ABSTRACT
Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.
Subject(s)
Cellular Senescence , RNA, Small Nucleolar , Ribosomal Proteins , Ribosomes , Tumor Suppressor Protein p53 , Humans , RNA, Small Nucleolar/metabolism , RNA, Small Nucleolar/genetics , Cellular Senescence/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Ribosomes/metabolism , Animals , Mice , Ribosomal Proteins/metabolism , Ribosomal Proteins/geneticsABSTRACT
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.
Subject(s)
MicroRNAs , Mice , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Mammals/genetics , Base SequenceABSTRACT
Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.
Subject(s)
Intestinal Mucosa/physiology , MicroRNAs/metabolism , Animals , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dextran Sulfate , Humans , Insulin-Like Growth Factor Binding Protein 5/genetics , Intestinal Mucosa/cytology , Mesoderm/metabolism , Mice , MicroRNAs/genetics , Myofibroblasts/metabolism , Paracrine Communication , Regeneration , Somatomedins/metabolismABSTRACT
Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.
Subject(s)
Adipogenesis/genetics , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , MicroRNAs/metabolism , Obesity/genetics , Stem Cells/cytology , Animals , Diet, High-Fat , Gene Expression , Gene Knockdown Techniques , Mice , MicroRNAs/geneticsABSTRACT
Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.
Subject(s)
Exoribonucleases/genetics , Fetal Macrosomia/genetics , Insulin-Like Growth Factor II/genetics , Up-Regulation , Wilms Tumor/genetics , Animals , Cell Line , Disease Models, Animal , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , MicroRNAs/genetics , Mutation , Nephrons/cytology , Nephrons/physiopathology , Stem CellsABSTRACT
Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apc(min/+) mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.
Subject(s)
Adenoma/physiopathology , Carcinogenesis/genetics , Intestinal Neoplasms/physiopathology , MicroRNAs/metabolism , Adenoma/genetics , Animals , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/physiopathology , Intestinal Neoplasms/genetics , Mice , Mice, Transgenic , MicroRNAs/genetics , Tumor Cells, CulturedABSTRACT
The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.
Subject(s)
Cell Transdifferentiation , Cellular Reprogramming , Fibroblasts/cytology , Heart/physiology , Myocardial Infarction/therapy , Myocytes, Cardiac/cytology , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Lineage , Fibroblasts/physiology , Heart/physiopathology , Mice , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/cytology , Myocardium/pathology , Myocytes, Cardiac/physiology , Phenotype , Regenerative Medicine/methods , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , S100 Proteins/metabolism , Tail/cytology , Transcription Factors/geneticsABSTRACT
The lineage relationships of fetal adrenal cells and adrenal capsular cells to the differentiated adrenal cortex are not fully understood. Existing data support a role for each cell type as a progenitor for cells of the adult cortex. This report reveals that subsets of capsular cells are descendants of fetal adrenocortical cells that once expressed Nr5a1. These fetal adrenocortical cell descendants within the adrenal capsule express Gli1, a known marker of progenitors of steroidogenic adrenal cells. The capsule is also populated by cells that express Tcf21, a known inhibitor of Nr5a1 gene expression. We demonstrate that Tcf21-expressing cells give rise to Nr5a1-expressing cells but only before capsular formation. After the capsule has formed, capsular Tcf21-expressing cells give rise only to non-steroidogenic stromal adrenocortical cells, which also express collagen 1a1, desmin and platelet-derived growth factor (alpha polypeptide) but not Nr5a1. These observations integrate prior observations that define two separate origins of adult adrenocortical steroidogenic cells (fetal adrenal cortex and/or the adrenal capsule). Thus, these observations predict a unique temporal and/or spatial role of adult cortical cells that arise directly from either fetal cortical cells or from fetal cortex-derived capsular cells. Last, the data uncover the mechanism by which two populations of fetal cells (fetal cortex derived Gli1-expressing cells and mesenchymal Tcf21-expressing mesenchymal cells) participate in the establishment of the homeostatic capsular progenitor cell niche of the adult cortex.
Subject(s)
Adrenal Cortex/cytology , Adrenal Cortex/embryology , Cell Lineage , Fetus/cytology , Stem Cells/cytology , Steroids/metabolism , Aging/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Homeostasis , Mice , Models, Biological , Steroidogenic Factor 1/metabolism , Stromal Cells , Transcription Factors/metabolism , Zinc Finger Protein GLI1ABSTRACT
The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Lineage , Epithelial-Mesenchymal Transition , Fibroblasts/cytology , Myocardium/cytology , Stem Cells/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Coronary Vessels/cytology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Integrases/metabolism , Mice , Models, Biological , Muscle, Smooth, Vascular/cytology , Myocardium/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Phenotype , Stem Cells/metabolism , Time FactorsABSTRACT
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8 , which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.
ABSTRACT
The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA and cardamom-treated (DMBA+CARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral administration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glutathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in 'DMBA+CARD' mice. Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in the same 'DMBA+CARD' group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked NF-κB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas generated on the skin due to the DMBA treatment were reduced in the 'DMBA+CARD' group. Thus, the results from the present study suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.
Subject(s)
Antioxidants/therapeutic use , Elettaria/chemistry , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Signal Transduction , Skin Neoplasms/prevention & control , Spices , 9,10-Dimethyl-1,2-benzanthracene/antagonists & inhibitors , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Anticarcinogenic Agents/therapeutic use , Carcinogens/antagonists & inhibitors , Carcinogens/toxicity , Cyclooxygenase 2/chemistry , Cyclooxygenase 2/metabolism , Female , Metabolic Detoxication, Phase II , Mice , NF-kappa B/antagonists & inhibitors , Neoplasm Proteins/metabolism , Papilloma/chemically induced , Papilloma/metabolism , Papilloma/pathology , Papilloma/prevention & control , Seeds/chemistry , Skin/drug effects , Skin/metabolism , Skin/pathology , Skin Neoplasms/chemically induced , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tumor BurdenABSTRACT
Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Lineage , Integrases/metabolism , Kidney/metabolism , Recombination, Genetic , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Movement , Embryo, Mammalian/metabolism , Embryonic Development , Enzyme Activation , Female , Gene Expression Regulation, Developmental , Genes, Reporter , Genetic Loci , Gonads/metabolism , Heart/drug effects , Heart/embryology , Integrases/genetics , Kidney/drug effects , Kidney/embryology , Male , Mice , Mice, Transgenic , Pregnancy , Species Specificity , Tamoxifen/pharmacologyABSTRACT
To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.
Subject(s)
Adenocarcinoma, Sebaceous/metabolism , Carcinoma, Squamous Cell/metabolism , Skin Neoplasms/metabolism , Transcription Factor AP-1/metabolism , Adenocarcinoma, Sebaceous/pathology , Animals , Carcinoma, Squamous Cell/pathology , DNA, Neoplasm/metabolism , Hyperplasia , Keratinocytes/metabolism , Mice , Mice, Transgenic , Precancerous Conditions/genetics , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-fos/biosynthesis , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/metabolism , Sebaceous Glands/pathology , Skin Neoplasms/pathology , Transcription Factor AP-1/antagonists & inhibitors , Transcription Factor AP-1/genetics , Transcriptional Activation , Wnt Proteins/geneticsABSTRACT
Over the last 15 years, numerous studies have addressed the structural rules that regulate dimerization stability and dimerization specificity of the leucine zipper, a dimeric parallel coiled-coil domain that can either homodimerize or heterodimerize. Initially, these studies were performed with a limited set of B-ZIP proteins, sequence-specific DNA binding proteins that dimerize using the leucine zipper domain to bind DNA. A global analysis of B-ZIP leucine zipper dimerization properties can be rationalized using a limited number of structural rules [J.R. Newman, A.E. Keating, Comprehensive identification of human bZIP interactions with coiled-coil arrays, Science 300 (2003) 2097-2101]. Today, however, access to the genomic sequences of many different organisms has made possible the annotation of all B-ZIP proteins from several species and has generated a bank of data that can be used to refine, and potentially expand, these rules. Already, a comparative analysis of the B-ZIP proteins from Arabidopsis thaliana and Homo sapiens has revealed that the same amino acids are used in different patterns to generate diverse B-ZIP dimerization patterns [C.D. Deppmann, A. Acharya, V. Rishi, B. Wobbes, S. Smeekens, E.J. Taparowsky, C. Vinson, Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs, Nucleic Acids Res. 32 (2004) 3435-3445]. The challenge ahead is to investigate the biological significance of different B-ZIP protein-protein interactions. Gaining insight at this level will rely on ongoing investigations to (a) define the role of target DNA on modulating B-ZIP dimerization partners, (b) characterize the B-ZIP transcriptome in various cells and tissues through mRNA microarray analysis, (c) identify the genomic localization of B-ZIP binding at a genomic level using the chromatin immunoprecipitation assay, and (d) develop more sophisticated imaging technologies to visualize dimer dynamics in single cells and whole organisms. Studies of B-ZIP family leucine zipper dimerization and the regulatory mechanisms that control their biological activities could serve as a paradigm for deciphering the biophysical and biological parameters governing other well-characterized protein-protein interaction motifs. This review will focus on the dimerization specificity of coiled-coil proteins, particularly the human B-ZIP transcription family that consists of 53 proteins that use the leucine zipper coiled-coil as a dimerization motif.
Subject(s)
Nuclear Proteins/chemistry , RNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Motifs , Amino Acid Sequence , DNA/metabolism , Dimerization , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Thermodynamics , Transcription Factors/metabolismABSTRACT
We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5'-TAACGTTA-3'), C/EBP (5'-TTGCGCAA-3') and AP-1 (5'-TGAGTCA-3') sites. The addition of 10 mM MgCl2 diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using 'standard' EMSA conditions (0.25x TBE), CREB bound all the DNA sequences examined. The CREB-CRE complex had an apparent Kd of approximately 300 pM, PAR of approximately 1 nM, C/EBP and AP-1 of approximately 3 nM and SP1 of approximately 30 nM. The addition of 10 mM MgCl2 to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl2. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.
Subject(s)
Avian Proteins , Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Magnesium/physiology , Animals , Basic-Leucine Zipper Transcription Factors , CCAAT-Enhancer-Binding Proteins/metabolism , Carrier Proteins/metabolism , Circular Dichroism , DNA/metabolism , Electrophoretic Mobility Shift Assay , Magnesium/pharmacology , Protein Denaturation , Protein Structure, Tertiary , Temperature , Transcription Factor AP-1/metabolism , Transcription Factors/metabolismABSTRACT
Basic region-leucine zipper (B-ZIP) proteins are a class of dimeric sequence-specific DNA-binding proteins unique to eukaryotes. We have identified 67 B-ZIP proteins in the Arabidopsis thaliana genome. No A.thaliana B-ZIP domains are homologous with any Homo sapiens B-ZIP domains. Here, we predict the dimerization specificity properties of the 67 B-ZIP proteins in the A.thaliana genome based on three structural properties of the dimeric alpha-helical leucine zipper coiled coil structure: (i) length of the leucine zipper, (ii) placement of asparagine or a charged amino acid in the hydrophobic interface and (iii) presence of interhelical electrostatic interactions. Many A.thaliana B-ZIP leucine zippers are predicted to be eight or more heptads in length, in contrast to the four or five heptads typically found in H.sapiens, a prediction experimentally verified by circular dichroism analysis. Asparagine in the a position of the coiled coil is typically observed in the second heptad in H.sapiens. In A.thaliana, asparagine is abundant in the a position of both the second and fifth heptads. The particular placement of asparagine in the a position helps define 14 families of homodimerizing B-ZIP proteins in A.thaliana, in contrast to the six families found in H.sapiens. The repulsive interhelical electrostatic interactions that are used to specify heterodimerizing B-ZIP proteins in H.sapiens are not present in A.thaliana. Instead, we predict that plant leucine zippers rely on charged amino acids in the a position to drive heterodimerization. It appears that A.thaliana define many families of homodimerizing B-ZIP proteins by having long leucine zippers with asparagine judiciously placed in the a position of different heptads.
Subject(s)
Arabidopsis/genetics , DNA-Binding Proteins/chemistry , Plant Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Asparagine/analysis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Genome, Human , Genome, Plant , Humans , Leucine Zippers , Molecular Sequence Data , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Alignment , Sequence Analysis, Protein , Static Electricity , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, and have important implications for let-7 based therapeutics.
Subject(s)
Gene Expression Regulation , Genes, Tumor Suppressor , MicroRNAs/biosynthesis , Neoplasms/pathology , Regeneration , Animals , MiceSubject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/classification , Transcription Factors/chemistry , Transcription Factors/classification , Amino Acid Motifs , Amino Acid Sequence , Animals , Crystallography, X-Ray , Dimerization , Humans , Leucine Zippers , Molecular Sequence Data , Protein Binding , ThermodynamicsABSTRACT
The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRß, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.