RESUMO
Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure. SIGNIFICANCE: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging. This article is highlighted in the In This Issue feature, p. 1825.
Assuntos
Antineoplásicos , Segunda Neoplasia Primária , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Criança , Células-Tronco Hematopoéticas/patologia , Humanos , Mieloma Múltiplo/induzido quimicamente , Mieloma Múltiplo/genética , Mutação , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/genética , Segunda Neoplasia Primária/induzido quimicamente , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/patologia , FilogeniaRESUMO
BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .
Assuntos
Genoma Humano , Neoplasias , Análise Mutacional de DNA , Reparo do DNA , Humanos , Mutação , Acúmulo de Mutações , Neoplasias/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, but genetic instability is a major concern. Embryonic pluripotent cells also accumulate mutations during early development, but how this relates to the mutation burden in iPSCs remains unknown. Here, we directly compared the mutation burden of cultured iPSCs with their isogenic embryonic cells during human embryogenesis. We generated developmental lineage trees of human fetuses by phylogenetic inference from somatic mutations in the genomes of multiple stem cells, which were derived from different germ layers. Using this approach, we characterized the mutations acquired pre-gastrulation and found a rate of 1.65 mutations per cell division. When cultured in hypoxic conditions, iPSCs generated from fetal stem cells of the assessed fetuses displayed a similar mutation rate and spectrum. Our results show that iPSCs maintain a genomic integrity during culture at a similar degree as their pluripotent counterparts do in vivo.
RESUMO
Barrett's esophagus (BE) is categorized, based on morphological appearance, into different stages, which correlate with the risk of developing esophageal adenocarcinoma. More advanced stages are more likely to acquire chromosomal instabilities, but stage-specific markers remain elusive. Here, we performed single-cell DNA-sequencing experiments (scDNAseq) with fresh BE biopsies. Dysplastic BE cells frequently contained chromosomal instability (CIN) regions, and these CIN cells carried mutations corresponding to the COSMIC mutational signature SBS17, which were not present in biopsy-matched chromosomally stable (CS) cells or patient-matched nondiseased control cells. CS cells were predominantly found in nondysplastic BE biopsies. The single-base substitution (SBS) signatures of all CS BE cells analyzed were indistinguishable from those of nondiseased esophageal or gastric cells. Single-cell RNA-sequencing (scRNAseq) experiments with BE biopsies identified two sets of marker genes which facilitate the distinction between columnar BE epithelium and nondysplastic/dysplastic stages. Moreover, histological validation confirmed a correlation between increased CLDN2 expression and the presence of dysplastic BE stages. Our scDNAseq and scRNAseq datasets, which are a useful resource for the community, provide insight into the mutational landscape and gene expression pattern at different stages of BE development.
Assuntos
Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Análise de Célula Única/métodos , Adenocarcinoma/genética , Esôfago de Barrett/diagnóstico , Biomarcadores , Biópsia , Instabilidade Cromossômica , Epitélio , Esôfago , Expressão Gênica , Humanos , Hiperplasia/patologia , Mutação , Análise de Sequência de DNA , Sequenciamento do ExomaRESUMO
Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.
Assuntos
Leucemia Mieloide Aguda , Medula Óssea/patologia , Criança , Hematopoese , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Adulto JovemRESUMO
Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.
Assuntos
Reparo do DNA/genética , Guanosina/análogos & derivados , Neuroblastoma/genética , Adenina/metabolismo , Criança , Citosina/metabolismo , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Feminino , Guanina/metabolismo , Guanosina/genética , Guanosina/metabolismo , Humanos , Masculino , Mutagênese , Recidiva Local de Neoplasia/genética , Neuroblastoma/metabolismo , Estresse Oxidativo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.
Assuntos
Antivirais/efeitos adversos , Infecções por Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Mutação , Neoplasias , Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Humanos , Neoplasias/genética , TransplantadosRESUMO
Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell-derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing-repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.
Assuntos
Sistemas CRISPR-Cas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Edição de Genes , Mutação , Oncogenes/genética , Organoides , Alelos , Substituição de Aminoácidos , Animais , Células Epiteliais/metabolismo , Terapia Genética/métodos , Hepatócitos/metabolismo , Humanos , Análise de Sequência de DNARESUMO
Most known pathogenic point mutations in humans are Câ¢G to Tâ¢A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.
Assuntos
Adenina , LDL-Colesterol , Edição de Genes/métodos , Pró-Proteína Convertase 9/genética , Animais , LDL-Colesterol/sangue , LDL-Colesterol/genética , Fígado/metabolismo , Macaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.
Assuntos
Neoplasias do Colo do Útero , Animais , Carcinogênese , Epitélio , Feminino , Humanos , Camundongos , Organoides , PapillomaviridaeRESUMO
Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.
Assuntos
Citidina/genética , DNA/genética , Edição de Genes/métodos , Hepatócitos/metabolismo , RNA/genética , Adenoviridae/fisiologia , Animais , Vetores Genéticos/fisiologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BLRESUMO
Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.
Assuntos
Edição de Genes/métodos , Organoides/metabolismo , beta Catenina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células , ATPases Transportadoras de Cobre/genética , Desoxirribonuclease I/metabolismo , Diacilglicerol O-Aciltransferase/genética , Células HEK293 , Degeneração Hepatolenticular/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Reparo de DNA por Recombinação , Células-Tronco , Reparo Gênico Alvo-Dirigido/métodosRESUMO
Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.
Assuntos
Bancos de Espécimes Biológicos , Neoplasias Renais/genética , Rim/patologia , Organoides/patologia , Adolescente , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Técnicas de Cultura de Células/métodos , Criança , Pré-Escolar , Metilação de DNA , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Técnicas de Genotipagem , Humanos , Lactente , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Masculino , Nefroma Mesoblástico/tratamento farmacológico , Nefroma Mesoblástico/genética , Nefroma Mesoblástico/patologia , Países Baixos , Medicina de Precisão/métodos , RNA-Seq , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Análise de Célula Única , Transfecção , Células Tumorais Cultivadas , Sequenciamento Completo do Genoma , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/genética , Tumor de Wilms/patologia , Adulto JovemRESUMO
Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fibrose Cística , Adenina , Bancos de Espécimes Biológicos , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Códon sem Sentido , Fibrose Cística/genética , Edição de Genes , Humanos , Organoides/metabolismoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.
Assuntos
Sangue/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Cultivadas , Humanos , MutaçãoRESUMO
Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Mucosa Bucal/patologia , Organoides/patologia , Medicina de Precisão/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Animais , Carboplatina/administração & dosagem , Cetuximab/administração & dosagem , Quimiorradioterapia , Cisplatino/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/efeitos da radiação , Organoides/efeitos dos fármacos , Organoides/efeitos da radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.
Assuntos
Colangiocarcinoma/genética , Cromatina/metabolismo , Células Epiteliais/fisiologia , Neoplasias Hepáticas/genética , Fígado/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Bioengenharia , Carcinogênese , Células Cultivadas , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citoesqueleto/metabolismo , Feminino , Humanos , Mutação com Perda de Função/genética , Camundongos , Camundongos SCID , Organoides , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.
Assuntos
Linhagem da Célula/genética , Senescência Celular/genética , Hematopoese/genética , Mutagênese/genética , Mutação/genética , Adulto , Embrião de Mamíferos/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Especificidade de ÓrgãosRESUMO
When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2-0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.
Assuntos
Vetores Genéticos/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Transformação Genética/genéticaRESUMO
Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance; however, it remains unclear whether epigenetic changes are a cause or a consequence of heterosis. Here, we analyze a large panel of over 500 Arabidopsis (Arabidopsis thaliana) epigenetic hybrid plants (epiHybrids), which we derived from near-isogenic but epigenetically divergent parents. This proof-of-principle experimental system allowed us to quantify the contribution of parental methylation differences to heterosis. We measured traits such as leaf area, growth rate, flowering time, main stem branching, rosette branching, and final plant height and observed several strong positive and negative heterotic phenotypes among the epiHybrids. Using an epigenetic quantitative trait locus mapping approach, we were able to identify specific differentially methylated regions in the parental genomes that are associated with hybrid performance. Sequencing of methylomes, transcriptomes, and genomes of selected parent-epiHybrid combinations further showed that these parental differentially methylated regions most likely mediate the remodeling of methylation and transcriptional states at specific loci in the hybrids. Taken together, our data suggest that locus-specific epigenetic divergence between the parental lines can directly or indirectly trigger heterosis in Arabidopsis hybrids independent of genetic changes. These results add to a growing body of evidence that points to epigenetic factors as one of the key determinants of hybrid performance.