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1.
Mol Cell ; 58(2): 323-38, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25843623

RESUMO

Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.


Assuntos
Cromatina/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Células HeLa , Humanos , Hidroxiureia/farmacologia , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
2.
J Biol Chem ; 296: 100349, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33524394

RESUMO

The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Descoberta de Drogas , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Células HeLa , Humanos , Camundongos SCID , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Nat Rev Mol Cell Biol ; 9(10): 815-20, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18784729

RESUMO

Covalent post-translational modifications (PTMs) provide vast indexing potential and expanded protein use. The 'histone code' hypothesis has inspired rapid advances throughout chromatin biology, and has recently been tapped for its relevance to non-histone proteins. Comprehensive analyses suggest that rather than constituting a general code, the covalent modifications of proteins (including histones) provide surfaces that are recognized by effectors that can give rise to intricate interactions and downstream events. These are reminiscent of other regulatory cascades in transcription and cell signalling.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Animais , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Modelos Biológicos , Fosforilação , Proteínas/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
4.
J Biol Chem ; 291(25): 13014-27, 2016 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-27056325

RESUMO

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.


Assuntos
Proteína de Ligação a CREB/antagonistas & inibidores , Proteína p300 Associada a E1A/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Transcriptoma/efeitos dos fármacos
5.
Bioorg Med Chem Lett ; 25(9): 1842-8, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25851940

RESUMO

In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.


Assuntos
Azepinas/farmacologia , Desenho de Fármacos , Proteínas Nucleares/antagonistas & inibidores , Oxazóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Azepinas/síntese química , Azepinas/química , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Relação Estrutura-Atividade
6.
Proc Natl Acad Sci U S A ; 109(18): 6927-32, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22509028

RESUMO

The bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (CTD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other CTD kinases are inactive. Phosphorylation of the CTD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II CTD Ser2 kinase implicates it as a regulator of eukaryotic transcription.


Assuntos
Proteínas Nucleares/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Ciclo Celular , Células Cultivadas , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Estrutura Terciária de Proteína , RNA Polimerase II/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica
7.
Proc Natl Acad Sci U S A ; 108(40): 16669-74, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21949397

RESUMO

The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G(1) arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.


Assuntos
Apoptose/fisiologia , Linfoma de Burkitt/tratamento farmacológico , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Azepinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologia
8.
Nat Genet ; 31(1): 25-32, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11923873

RESUMO

Many transcription factors regulate specific temporal-spatial events during cardiac differentiation; however, the mechanisms that regulate such events are largely unknown. Using a modified subtractive hybridization method to identify specific genes that influence early cardiac development, we found that Bop is expressed specifically in cardiac and skeletal muscle precursors before differentiation of these lineages. Bop encodes a protein containing MYND and SET domains, which have been shown to regulate transcription by mediating distinct chromatin modifications. We show that m-Bop is a histone deacetylase-dependent transcriptional repressor. Targeted deletion of Bop in mice disrupted maturation of ventricular cardiomyocytes and interfered with formation of the right ventricle. Normal expression of Hand2, a transcription factor essential for right ventricular development, in cardiomyocyte precursors is dependent upon m-Bop. These results indicate that m-Bop is essential for cardiomyocyte differentiation and cardiac morphogenesis.


Assuntos
Proteínas Musculares , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Embrião de Galinha , Clonagem Molecular , Proteínas de Ligação a DNA , Coração/embriologia , Coração/crescimento & desenvolvimento , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Histona Desacetilases/metabolismo , Hibridização In Situ , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Miocárdio/citologia , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Transfecção
9.
Cells ; 12(13)2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37443729

RESUMO

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.


Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase , Chaperonas Moleculares , Desenvolvimento Muscular , Proteínas Musculares , Fatores de Transcrição , Ativação Transcricional , Humanos , Catálise , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Chaperonas Moleculares/metabolismo , Desenvolvimento Muscular/genética , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Nat Struct Mol Biol ; 14(6): 564-7, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17460694

RESUMO

Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.


Assuntos
Reparo do DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Biologia Computacional , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação/genética , Análise de Sequência de DNA , Ubiquitinação
11.
PLoS One ; 17(3): e0262378, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35353838

RESUMO

Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer.


Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Acetiltransferases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteína p300 Associada a E1A/genética , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Células MCF-7 , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo
12.
J Med Chem ; 65(16): 11177-11186, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-35930799

RESUMO

Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.


Assuntos
DNA Helicases , Fatores de Transcrição , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares , Domínios Proteicos
13.
Curr Opin Cell Biol ; 16(3): 263-71, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15145350

RESUMO

Considerable advances into the basis of RNA-polymerase-II-mediated transcriptional regulation have recently emerged. Biochemical, genetic and structural studies have contributed to novel insights into transcription, as well as the functional significance of covalent histone modifications. New details regarding transcription elongation through chromatin have further defined the mechanism behind this action, and identified how chromatin structure may be maintained after RNAP II traverses a nucleosome. ATP-dependent chromatin remodeling complexes, along with histone chaperone complexes, were recently discovered to facilitate histone exchange. In addition, it has become increasingly clear that transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm.


Assuntos
RNA Polimerase II/genética , Transcrição Gênica , Animais , Cromatina/genética , Histonas/metabolismo , Humanos , Nucleossomos/genética , RNA Mensageiro/metabolismo , Fatores de Elongação da Transcrição/genética
15.
ACS Med Chem Lett ; 11(6): 1324-1329, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32551019

RESUMO

The histone acetyltransferases, CREB binding protein (CBP) and EP300, are master transcriptional co-regulators that have been implicated in numerous diseases, such as cancer, inflammatory disorders, and neurodegeneration. A novel, highly potent, orally bioavailable EP300/CBP histone acetyltransferase (HAT) inhibitor, CPI-1612 or 17, was developed from the lead compound 3. Replacement of the indole scaffold of 3 with the aminopyridine scaffold of 17 led to improvements in potency, solubility, and bioavailability. These characteristics resulted in a 20-fold lower efficacious dose for 17 relative to lead 3 in a JEKO-1 tumor mouse xenograft study.

16.
ACS Med Chem Lett ; 11(6): 1205-1212, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32551002

RESUMO

Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27-a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.

17.
Nat Rev Drug Discov ; 18(8): 609-628, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31273347

RESUMO

Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-molecule targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chemical probe discovery to understand the biological potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compounds have since advanced to human clinical trials. Here, we review the current state of BET inhibitor biology in relation to clinical development, and we discuss the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications. The lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.


Assuntos
Desenvolvimento de Medicamentos/métodos , Fatores de Transcrição/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Epigênese Genética , Humanos , Terapia de Alvo Molecular , Fatores de Transcrição/genética
18.
Fluids Barriers CNS ; 15(1): 22, 2018 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-30111340

RESUMO

BACKGROUND: Choroid plexus epithelial cells express high levels of transthyretin, produce cerebrospinal fluid and many of its proteins, and make up the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells are vital to brain health and may be involved in neurological diseases. Transgenic mice containing fluorescent and luminescent reporters of these cells would facilitate their study in health and disease, but prior transgenic reporters lost expression over the early postnatal period. METHODS: Human bacterial artificial chromosomes in which the transthyretin coding sequence was replaced with DNA for tdTomato or luciferase 2 were used in pronuclear injections to produce transgenic mice. These mice were characterized by visualizing red fluorescence, immunostaining, real-time reverse transcription polymerase chain reaction, and luciferase enzyme assay. RESULTS: Reporters were faithfully expressed in cells that express transthyretin constitutively, including choroid plexus epithelial cells, retinal pigment epithelium, pancreatic islets, and liver. Expression of tdTomato in choroid plexus began at the appropriate embryonic age, being detectable by E11.5. Relative levels of tdTomato transcript in the liver and choroid plexus paralleled relative levels of transcripts for transthyretin. Expression remained robust over the first postnatal year, although choroid plexus transcripts of tdTomato declined slightly with age whereas transthyretin remained constant. TdTomato expression patterns were consistent across three founder lines, displayed no sex differences, and were stable across several generations. Two of the tdTomato lines were bred to homozygosity, and homozygous mice are healthy and fertile. The usefulness of tdTomato reporters in visualizing and analyzing live Transwell cultures was demonstrated. Luciferase activity was very high in homogenates of choroid plexus and continued to be expressed through adulthood. Luciferase also was detectable in eye and pancreas. CONCLUSIONS: Transgenic mice bearing fluorescent and luminescent reporters of transthyretin should prove useful for tracking transplanted choroid plexus epithelial cells, for purifying the cells, and for reporting their derivation from stem cells. They also should prove useful for studying transthyretin synthesis by other cell types, as transthyretin has been implicated in many functions and conditions, including clearance of ß-amyloid peptides associated with Alzheimer's disease, heat shock in neurons, processing of neuropeptides, nerve regeneration, astrocyte metabolism, and transthyretin amyloidosis.


Assuntos
Plexo Corióideo/citologia , Células Epiteliais/citologia , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos , Modelos Animais , Pré-Albumina/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Plexo Corióideo/crescimento & desenvolvimento , Plexo Corióideo/metabolismo , Cromossomos Artificiais Bacterianos , Células Epiteliais/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Fígado/citologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Proteínas Luminescentes/genética , Pré-Albumina/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo
19.
ACS Med Chem Lett ; 8(7): 737-741, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28740608

RESUMO

The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.

20.
Mol Cancer ; 5: 26, 2006 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-16805913

RESUMO

BACKGROUND: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. RESULTS: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. CONCLUSION: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.


Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/química , Metiltransferases/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/química , Histona-Lisina N-Metiltransferase/química , Humanos , Lisina/química , Metiltransferases/biossíntese , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Homologia de Sequência de Aminoácidos , Complexo Correpressor Histona Desacetilase e Sin3
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