Showing papers in "Genes & Development in 2011"
TL;DR: It is found that lincRNA expression is strikingly tissue-specific compared with coding genes, and that l incRNAs are typically coexpressed with their neighboring genes, albeit to an extent similar to that of pairs of neighboring protein-coding genes.
Abstract: Large intergenic noncoding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an unprecedented analysis of such transcripts. Here, we present an integrative approach to define a reference catalog of >8000 human lincRNAs. Our catalog unifies previously existing annotation sources with transcripts we assembled from RNA-seq data collected from ~4 billion RNA-seq reads across 24 tissues and cell types. We characterize each lincRNA by a panorama of >30 properties, including sequence, structural, transcriptional, and orthology features. We found that lincRNA expression is strikingly tissue-specific compared with coding genes, and that lincRNAs are typically coexpressed with their neighboring genes, albeit to an extent similar to that of pairs of neighboring protein-coding genes. We distinguish an additional subset of transcripts that have high evolutionary conservation but may include short ORFs and may serve as either lincRNAs or small peptides. Our integrated, comprehensive, yet conservative reference catalog of human lincRNAs reveals the global properties of lincRNAs and will facilitate experimental studies and further functional classification of these genes.
3,114 citations
TL;DR: Vertebrate CpG islands are generically equipped to influence local chromatin structure and simplify regulation of gene activity.
Abstract: Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, CpG-rich, and predominantly nonmethylated. Most, perhaps all, CGIs are sites of transcription initiation, including thousands that are remote from currently annotated promoters. Shared DNA sequence features adapt CGIs for promoter function by destabilizing nucleosomes and attracting proteins that create a transcriptionally permissive chromatin state. Silencing of CGI promoters is achieved through dense CpG methylation or polycomb recruitment, again using their distinctive DNA sequence composition. CGIs are therefore generically equipped to influence local chromatin structure and simplify regulation of gene activity.
2,710 citations
TL;DR: The field is reviewed and how pioneer factors may enable cellular reprogramming is described, which can passively enhance transcription by reducing the number of additional factors that are needed to bind the DNA, culminating in activation.
Abstract: Transcription factors are adaptor molecules that detect regulatory sequences in the DNA and target the assembly of protein complexes that control gene expression. Yet much of the DNA in the eukaryotic cell is in nucleosomes and thereby occluded by histones, and can be further occluded by higher-order chromatin structures and repressor complexes. Indeed, genome-wide location analyses have revealed that, for all transcription factors tested, the vast majority of potential DNA-binding sites are unoccupied, demonstrating the inaccessibility of most of the nuclear DNA. This raises the question of how target sites at silent genes become bound de novo by transcription factors, thereby initiating regulatory events in chromatin. Binding cooperativity can be sufficient for many kinds of factors to simultaneously engage a target site in chromatin and activate gene expression. However, in cases in which the binding of a series of factors is sequential in time and thus not initially cooperative, special "pioneer transcription factors" can be the first to engage target sites in chromatin. Such initial binding can passively enhance transcription by reducing the number of additional factors that are needed to bind the DNA, culminating in activation. In addition, pioneer factor binding can actively open up the local chromatin and directly make it competent for other factors to bind. Passive and active roles for the pioneer factor FoxA occur in embryonic development, steroid hormone induction, and human cancers. Herein we review the field and describe how pioneer factors may enable cellular reprogramming.
1,452 citations
TL;DR: Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation.
Abstract: AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth.
1,347 citations
TL;DR: Inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on Autophagy.
Abstract: Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.
1,260 citations
TL;DR: This "autophagy addiction" suggests that targeting autophagy and mitochondrial metabolism are valuable new approaches to treat these aggressive cancers.
Abstract: Autophagy is a catabolic pathway used by cells to support metabolism in response to starvation and to clear damaged proteins and organelles in response to stress. We report here that expression of a H-rasV12 or K-rasV12 oncogene up-regulates basal autophagy, which is required for tumor cell survival in starvation and in tumorigenesis. In Ras-expressing cells, defective autophagosome formation or cargo delivery causes accumulation of abnormal mitochondria and reduced oxygen consumption. Autophagy defects also lead to tricarboxylic acid (TCA) cycle metabolite and energy depletion in starvation. As mitochondria sustain viability of Ras-expressing cells in starvation, autophagy is required to maintain the pool of functional mitochondria necessary to support growth of Ras-driven tumors. Human cancer cell lines bearing activating mutations in Ras commonly have high levels of basal autophagy, and, in a subset of these, down-regulating the expression of essential autophagy proteins impaired cell growth. As cancers with Ras mutations have a poor prognosis, this “autophagy addiction” suggests that targeting autophagy and mitochondrial metabolism are valuable new approaches to treat these aggressive cancers.
1,154 citations
TL;DR: The results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.
Abstract: Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.
1,121 citations
TL;DR: How the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells is outlined.
Abstract: Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR.
1,051 citations
TL;DR: It is found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined, and a tumor-suppressive function of NF-σB that contributes to the outcome of cancer therapy is identified.
Abstract: Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
729 citations
TL;DR: Two of the core machineries that initiate recombinational DNA repair in human cells are established: Bloom helicase and DNA2 nuclease, and the complex comprising MRE11, RAD50, and NBS1 (MRN).
Abstract: Repair of dsDNA breaks requires processing to produce 3′-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5′ → 3′ resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.
640 citations
TL;DR: New biochemical, genetic, and functional evidence suggests that Tet proteins are crucial for diverse biological processes, including zygotic epigenetic reprogramming, pluripotent stem cell differentiation, hematopoiesis, and development of leukemia.
Abstract: Ten-eleven translocation 1–3 (Tet1–3) proteins have recently been discovered in mammalian cells to be members of a family of DNA hydroxylases that possess enzymatic activity toward the methyl mark on the 5-position of cytosine (5-methylcytosine [5mC]), a well-characterized epigenetic modification that has essential roles in regulating gene expression and maintaining cellular identity. Tet proteins can convert 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) through three consecutive oxidation reactions. These modified bases may represent new epigenetic states in genomic DNA or intermediates in the process of DNA demethylation. Emerging biochemical, genetic, and functional evidence suggests that Tet proteins are crucial for diverse biological processes, including zygotic epigenetic reprogramming, pluripotent stem cell differentiation, hematopoiesis, and development of leukemia. Insights into how Tet proteins contribute to dynamic changes in DNA methylation and gene expression will greatly enhance our understanding of epigenetic regulation of normal development and human diseases.
TL;DR: The results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization.
Abstract: The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.
TL;DR: A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.
Abstract: Polyadenylation [poly(A)] signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid-1970s and subsequently shown to require flanking, auxiliary elements for both 3′-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA. Evidence for the mechanism of mRNA 3′-end formation is outlined, as is the way this RNA processing reaction communicates with RNA polymerase II to terminate transcription. The widespread phenomenon of alternative poly(A) site usage and how this interrelates with pre-mRNA splicing is then reviewed. This shows that gene expression can be drastically affected by how the message is ended. A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.
TL;DR: The data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.
Abstract: Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) ‘‘phased’’ intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ~540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent ‘‘two-hit’’ phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.
TL;DR: The underlying basis for these opposing functions and a model suggesting a dynamic role for autophagy in malignancy are discussed and it seems clear that modulation of this process will be an attractive avenue for future cancer therapeutic approaches.
Abstract: Macroautophagy (referred to hereafter as autophagy) is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell. Autophagy contributes to an array of normal and pathological processes, and has recently emerged as a key regulator of multiple aspects of cancer biology. The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. This complexity is illustrated by the identification of settings where autophagy acts potently to either promote or inhibit tumorigenesis. In this review, I discuss the underlying basis for these opposing functions and propose a model suggesting a dynamic role for autophagy in malignancy. Collectively, the data point to autophagy as serving as a barrier to limit tumor initiation. Once neoplastic lesions are established, it appears that adaptive changes occur that now result in positive roles for autophagy in malignant progression and in subsequent tumor maintenance. Remarkably, constitutive activation of autophagy is critical for continued growth of some tumors, serving to both reduce oxidative stress and provide key intermediates to sustain cell metabolism. Autophagy is also induced in response to cancer therapies where it can function as a survival mechanism that limits drug efficacy. These findings have inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. It is now apparent that aberrant control of autophagy is among the key hallmarks of cancer. While much needs to be learned about the regulation and context-dependent biological functions of autophagy, it seems clear that modulation of this process will be an attractive avenue for future cancer therapeutic approaches.
TL;DR: This study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.
Abstract: Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.
TL;DR: The involvement of DOT1L enzymatic activity in leukemogenesis driven by a subset of MLL (mixed-lineage leukemia) fusion proteins raises the possibility of targeting DOT1S for therapeutic intervention and reveals that mouse DOT1 L plays an essential role in embryonic development, hematopoiesis, cardiac function, and the development of leukemia.
Abstract: DOT1 (disruptor of telomeric silencing; also called Kmt4) was initially discovered in budding yeast in a genetic screen for genes whose deletion confers defects in telomeric silencing. Since the discovery ∼10 years ago that Dot1 and its mammalian homolog, DOT1L (DOT1-Like), possess histone methyltransferase activity toward histone H3 Lys 79, great progress has been made in characterizing their enzymatic activities and the role of Dot1/DOT1L-mediated H3K79 methylation in transcriptional regulation, cell cycle regulation, and the DNA damage response. In addition, gene disruption in mice has revealed that mouse DOT1L plays an essential role in embryonic development, hematopoiesis, cardiac function, and the development of leukemia. The involvement of DOT1L enzymatic activity in leukemogenesis driven by a subset of MLL (mixed-lineage leukemia) fusion proteins raises the possibility of targeting DOT1L for therapeutic intervention.
TL;DR: The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000, and recently, many important studies have reported that G9a and GLP play critical roles in various biological processes.
Abstract: The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000. Since then, research on histone methylation has progressed rapidly. Among the identified HKMTs in mammals, G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9me1 and H3K9me2), and exist predominantly as a G9a–GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. Recently, many important studies have reported that G9a and GLP play critical roles in various biological processes. The physiological relevance of G9a/GLP-mediated epigenetic gene regulation is discussed.
TL;DR: It is shown that maintenance of the WUS gradient is essential to regulate stem cell number and Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.
Abstract: WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.
TL;DR: Increased macrophage infiltration and cathepsin protease levels in mammary tumors following paclitaxel (Taxol) chemotherapy are found and this study highlights the importance of integrated targeting of the tumor and its microenvironment and implicates macrophages andCathepsins in blunting chemotherapeutic response.
Abstract: The microenvironment is known to critically modulate tumor progression, yet its role in regulating treatment response is poorly understood. Here we found increased macrophage infiltration and cathepsin protease levels in mammary tumors following paclitaxel (Taxol) chemotherapy. Cathepsin-expressing macrophages protected against Taxol-induced tumor cell death in coculture, an effect fully reversed by cathepsin inhibition and mediated partially by cathepsins B and S. Macrophages were also found to protect against tumor cell death induced by additional chemotherapeutics, specifically etoposide and doxorubicin. Combining Taxol with cathepsin inhibition in vivo significantly enhanced efficacy against primary and metastatic tumors, supporting the therapeutic relevance of this effect. Additionally incorporating continuous low-dose cyclophosphamide dramatically impaired tumor growth and metastasis and improved survival. This study highlights the importance of integrated targeting of the tumor and its microenvironment and implicates macrophages and cathepsins in blunting chemotherapeutic response.
TL;DR: It is demonstrated that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible, and it is shown that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells.
Abstract: Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.
TL;DR: It is reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation and may be a new therapeutic target for the treatment of hypoxic tumors.
Abstract: Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.
TL;DR: The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence and overexpression of LB1 increases the proliferation rate and delays the onset of senescences of human diploid cells through a ROS signaling pathway.
Abstract: Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.
TL;DR: It is concluded that through regulation of expression of DNA damage response genes, CycK/Cdk12 protects cells from genomic instability and the essential role of Cyck for organisms in vivo is further supported.
Abstract: Various cyclin-dependent kinase (Cdk) complexes have been implicated in the regulation of transcription. In this study, we identified a 70-kDa Cyclin K (CycK) that binds Cdk12 and Cdk13 to form two different complexes (CycK/Cdk12 or CycK/Cdk13) in human cells. The CycK/Cdk12 complex regulates phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and expression of a small subset of human genes, as revealed in expression microarrays. Depletion of CycK/Cdk12 results in decreased expression of predominantly long genes with high numbers of exons. The most prominent group of down-regulated genes are the DNA damage response genes, including the critical regulators of genomic stability: BRCA1 (breast and ovarian cancer type 1 susceptibility protein 1), ATR (ataxia telangiectasia and Rad3-related), FANCI, and FANCD2. We show that CycK/Cdk12, rather than CycK/Cdk13, is necessary for their expression. Nuclear run-on assays and chromatin immunoprecipitations with RNA polymerase II on the BRCA1 and FANCI genes suggest a transcriptional defect in the absence of CycK/Cdk12. Consistent with these findings, cells without CycK/Cdk12 induce spontaneous DNA damage and are sensitive to a variety of DNA damage agents. We conclude that through regulation of expression of DNA damage response genes, CycK/Cdk12 protects cells from genomic instability. The essential role of CycK for organisms in vivo is further supported by the result that genetic inactivation of CycK in mice causes early embryonic lethality.
TL;DR: A technology called iPOND (isolation of proteins on nascent DNA) is described to analyze proteins at active and damaged replication forks at high resolution and is established as a useful methodology to study DNA replication and chromatin maturation.
Abstract: Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.
TL;DR: A novel and unexpected activity of the proneural gene Ascl1 is identified, and a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation is revealed.
Abstract: Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.
TL;DR: It is shown that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes, and that this underlies the effects of R loops on genomic stability.
Abstract: Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.
TL;DR: Conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis and reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3k27me3.
Abstract: Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. Here, we conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. Our findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3.
TL;DR: Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.
Abstract: Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective toward the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells and mouse epiblast stem cells to study specification of definitive endoderm in vitro. Using a combination of whole-genome expression and chromatin immunoprecipitation (ChIP) deep sequencing (ChIP-seq) analyses, we established an hierarchy of transcription factors regulating endoderm specification. Importantly, the pluripotency factors NANOG, OCT4, and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMESODERMIN (EOMES), which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.
TL;DR: These studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.
Abstract: Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.