their possible side effects on normally methylated sequences such as long interspersed nuclear element (LINE)-1 retroelements. It has been shown that demethylation and activation of the LINE-1 antisense promoter can drive the transcription of neighbouring sequences. In this study, we show that demethylation of the colon carcinoma cell line HCT116, either by treatment with DNMT inhibitors or by genetic disruption of the major DNMTs, induces the expression of an illegitimate fusion transcript between an intronic LINE-1 element and the proto-oncogene cMet (L1-cMet). Similar findings were also obtained with myeloid leukaemia cells, an established cellular model for the approved indication of azacytidine and decitabine. Interestingly, upregulation of L1-cMet transcription resulted in reduced cMet expression, which in turn led to decreased cMet receptor signalling. Our results thus provide an important paradigm for demethylation-dependent modulation of gene expression, even if the promoter of the corresponding gene is unmethylated.

Genomic imprinting is an epigenetic phenomenon whereby genes are expressed in a monoallelic manner, which is inherited either maternally or paternally. Expression of imprinted genes has been examined in human embryonic stem (ES) cells, and the cells show a substantial degree of genomic imprinting stability. Recently, human somatic cells were reprogrammed to a pluripotent state using various defined factors. These induced pluripotent stem (iPS) cells are thought to have a great potential for studying genetic diseases and to be a source of patient-specific stem cells. Thus, studying the expression of imprinted genes in these cells is important. We examined the allelic expression of various imprinted genes in several iPS cell lines and found polymorphisms in four genes. After analyzing parent-specific expression of these genes, we observed overall normal monoallelic expression in the iPS cell lines. However, we found biallelic expression of the H19 gene in one iPS cell line and biallelic expression of the KCNQ10T1 gene in another iPS cell line. We further analyzed the DNA methylation levels of the promoter region of the H19 gene and found that the cell line that showed biallelic expression had undergone extensive DNA demethylation. Additionally we studied the imprinting gene expression pattern of multiple human iPS cell lines via DNA microarray analyses and divided the pattern of expression into three groups: (a) genes that showed significantly stable levels of expression in iPS cells, (b) genes that showed a substantial degree of variability in expression in both human ES and iPS cells, and (c) genes that showed aberrant expression levels in some human iPS cell lines, as compared with human ES cells. In general, iPS cells have a rather stable expression of their imprinted genes. However, we found a significant number of cell lines with abnormal expression of imprinted genes, and thus we believe that imprinted genes should be examined for each cell line if it is to be used for studying genetic diseases or for the purpose of regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

Summary Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFβ required for TGFβ-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16 ink4a /Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.

Genome-wide DNA hypomethylation and concomitant promoter-specific tumor suppressor gene hypermethylation are among the most common molecular alterations in human neoplasia. Consistent with the notion that both promoter hypermethylation and genome-wide hypomethylation are functionally important in tumorigenesis, genetic and/or pharmacologic reduction of DNA methylation levels results in suppression or promotion of tumor incidence, respectively, depending on the tumor cell type. For instance, DNA hypomethylation promotes tumors that rely predominantly on loss of heterozygosity (LOH) or chromosomal instability mechanisms, whereas loss of DNA methylation suppresses tumors that rely on epigenetic silencing. Mutational and epigenetic silencing events in Wnt pathway genes have been identified in human colon tumors. We used Apc Min/+ mice to investigate the effect of hypomethylation on intestinal and liver tumor formation. Intestinal carcinogenesis in Apc Min/+ mice occurs in two stages, with the formation of microadenomas leading to the development of macroscopic polyps. Using Dnmt1 hypomorphic alleles to reduce genomic methylation, we observed elevated incidence of microadenomas that were associated with LOH at Apc . In contrast, the incidence and growth of macroscopic intestinal tumors in the same animals was strongly suppressed. In contrast to the overall inhibition of intestinal tumorigenesis in hypomethylated Apc Min/+ mice, hypomethylation caused development of multifocal liver tumors accompanied by Apc LOH. These findings support the notion of a dual role for DNA hypomethylation in suppressing later stages of intestinal tumorigenesis, but promoting early lesions in the colon and liver through an LOH mechanism.

ECA39 was isolated as a target gene for c-Myc regulation in mice. We identified two homologs for the murine ECA39 in the yeast Saccharomyces cerevisiae, ECA39 and ECA40, as well as two human homologs. These genes show a significant homology to prokaryotic branched-chain amino acid aminotransferase (BCAT) (EC). To understand the function of eukaryotic ECA39 and ECA40, we deleted either gene from the yeast genome. Activity of branched-chain amino acid aminotransferase was measured in the wild-type and mutants with either leucine, isoleucine, or valine as substrates. The results demonstrate that in S. cerevisiae ECA39 and ECA40 code for mitochondrial and cytosolic branched-chain amino acid aminotransferases, respectively. ECA39 is highly expressed during log phase and is down-regulated during the stationary phase of growth, while ECA40 shows an inverse pattern of gene expression. In agreement with these results, while we previously showed that deletion of ECA39 affected the cell cycle in proliferating cells, we do not observe a growth phenotype in eca40Delta cells. We suggest that BCAT is a target for c-Myc activity and discuss the evolutionary conservation of prokaryotic and eukaryotic BCATs and their possible involvement in regulation of cell proliferation.

The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis. The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation. Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated. To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences. We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene. Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively. Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog. This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc. To understand the function of ECA39, we deleted the gene from the yeast genome. Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle. Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains. Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle. Moreover, mutant strains show higher rates of UV-induced mutations. The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.

We have recently shown that genome-wide DNA hypomethylation induces genomic instability and tumors in animals ([ 1 ][1], [ 2 ][2]). Based on these results, we cautioned that long-term treatment with a demethylating agent such as 5-aza-2′deoxycytidine (5-aza-dC) may produce unwanted side effects

Human tumors often display changes in DNA methylation, including both genome-wide hypomethylation and site-specific hypermethylation ( [1][1], [2][2] ). In mice, DNA hypomethylation is sufficient to induce T cell lymphomas with consistent gain of chromosome 15 ( [3][3] ), indicating that genome-wide

SummaryWe report on the establishment of a human embryonic stem cell (HESC) line from a preimplantation fragile X-affected embryo and demonstrate its value as an appropriate model to study developmentally regulated events that are involved in the pathogenesis of this disorder. Fragile X syndrome results from FMR1 gene inactivation due to a CGG expansion at the 5′UTR region of the gene. Early events in FMR1 silencing have not been fully characterized due to the lack of appropriate animal or cellular models. Here we show that, despite the presence of a full mutation, affected undifferentiated HESCs express FMR1 and are DNA unmethylated. However, epigenetic silencing by DNA methylation and histone modification occurs upon differentiation. Our unique cell system allows the dissection of the sequence by which these epigenetic changes are acquired and illustrates the importance of HESCs in unraveling developmentally regulated mechanisms associated with human genetic disorders.

The branched-chain amino acid aminotransferase, Bcat1/Eca39, catalyzes the first step of branched-chain amino acid catabolism. Bcat1/Eca39 was originally isolated from a c-myc-induced tumor and was proven to be a direct target for c-Myc regulation. The gene is highly conserved in evolution and disruption of its yeast homolog affects cell growth. To assess the role of Bcat1/Eca39 in mammalian cells, we overexpressed Bcat1/Eca39 in murine cells and studied effects on cell growth. Overexpression of Bcat1/Eca39 had no apparent effect on the proliferation of cells grown with high serum concentrations, but under serum deprivation conditions, led to a decrease in cell viability. Cell death under these conditions displayed apoptotic features. The branched-chain keto acid, alpha-ketoisocaproate, a metabolite of leucine catabolism produced by BCAT1/ECA39, was previously found to inhibit cell growth. We show that alpha-ketoisocaproate can induce rapid apoptotic cell death. This observation suggests that the growth inhibitory effect of BCAT1/ECA39 and its apoptosis promoting effect may be mediated by the levels of the products of BCAT1/ECA39 activity, namely, branched-chain keto acids.