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1.
bioRxiv ; 2023 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-37873380

RESUMO

Physical activity is a modifiable lifestyle factor that is associated with a decreased risk for the development of breast cancer. While the exact mechanisms for the reduction in cancer risk due to physical activity are largely unknown, it is postulated that the biological reduction in cancer risk is driven by improvements in inflammation and immune function with exercise. Hematopoietic stem cells (HSCs) are the progenitor for all of the cells of the immune system and are involved in cancer immunosurveillance through differentiation into cytotoxic cell population. In this study, we investigate the role of physical activity (PA) in a spontaneously occurring model of breast cancer over time, with a focus on tumor incidence, circulating and tumor-infiltrating immune cells as well gene expression profiles of tumors and hematopoietic stem cells. Furthermore, we show that, in addition to a direct effect of PA on the immune cells of tumor-bearing mice, PA reduces the oxidative stress in HSCs of wildtype and tumor-bearing mice, and by doing so, alters the differentiation of the HSCs towards T cells in order to enhance cancer immunosurveillance.

2.
bioRxiv ; 2023 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-37693594

RESUMO

Aging and metabolic diseases are accompanied by systemic inflammation, but the mechanisms that induce this state are not known. We developed a human bone-marrow organoid system to explore mechanisms underlying metabolic-disease associated systemic inflammation. We find that a distinct type of hematopoietic stem cell (HSC) develops in the adipose-rich, yellow bone marrow, which is known to gradually replace the hematopoietic red marrow as we age and during metabolic disease. Unlike HSCs derived from the red bone marrow, HSCs derived from the yellow bone marrow have higher proliferation rates, increase myeloid differentiation, skew towards pro-inflammatory M1 macrophage differentiation, and express a distinct transcriptomic profile associated with responsiveness to wounding. Yellow marrow-derived HSCs express higher levels of the leptin receptor, which we find to be further increased in patients with type 2 diabetes. Our work demonstrates that the human long bone yellow marrow is a niche for a distinct class of HSCs which could underlie hematopoietic dysfunction during aging and metabolic disease processes suggesting a shared inflammaging mechanism.

3.
Sci Rep ; 10(1): 3567, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107419

RESUMO

Hypercholesterolemia accelerates the phenotypes of aging in hematopoietic stem cells (HSCs). As yet, little is known about the underlying mechanism. We found that hypercholesterolemia downregulates Ten eleven translocation 1 (Tet1) in HSCs. The total HSC population was increased, while the long-term (LT) population, side population and reconstitution capacity of HSCs were significantly decreased in Tet1-/- mice. Expression of the Tet1 catalytic domain in HSCs effectively restored the LT population and reconstitution capacity of HSCs isolated from Tet1-/- mice. While Tet1 deficiency upregulated the expression of p19 and p21 in HSCs by decreasing the H3K27me3 modification, the restoration of Tet1 activity reduced the expression of p19, p21 and p27 by restoring the H3K27me3 and H3K36me3 modifications on these genes. These results indicate that Tet1 plays a critical role in maintaining the quiescence and reconstitution capacity of HSCs and that hypercholesterolemia accelerates HSC aging phenotypes by decreasing Tet1 expression in HSCs.


Assuntos
Envelhecimento/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hipercolesterolemia/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , Hipercolesterolemia/genética , Masculino , Metilação , Camundongos , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas/genética
4.
Epigenetics Chromatin ; 12(1): 24, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992049

RESUMO

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.


Assuntos
Replicação do DNA , Histona Acetiltransferases/metabolismo , Acetilação , Camptotecina/toxicidade , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Inibidores da Topoisomerase I/toxicidade
5.
Nat Commun ; 9(1): 33, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29295997

RESUMO

People with type 2 diabetes mellitus (T2DM) have a 25-fold higher risk of limb loss than non-diabetics due in large part to impaired wound healing. Here, we show that the impaired wound healing phenotype found in T2D mice is recapitulated in lethally irradiated wild type recipients, whose hematopoiesis is reconstituted with hematopoietic stem cells (HSCs) from T2D mice, indicating an HSC-autonomous mechanism. This impaired wound healing phenotype of T2D mice is due to a Nox-2-dependent increase in HSC oxidant stress that decreases microRNA let-7d-3p, which, in turn, directly upregulates Dnmt1, leading to the hypermethylation of Notch1, PU.1, and Klf4. This HSC-autonomous mechanism reduces the number of wound macrophages and skews their polarization towards M1 macrophages. These findings reveal a novel inflammatory mechanism by which a metabolic disorder induces an epigenetic mechanism in HSCs, which predetermines the gene expression of terminally differentiated inflammatory cells that controls their number and function.


Assuntos
Diferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Cicatrização/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Células-Tronco Hematopoéticas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/citologia , Camundongos , MicroRNAs/genética , NADPH Oxidase 2/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas/genética , Receptor Notch1/genética , Transativadores/genética
6.
Cancer Res ; 77(9): 2351-2362, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28249902

RESUMO

Obesity will soon surpass smoking as the most preventable cause of cancer. Hypercholesterolemia, a common comorbidity of obesity, has been shown to increase cancer risk, especially colorectal cancer. However, the mechanism by which hypercholesterolemia or any metabolic disorder increases cancer risk remains unknown. In this study, we show that hypercholesterolemia increases the incidence and pathologic severity of colorectal neoplasia in two independent mouse models. Hypocholesterolemia induced an oxidant stress-dependent increase in miR101c, which downregulated Tet1 in hematopoietic stem cells (HSC), resulting in reduced expression of genes critical to natural killer T cell (NKT) and γδ T-cell differentiation. These effects reduced the number and function of terminally differentiated NKT and γδ T cells in the thymus, the colon submucosa, and during early tumorigenesis. These results suggest a novel mechanism by which a metabolic disorder induces epigenetic changes to reduce lineage priming of HSC toward immune cells, thereby compromising immunosurveillance against cancer. Cancer Res; 77(9); 2351-62. ©2017 AACR.


Assuntos
Neoplasias Colorretais/genética , Hipercolesterolemia/genética , MicroRNAs/genética , Oxigenases de Função Mista/genética , Obesidade/genética , Proteínas Proto-Oncogênicas/genética , Animais , Carcinogênese , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Ativação Linfocitária/genética , Camundongos , MicroRNAs/biossíntese , Oxigenases de Função Mista/biossíntese , Células T Matadoras Naturais/patologia , Obesidade/complicações , Obesidade/patologia , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS Genet ; 11(8): e1005438, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26263206

RESUMO

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citidina Desaminase/fisiologia , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Imunoprecipitação da Cromatina , DNA Intergênico/genética , Proteínas de Ligação a DNA , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Baço/citologia , Baço/enzimologia , Sequências de Repetição em Tandem
8.
J Immunol ; 193(3): 1440-50, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24973444

RESUMO

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR, but not for S-region DNA double-strand breaks (DSBs) during CSR, and it is not required for SHM. AID lacking the C terminus (ΔAID) is a dominant negative (DN) mutant, because human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that ΔAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. To have DN function, ΔAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have been shown to contribute to DSB formation in S regions, and we find in this study that Msh2 is required for the DN activity, because ΔAID is not a DN mutant in msh2(-/-) cells. Our results suggest that the DNA DSBs induced by ΔAID are unable to participate in CSR and might interfere with the ability of full-length AID to participate in CSR. We propose that ΔAID is impaired in its ability to recruit nonhomologous end joining repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by ΔAID have longer microhomologies than do those induced by full-length AID. In addition, our data suggest that AID binds Sµ regions in vivo as a monomer.


Assuntos
Citidina Desaminase/fisiologia , Reparo de Erro de Pareamento de DNA/imunologia , Rearranjo Gênico/imunologia , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fragmentos de Peptídeos/genética , Cultura Primária de Células
9.
J Immunol ; 192(10): 4887-96, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24729610

RESUMO

Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sµ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sµ DSBs accumulate as they lack a recombination partner.


Assuntos
Citidina Desaminase/imunologia , Rearranjo Gênico do Linfócito B/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Citidina Desaminase/genética , Dano ao DNA/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Rearranjo Gênico do Linfócito B/genética , Histonas/genética , Histonas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
10.
Genetics ; 190(2): 487-500, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22095079

RESUMO

Bromodomain proteins bind acetylated histones to regulate transcription. Emerging evidence suggests that histone acetylation plays an important role in DNA replication and repair, although its precise mechanisms are not well understood. Here we report studies of two double bromodomain-containing proteins, Bdf1 and Bdf2, in fission yeast. Loss of Bdf1 or Bdf2 led to a reduction in the level of histone H4 acetylation. Both bdf1Δ and bdf2Δ cells showed sensitivity to DNA damaging agents, including camptothecin, that cause replication fork breakage. Consistently, Bdf1 and Bdf2 were important for recovery of broken replication forks and suppression of DNA damage. Surprisingly, deletion of bdf1 or bdf2 partially suppressed sensitivity of various checkpoint mutants including swi1Δ, mrc1Δ, cds1Δ, crb2Δ, chk1Δ, and rad3Δ, to hydroxyurea, a compound that stalls replication forks and activates the Cds1-dependent S-phase checkpoint. This suppression was not due to reactivation of Cds1. Instead, we found that bdf2 deletion alleviates DNA damage accumulation caused by defects in the DNA replication checkpoint. We also show that hydroxyurea sensitivity of mrc1Δ and swi1Δ was suppressed by mutations in histone H4 acetyltransferase subunits or histone H4. These results suggest that the double bromodomain-containing proteins modulate chromatin structure to coordinate DNA replication and S-phase stress response.


Assuntos
Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Fase S/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Estresse Fisiológico/genética , Acetilação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Deleção de Genes , Histonas/metabolismo , Hidroxiureia/farmacologia , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
11.
J Immunol ; 187(5): 2464-75, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21804017

RESUMO

Activation-induced cytidine deaminase (AID) is induced in B cells during an immune response and is essential for both class-switch recombination (CSR) and somatic hypermutation of Ab genes. The C-terminal 10 aa of AID are required for CSR but not for somatic hypermutation, although their role in CSR is unknown. Using retroviral transduction into mouse splenic B cells, we show that the C terminus is not required for switch (S) region double-strand breaks (DSBs) and therefore functions downstream of DSBs. Using chromatin immunoprecipitation, we show that AID binds cooperatively with UNG and the mismatch repair proteins Msh2-Msh6 to Ig Sµ and Sγ3 regions, and this depends on the C terminus and the deaminase activity of AID. We also show that mismatch repair does not contribute to the efficiency of CSR in the absence of the AID C terminus. Although it has been demonstrated that both UNG and Msh2-Msh6 are important for introduction of S region DSBs, our data suggest that the ability of AID to recruit these proteins is important for DSB resolution, perhaps by directing the S region DSBs toward accurate and efficient CSR via nonhomologous end joining.


Assuntos
Citidina Desaminase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina/fisiologia , Região de Troca de Imunoglobulinas/fisiologia , Proteína 2 Homóloga a MutS/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Separação Celular , Imunoprecipitação da Cromatina , Citidina Desaminase/química , Citometria de Fluxo , Imunoglobulina G , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Nature ; 467(7312): 228-32, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20829797

RESUMO

Telomeres protect the normal ends of chromosomes from being recognized as deleterious DNA double-strand breaks. Recent studies have uncovered an apparent paradox: although DNA repair is prevented, several proteins involved in DNA damage processing and checkpoint responses are recruited to telomeres in every cell cycle and are required for end protection. It is currently not understood how telomeres prevent DNA damage responses from causing permanent cell cycle arrest. Here we show that fission yeast (Schizosaccharomyces pombe) cells lacking Taz1, an orthologue of human TRF1 and TRF2 (ref. 2), recruit DNA repair proteins (Rad22(RAD52) and Rhp51(RAD51), where the superscript indicates the human orthologue) and checkpoint sensors (RPA, Rad9, Rad26(ATRIP) and Cut5/Rad4(TOPBP1)) to telomeres. Despite this, telomeres fail to accumulate the checkpoint mediator Crb2(53BP1) and, consequently, do not activate Chk1-dependent cell cycle arrest. Artificially recruiting Crb2(53BP1) to taz1Δ telomeres results in a full checkpoint response and cell cycle arrest. Stable association of Crb2(53BP1) to DNA double-strand breaks requires two independent histone modifications: H4 dimethylation at lysine 20 (H4K20me2) and H2A carboxy-terminal phosphorylation (γH2A). Whereas γH2A can be readily detected, telomeres lack H4K20me2, in contrast to internal chromosome locations. Blocking checkpoint signal transduction at telomeres requires Pot1 and Ccq1, and loss of either Pot1 or Ccq1 from telomeres leads to Crb2(53BP1) foci formation, Chk1 activation and cell cycle arrest. Thus, telomeres constitute a chromatin-privileged region of the chromosomes that lack essential epigenetic markers for DNA damage response amplification and cell cycle arrest. Because the protein kinases ATM and ATR must associate with telomeres in each S phase to recruit telomerase, exclusion of Crb2(53BP1) has a critical role in preventing telomeres from triggering cell cycle arrest.


Assuntos
Reparo do DNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais , Telômero/metabolismo , Ciclo Celular , Dano ao DNA , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
13.
Cell Cycle ; 9(11): 2237-48, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20505337

RESUMO

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.


Assuntos
Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Alelos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Quinase do Ponto de Checagem 2 , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endonucleases/metabolismo , Endonucleases/fisiologia , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
14.
J Biol Chem ; 285(8): 5327-37, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20040595

RESUMO

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Delta cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-DeltaT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-DeltaT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-DeltaT/RT to telomeres, these factors are likely to enhance the ability of Trt1-DeltaT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.


Assuntos
Cromossomos Fúngicos/metabolismo , Heterocromatina/metabolismo , Recombinação Genética/fisiologia , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/genética , Deleção de Genes , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Deleção de Sequência , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
15.
PLoS Genet ; 5(8): e1000622, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19714219

RESUMO

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1(ATM) and Rad3(ATR) kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Delta rad3Delta cells. Thus, Tel1(ATM) and Rad3(ATR) are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1(ATM)/Rad3(ATR) kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/genética , Telômero/genética
16.
Mol Cell Biol ; 26(7): 2531-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16537899

RESUMO

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.


Assuntos
Proteínas Culina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Quinases Associadas a Fase S/metabolismo , Complexo do Signalossomo COP9 , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética
17.
J Virol ; 78(20): 11077-83, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15452228

RESUMO

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are complex retroviruses that persist in the host, eventually causing leukemia and neurological disease in a small percentage of infected individuals. In addition to structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (open reading frame I and II) proteins. The viral Tax and Rex proteins positively regulate virus production. Tax activates viral and cellular transcription to promote T-cell growth and, ultimately, malignant transformation. Rex acts posttranscriptionally to facilitate cytoplasmic expression of viral mRNAs that encode the structural and enzymatic gene products, thus positively controlling virion expression. Here, we report that both HTLV-1 and HTLV-2 have evolved accessory genes to encode proteins that act as negative regulators of both Tax and Rex. HTLV-1 p30(II) and the related HTLV-2 p28(II) inhibit virion production by binding to and retaining tax/rex mRNA in the nucleus. Reduction of viral replication in a cell carrying the provirus may allow escape from immune recognition in an infected individual. These data are consistent with the critical role of these proteins in viral persistence and pathogenesis in animal models of HTLV-1 and HTLV-2 infection.


Assuntos
Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Proteínas dos Retroviridae/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Produtos do Gene rex/metabolismo , Produtos do Gene tax/metabolismo , Genes pX , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/genética , Humanos , RNA Mensageiro/metabolismo , RNA Viral/metabolismo
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