RESUMO
We show that the time required to transcribe human genes larger than 800 kb spans more than one complete cell cycle, while their transcription speed equals that of smaller genes. Independently of their expression status, we find the long genes to replicate late. Regions of concomitant transcription and replication in late S phase exhibit DNA break hot spots known as common fragile sites (CFSs). This CFS instability depends on the expression of the underlying long genes. We show that RNA:DNA hybrids (R-loops) form at sites of transcription/replication collisions and that RNase H1 functions to suppress CFS instability. In summary, our results show that, on the longest human genes, collisions of the transcription machinery with a replication fork are inevitable, creating R-loops and consequent CFS formation. Functional replication machinery needs to be involved in the resolution of conflicts between transcription and replication machineries to ensure genomic stability.
Assuntos
Sítios Frágeis do Cromossomo/genética , Replicação do DNA , Genes/genética , Instabilidade Genômica/genética , Transcrição Gênica , Ciclo Celular/genética , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Humanos , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , Ribonuclease H/metabolismo , Fatores de TempoRESUMO
Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5-E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development.
Assuntos
Inversão Cromossômica , Deformidades Congênitas do Pé , Pé , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Animais , Cromossomos Humanos Par 5 , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Pé/anatomia & histologia , Pé/crescimento & desenvolvimento , Proteínas Hedgehog , Humanos , Hibridização In Situ , Articulações/anatomia & histologia , Articulações/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Morfogênese , Osteogênese/fisiologia , FenótipoRESUMO
Predicting responsiveness to anticancer therapy based on molecular findings at diagnosis is important to optimize treatment decisions. Although clinical outcome correlates with distinct mutations in some cancer entities, treatment responses within these lesion-stratified subgroups still remain heterogeneous, underscoring the need for additional prognosticators. We previously demonstrated that defined genetic defects at the INK4a/ARF locus, which encodes the tumor suppressors p16INK4a and ARF, not only accelerated lymphomagenesis in the Emu-myc transgenic mouse but also interfered with treatment sensitivity. In this study, we take a nonbiased genome-wide approach to examine whether the responsiveness of these lymphomas can be further stratified based on cytogenetic information at diagnosis. Indeed, using spectral karyotyping, comparative genomic hybridization, and fluorescence in situ hybridization in 38 primary lymphomas, we find recurrent cytogenetic alterations that refine the predictive value of INK4a/ARF lesions on drug responses in vivo: gain of chromosome 14, which was never detected in INK4a/ARFnull lymphomas, defined an ARFnull subgroup with superior treatment outcome. Gain of chromosome 6 was identified as a recurrent chromosomal aberration that predisposed ARFnull tumors to their subsequent INK4a loss during therapy. These data illustrate how cytogenetic information from cancer specimens might complement established prognostic markers and may improve anticancer treatment strategies.
Assuntos
Aberrações Cromossômicas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Animais , Tomada de Decisões , Modelos Animais de Doenças , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Camundongos Transgênicos , Valor Preditivo dos Testes , Resultado do TratamentoRESUMO
To ensure accurate duplication of genetic material, the replication fork must overcome numerous natural obstacles on its way, including transcription complexes engaged along the same template. Here we review the various levels of interdependence between transcription and replication processes and how different types of encounters between RNA- and DNA-polymerase complexes may result in clashes of those machineries on the DNA template and thus increase genomic instability. In addition, we summarize strategies evolved in bacteria and eukaryotes to minimize the consequences of collisions, including R-loop formation and topological stresses.
Assuntos
Replicação do DNA , Instabilidade Genômica , Transcrição GênicaRESUMO
The Chromatin, Replication and Chromosomal Stability Conference took place on June 20-21 in Stockholm, Sweden. In this article, I outline the broad scientific program of the meeting which reflected the wide diversity in epigenetics research. Distinct histone modifications are linked with specific chromatin structures and intranuclear positioning, thereby impacting replication timing and replication initiation, which in turn are related to gene expression and cell differentiation. Interference in any of these interconnected mechanisms can result in DNA breakage and lead to the activation of repair pathways. The DNA repair mechanisms again are influenced by the chromatin structure. In summary, the conference highlighted the functional implication of epigenetics in chromatin compaction, transcription regulation, replication control and DNA repair. The tight control of all these mechanisms defines the final cellular character.
Assuntos
Cromatina/fisiologia , Instabilidade Cromossômica , Congressos como Assunto , Reparo do DNA/fisiologia , Período de Replicação do DNA/fisiologia , Replicação do DNA/fisiologia , Epigenômica/métodos , Epigenômica/tendênciasRESUMO
The cells in the preimplantation mammalian embryo undergo several rounds of fast cell division. Whether the known DNA repair pathways are active during these early stages of development where cell division is of primary importance, has not been fully established. Because of the important role of phosphorylated H2A.X (gammaH2A.X) in the DNA damage response as well as its putative role in assembly of embryonic chromatin, we analysed its distribution in the preimplantation mouse embryo. We found that H2A.X is highly phosphorylated throughout preimplantation development in the absence of any induced DNA damage. Moreover, gammaH2A.X levels vary significantly throughout the cell cycle. Interestingly, after the 4-cell stage, we detected high levels of H2A.X phosphorylation in mitosis, where telomeres appeared focally enriched with gammaH2A.X. In contrast, 53BP1, which is known to be recruited to DNA damage sites, is undetectable at mitotic chromosomes at these stages and its localisation changes upon blastocyst formation from mainly nuclear to cytoplasmic. We also show that 53BP1 and gammaH2A.X rarely colocalise, suggesting that the high levels of phosphorylation of H2A.X in the embryo might not be directly linked to the DNA damage response in the embryo. Our data suggest that phosphorylation of H2A.X is an important event in the fast dividing cells of the early embryo in the absence of any induced DNA damage. We discuss the possible consequences of these findings on the genome-wide chromatin remodelling that ocurs in the preimplantation mammalian embryo.
Assuntos
Dano ao DNA , Desenvolvimento Embrionário/fisiologia , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Proteínas Cromossômicas não Histona , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Proteínas de Ligação a DNA , Feminino , Masculino , Camundongos , Mitose , Mórula/citologia , Mórula/metabolismo , Fosforilação , Gravidez , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Zigoto/metabolismoRESUMO
Common fragile sites (CFSs) are expressed as chromosome gaps in cells of different species including human and mouse as a result of the inhibition of DNA replication. They may serve as hot spots for DNA breakage in processes such as tumorigenesis and chromosome evolution. Using multicolor fluorescence in situ hybridization mapping, the authors describe here human CFS FRA7K on chromosome band 7q31.1 and its murine homolog Fra12C1. Within the syntenic FRA7K/Fra12C1 region lies the IMMP2L/Immp2l gene with a size of 899/983 kb. The authors further mapped 2 amplification breakpoints of the breast cancer cell line SKBR3 to the CFSs FRA7G and FRA7H. The 5 molecularly defined CFSs on chromosome 7 do not preferentially colocalize with synteny breaks between the human and mouse genomes and with intragenomic duplications that have occurred during chromosome evolution. In addition, in contrast to all currently reported data, CFSs in chromosome band 7q31 do not show increased DNA helix flexibility in comparison with control regions without CFS expression.
Assuntos
Quebra Cromossômica , Sítios Frágeis do Cromossomo/genética , Cromossomos Humanos Par 7/genética , Evolução Molecular , Sintenia , Animais , Neoplasias da Mama/genética , Fragilidade Cromossômica , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Bases de Dados Genéticas , Amplificação de Genes , Genoma , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/metabolismoRESUMO
Common fragile sites (CFSs) are seen as chromosomal gaps and breaks brought about by inhibition of replication, and it is thought that they cluster with tumor breakpoints. This study presents a comprehensive analysis using conventional and molecular cytogenetic mapping of CFSs and their expression frequencies in two mouse strains, BALB/c and C57BL/6, and in human probands. Here we show that induced mouse CFSs relate to sites of spontaneous gaps and breaks and that CFS expression levels in chromosome bands are conserved between the two mouse strains and between syntenic mouse and human DNA segments. Furthermore, four additional mouse CFSs were found to be homologous to human CFSs on the molecular cytogenetic level (Fra2D-FRA2G, Fra4C2-FRA9E, Fra6A3.1-FRA7G, and Fra6B1-FRA7H), increasing the number of such CFSs already described in the literature to eight. Contrary to previous reports, DNA helix flexibility is not increased in the 15 human and eight mouse CFSs molecularly defined so far, compared to large nonfragile control regions. Our findings suggest that the mechanisms that provoke instability at CFSs are evolutionarily conserved. The role that large transcriptionally active genes may play in CFS expression is discussed.