ABSTRACT
A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.
Subject(s)
Amino Acid Metabolism, Inborn Errors , Tandem Mass Spectrometry , Amino Acid Metabolism, Inborn Errors/metabolism , Chromatography, Liquid , Humans , Hydroxybutyrates/urine , Oxidoreductases , ValineABSTRACT
R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a "looped-out" nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes.
Subject(s)
DEAD-box RNA Helicases/metabolism , Genomic Instability/genetics , Nucleic Acid Conformation , Sirtuins/metabolism , Acetylation , DEAD-box RNA Helicases/genetics , DNA/chemistry , DNA/genetics , DNA Damage/genetics , DNA Helicases/metabolism , DNA Repair/genetics , DNA-Directed RNA Polymerases/metabolism , Enzyme Activation , Gene Knockdown Techniques , HEK293 Cells , Humans , MCF-7 Cells , Sirtuins/geneticsABSTRACT
Our aim was to search for new cellular binding partners for the E6 and E7 oncogenes of beta human papillomaviruses (HPV), whose direct role in skin carcinogenesis has not been thoroughly investigated. By employing glutathione S-transferase pulldown and coimmunoprecipitation, we identified nuclear myosin 1c as a binding partner of HPV 8 E7 protein. As nuclear myosin 1c is an essential component of the RNA polymerase I transcription complex, we studied the effects of HPV 8 E7 protein expression on ribosomal RNA (rRNA) expression. Here we show that the activity of RNA polymerase I is decreased and that pre-rRNA expression is consequently reduced due to HPV 8 E7 expression. However, the expression levels of mature cytoplasmic 18S and 28S rRNA are retained. We propose that by relieving their resources from the energy-consuming process of rRNA transcription, HPV 8 E7 expressing cells might support more efficient virus replication in the differentiating epithelium.
Subject(s)
Cell Nucleus/metabolism , Down-Regulation/physiology , Myosins/metabolism , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , Animals , COS Cells , Cell Differentiation/physiology , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Epithelium/metabolism , Epithelium/virology , HEK293 Cells , Humans , Papillomaviridae/metabolism , RNA Polymerase I/metabolism , Virus Replication/physiologyABSTRACT
SIRT7 is an NAD+-dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation.
Subject(s)
Cyclin-Dependent Kinase 9/metabolism , RNA Polymerase II/metabolism , Sirtuins/metabolism , Transcriptional Activation , Cell Line , Humans , Positive Transcriptional Elongation Factor B/metabolism , RNA/metabolism , RNA, Small Nucleolar/biosynthesis , Ribonucleoproteins, Small Nuclear/metabolism , Sirtuins/chemistryABSTRACT
SIRT7 is an NAD(+)-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-ribosomal RNA (rRNA) and promotes rRNA synthesis. Here we show that SIRT7 is also associated with small nucleolar RNP (snoRNPs) that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of pre-rRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA.
Subject(s)
RNA Precursors/metabolism , RNA Processing, Post-Transcriptional/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Small Nucleolar/metabolism , Ribonucleoproteins, Small Nucleolar/metabolism , Sirtuins/genetics , Blotting, Northern , Blotting, Western , Cell Line, Tumor , Chromatin Immunoprecipitation , Fluorescent Antibody Technique , Gene Knockdown Techniques , Gene Knockout Techniques , HEK293 Cells , Humans , Immunoprecipitation , In Vitro TechniquesABSTRACT
Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.
Subject(s)
Cell Movement , Neoplasms/pathology , Phosphoric Monoester Hydrolases/physiology , Cell Adhesion , HCT116 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/physiology , Neoplasm Metastasis , Phosphoproteins/physiology , Protein Tyrosine Phosphatases , Stress Fibers/physiologyABSTRACT
Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.
Subject(s)
Cell Nucleolus/metabolism , Genetic Loci , RNA Precursors/metabolism , RNA, Untranslated/metabolism , Alu Elements , Cell Nucleolus/genetics , HeLa Cells , Humans , Nucleic Acid Conformation , Oligodeoxyribonucleotides, Antisense/pharmacology , RNA Polymerase II/antagonists & inhibitors , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA, Untranslated/geneticsABSTRACT
Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF(I)110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD(+)-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF(I)110 is prevented. The results demonstrate that reversible phosphorylation of TAF(I)110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.
Subject(s)
Dual-Specificity Phosphatases/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Sirtuin 1/genetics , Transcription Factor TFIID/genetics , Transcription, Genetic , Acetylation , CDC2 Protein Kinase , Cell Nucleolus/genetics , Cyclin B/genetics , Cyclin B/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins , Dual-Specificity Phosphatases/metabolism , HeLa Cells , Histone Chaperones/genetics , Humans , Mitosis , Phosphorylation , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA Polymerase I/genetics , RNA, Ribosomal/biosynthesis , RNA, Ribosomal/genetics , Sirtuin 1/metabolism , Transcription Factor TFIID/metabolism , Transcription Factors/geneticsABSTRACT
Sirtuins are NAD(+)-dependent protein deacetylases that connect metabolism and cellular homeostasis. Here we show that the nuclear Sirtuin SIRT7 targets PAF53, a subunit of RNA polymerase I (Pol I). Acetylation of PAF53 at lysine 373 by CBP and deacetylation by SIRT7 modulate the association of Pol I with DNA, hypoacetylation correlating with increased rDNA occupancy of Pol I and transcription activation. SIRT7 is released from nucleoli in response to different stress conditions, leading to hyperacetylation of PAF53 and decreased Pol I transcription. Nucleolar detention requires binding of SIRT7 to nascent pre-rRNA, linking the spatial distribution of SIRT7 and deacetylation of PAF53 to ongoing transcription. The results identify a nonhistone target of SIRT7 and uncover an RNA-mediated mechanism that adapts nucleolar transcription to stress signaling.
Subject(s)
RNA Polymerase I/genetics , Sirtuins/metabolism , Transcription Factors/metabolism , Transcriptional Activation/genetics , Acetylation , CREB-Binding Protein/metabolism , HEK293 Cells , Humans , Lysine/genetics , RNA Polymerase I/antagonists & inhibitors , RNA Precursors/metabolism , Sirtuins/genetics , Stress, Physiological/genetics , Transcription Factors/geneticsABSTRACT
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.
Subject(s)
CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Dual-Specificity Phosphatases/physiology , Mitosis , cdc25 Phosphatases/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle/physiology , Cells, Cultured , Dual-Specificity Phosphatases/antagonists & inhibitors , Dual-Specificity Phosphatases/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genomic Instability/genetics , HeLa Cells , Humans , Mitosis/drug effects , Mitosis/genetics , Mitosis/physiology , Models, Biological , Phosphorylation/drug effects , Phosphorylation/genetics , RNA, Small Interfering/pharmacology , Time FactorsABSTRACT
AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.
Subject(s)
AMP-Activated Protein Kinases/metabolism , RNA, Ribosomal/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Cell Line , Energy Metabolism , Glucose/metabolism , Humans , Mice , Models, Biological , NIH 3T3 Cells , Phosphorylation , Pol1 Transcription Initiation Complex Proteins/antagonists & inhibitors , Pol1 Transcription Initiation Complex Proteins/chemistry , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA Polymerase I/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/chemistry , Transcription, GeneticABSTRACT
Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD-/- cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
Subject(s)
CDC2 Protein Kinase/antagonists & inhibitors , Cyclin B/antagonists & inhibitors , Death Domain Receptor Signaling Adaptor Proteins/physiology , Mitosis/physiology , Animals , Body Size , CDC2 Protein Kinase/metabolism , Cells, Cultured , Cyclin B/metabolism , Cyclin B1 , Death Domain Receptor Signaling Adaptor Proteins/deficiency , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Fibroblasts , Interphase/physiology , Mice , Mice, Knockout , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Organ Size , Protein Binding , RNA, Ribosomal/biosynthesisABSTRACT
Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.
Subject(s)
Gene Expression Regulation/genetics , Genome, Human/genetics , TATA-Binding Protein Associated Factors/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factors/genetics , Binding Sites/genetics , Chromatin Immunoprecipitation/methods , CpG Islands/genetics , Humans , Microarray Analysis , Principal Component Analysis , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TATA-Binding Protein Associated Factors/geneticsABSTRACT
The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.
Subject(s)
Cell Cycle/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA Polymerase I/metabolism , Acetylation , Animals , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Mice , NIH 3T3 Cells , RNA, Ribosomal/biosynthesis , Transcriptional ActivationABSTRACT
We investigated the role of SIRT7, one of the seven members of the mammalian sirtuin family. We show that SIRT7 is a widely expressed nucleolar protein that is associated with active rRNA genes (rDNA), where it interacts with RNA polymerase I (Pol I) as well as with histones. Overexpression of SIRT7 increases Pol I-mediated transcription, whereas knockdown of SIRT7 or inhibition of the catalytic activity results in decreased association of Pol I with rDNA and a reduction of Pol I transcription. Depletion of SIRT7 stops cell proliferation and triggers apoptosis. Our findings suggest that SIRT7 is a positive regulator of Pol I transcription and is required for cell viability in mammals.
Subject(s)
RNA Polymerase I/metabolism , Sirtuins/metabolism , Transcriptional Activation , Animals , Apoptosis , Cell Line , Cell Proliferation , Cell Survival , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Genes, rRNA , Histones/metabolism , Humans , Mice , RNA Interference , RNA Polymerase I/genetics , Sirtuins/antagonists & inhibitors , Sirtuins/geneticsABSTRACT
Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Myosin Type I/metabolism , Nuclear Proteins/metabolism , RNA Polymerase I/genetics , Transcription Factors/metabolism , Transcription, Genetic , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Cell Nucleus/chemistry , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Myosin Type I/chemistry , Myosin Type I/genetics , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Protein Binding/genetics , RNA Polymerase I/biosynthesis , RNA Polymerase I/chemistry , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.
Subject(s)
Myeloid Cells/cytology , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/physiology , Proteins/genetics , Proteins/physiology , Animals , Base Sequence , Cell Cycle , Cell Cycle Proteins , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Division/genetics , Cell Division/physiology , Cell Line , DNA, Complementary/genetics , Insulin Receptor Substrate Proteins , Insulin-Like Growth Factor I/pharmacology , Leukemia, Myeloid/etiology , Mice , Models, Biological , Myeloid Cells/drug effects , Myeloid Cells/physiology , Phosphoproteins/genetics , Phosphoproteins/physiology , RNA-Binding Proteins , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/physiology , Transduction, GeneticABSTRACT
The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions.
Subject(s)
Protein Serine-Threonine Kinases , Serine/chemistry , cdc25 Phosphatases/chemistry , Alanine/chemistry , Blotting, Western , Cell Line , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Cyclin E/metabolism , DNA/metabolism , DNA Damage , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , HeLa Cells , Histidine/chemistry , Humans , Models, Biological , Mutation , Peptide Mapping , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , S Phase , Time Factors , Transfection , Tumor Cells, Cultured , Ultraviolet RaysABSTRACT
We describe the clinical and serological followup of a 9-year-old girl with anti-nucleolar organizing region 90/human upstream-binding factor (anti-NOR 90/hUBF) who had features of systemic sclerosis over a period of 17 years, from childhood into adulthood. We review the associations of anti-UBF autoantibodies, and provide evidence that anti-NOR 90/UBF immune response is antigen driven.