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1.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
3.
Mol Cell Proteomics ; 20: 100014, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33257503

RESUMO

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution MS. By combining meiotic labels annotated from the mouse genomics informatics mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder (https://github.com/sjq111/FuncProFinder), was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of 10 genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

4.
Nat Protoc ; 15(9): 3009-3029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32796939

RESUMO

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.


Assuntos
Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Animais , Feminino , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
5.
Nat Methods ; 17(6): 600-604, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424272

RESUMO

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.


Assuntos
Proteína 9 Associada à CRISPR/genética , Citosina/metabolismo , DNA/genética , Edição de Genes/métodos , RNA/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células HEK293 , Humanos , Mutação , Mutação Puntual
7.
Genome Biol ; 20(1): 101, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118069

RESUMO

Base editing installs a precise nucleotide change in specific gene loci without causing a double-strand break. Its efficiency in human embryos is generally low, limiting its utility in functional genetic studies. Here, we report that injecting base editors into human cleaving two-cell and four-cell embryos results in much higher (up to 13-fold) homozygotic nucleotide substitution efficiency as opposed to MII oocytes or zygotes. Furthermore, as a proof-of-principle study, a point mutation can be efficiently corrected by our method. Our study indicates that human cleaving embryos provide an efficient base editing window for robust gene disruption and correction.


Assuntos
Pesquisas com Embriões , Embrião de Mamíferos , Edição de Genes/métodos , Humanos
8.
Science ; 364(6437): 289-292, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819928

RESUMO

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.


Assuntos
Blastômeros , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citosina , Edição de Genes/métodos , Polimorfismo de Nucleotídeo Único , Animais , Análise Mutacional de DNA , Embrião de Mamíferos , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
9.
Cell Rep ; 26(2): 302-312.e4, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625312

RESUMO

The coactivator-associated arginine methyltransferase CARM1 catalyzes the methylation of histone H3 arginine 17/26 (H3R17/26me) and non-histone proteins at arginine residues to regulate gene transactivation through profiling or Carm1 overexpression assays. However, the direct relationship between H3R17/26me and its causal role in mouse embryo development remains largely unclear. Here, we use rAPOBEC1-XTEN-Cas9n-UGI (BE3) to efficiently introduce a point mutation (R17H) at multiple Hist1/2H3 loci and a premature-stop codon into the catalytic domain of CARM1 in mouse embryos, resulting in remarkable downregulation of H3R17me levels and developmental defects in pre-implantation and fetal embryos. Transcriptomic analysis reveals that Yap1 and cell cycle signaling pathways are dysregulated in Carm1 truncation and H3R17H substitution embryos, and Yap1 overexpression could rescue the base-editing-elicited defects. Our data establish the direct regulatory relationship between CARM1-mediated H3R17me and early mouse embryo development and demonstrate that Yap1 acts downstream of CARM1-mediated H3R17me to regulate the mouse embryo development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Transdução de Sinais , Animais , Domínio Catalítico , Ciclo Celular , Linhagem Celular Tumoral , Código das Histonas , Histonas/química , Histonas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Mutação de Sentido Incorreto , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transcriptoma
10.
Development ; 145(20)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30275281

RESUMO

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Zigoto/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Sequência de Bases , Cóclea/metabolismo , Surdez/genética , Surdez/fisiopatologia , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Motores Moleculares/metabolismo , Mutação/genética
11.
Dev Cell ; 45(4): 526-536.e5, 2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29787711

RESUMO

The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.


Assuntos
Sistemas CRISPR-Cas , DNA/administração & dosagem , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes/métodos , RNA Guia/administração & dosagem , Animais , Células Cultivadas , Eletroporação , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Feminino , Fertilização In Vitro , Recombinação Homóloga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
12.
Nat Neurosci ; 21(3): 440-446, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29335603

RESUMO

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.


Assuntos
Química Encefálica/genética , Sistemas CRISPR-Cas/genética , Ativação Transcricional/genética , Animais , Astrócitos/fisiologia , Proteínas de Ligação a DNA , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Cultura Primária de Células , RNA Longo não Codificante/genética
13.
Genome Biol ; 18(1): 224, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29178945

RESUMO

BACKGROUND: The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown. RESULTS: Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells. CONCLUSIONS: CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.


Assuntos
Sistemas CRISPR-Cas , Deleção Cromossômica , Marcação de Genes , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias , Feminino , Edição de Genes , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Camundongos , Microinjeções , Fenótipo , RNA Guia , Síndrome de Turner/genética , Cromossomo Y
14.
Cell Res ; 27(7): 933-945, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28585534

RESUMO

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Haplorrinos/genética , RNA Guia/genética , Fatores de Transcrição ARNTL/genética , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Embrião de Mamíferos/citologia , Endonucleases , Éxons/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosaicismo/embriologia , Recuperação de Oócitos , Fenótipo , RNA Mensageiro/genética , Sequenciamento Completo do Genoma , Cromossomo Y , Zigoto/citologia
15.
EBioMedicine ; 20: 19-26, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28527830

RESUMO

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah-/- liver failure mice, offering an efficient approach for precisely targeted gene therapies.


Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Reparo de DNA por Recombinação , Animais , Biomarcadores , Linhagem Celular , Feminino , Técnicas de Transferência de Genes , Engenharia Genética , Terapia Genética , Vetores Genéticos/genética , Genótipo , Hepatócitos/metabolismo , Humanos , Hidrolases/genética , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fenótipo
16.
Cell Res ; 27(6): 801-814, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28524166

RESUMO

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Animais , Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA por Junção de Extremidades/fisiologia , Técnicas de Introdução de Genes , Engenharia Genética/métodos , Células HEK293 , Hepatócitos/metabolismo , Humanos , Camundongos , RNA Guia/genética
17.
Cell Reprogram ; 16(4): 253-65, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24960409

RESUMO

Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 µM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro.


Assuntos
Clonagem de Organismos , Embrião de Mamíferos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Técnicas de Transferência Nuclear , Acetilação/efeitos dos fármacos , Animais , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/biossíntese , Histonas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Sus scrofa , Tubulina (Proteína)/metabolismo
19.
Stem Cells ; 27(2): 383-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056907

RESUMO

Most mouse embryonic stem (ES) cells are derived from a 129 or C57BL/6 background, whereas the derivation efficiency of ES cells is extremely low on certain refractory types of background for which ES cells are highly desired. Here we report an optimized, highly efficient protocol by combining pluripotin, a small molecule, and leukemia inhibitory factor (LIF) for the derivation of mouse ES cells. With this method, we successfully isolated ES cell lines from five strains of mice, with an efficiency of 57% for NOD-scid, 63% for SCID beige, 80% for CD-1, and 100% for two F1 strains from C57BL/6xCD-1. By tracking the Oct4-positive cells in the Oct4-green fluorescent protein embryos in the process of ES cell isolation, we found that pluripotin combined with LIF improved the efficiency of ES cell isolation by selectively maintaining the Oct4-positive cells in the outgrowth. To our knowledge, this is the first report of ES cells being efficiently derived from immunodeficient mice on refractory backgrounds (NOD-scid on a NOD background and SCID beige on a BALB/c background).


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Imuno-Histoquímica , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Cell Res ; 17(2): 117-34, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17287829

RESUMO

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors (CFs) disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naïve chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the "erase-and-rebuild" process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper (Gao T, et al., Cell Res 2007; 17: 135-150.) that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.


Assuntos
Núcleo Celular/genética , Reprogramação Celular/genética , Transcrição Genética/fisiologia , Zigoto/fisiologia , Animais , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células NIH 3T3
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