RESUMO
Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Linfoma de Células T/genética , Complexos Multiproteicos/genética , Mutação de Sentido Incorreto/genética , Neoplasias do Timo/genética , Adenosina Trifosfatases/metabolismo , Anáfase , Animais , Células Cultivadas , Estruturas Cromossômicas/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Linfoma de Células T/fisiopatologia , Masculino , Metáfase , Camundongos , Complexos Multiproteicos/metabolismo , Timócitos/patologia , Neoplasias do Timo/fisiopatologiaRESUMO
Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.
Assuntos
Reparo do DNA/fisiologia , Heterocromatina/genética , Heterocromatina/fisiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/fisiologia , Mutagênese Insercional , Mutagênicos , Mutação/genética , Reparo de DNA por Recombinação/fisiologia , Deleção de SequênciaRESUMO
Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator-PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip-located at the 5' end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5' Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5' end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5' Hoxa genes and that Hottip RNA binds to the 5' end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Homeodomínio/genética , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas Homeobox A10 , Humanos , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/biossíntese , Fatores de Transcrição/biossínteseRESUMO
Compared with histone H3, acetylation of H4 tails has not been well studied, especially in mammalian cells. Yet, H4K16 acetylation is of particular interest because of its ability to decompact nucleosomes in vitro and its involvement in dosage compensation in flies. Here we show that, surprisingly, loss of H4K16 acetylation does not alter higher-order chromatin compaction in vivo in mouse embryonic stem cells (ESCs). As well as peaks of acetylated H4K16 and KAT8 histone acetyltransferase at the transcription start sites of expressed genes, we report that acetylation of H4K16 is a new marker of active enhancers in ESCs and that some enhancers are marked by H3K4me1, KAT8, and H4K16ac, but not by acetylated H3K27 or EP300, suggesting that they are novel EP300 independent regulatory elements. Our data suggest a broad role for different histone acetylation marks and for different histone acetyltransferases in long-range gene regulation.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Células Cultivadas , Mecanismo Genético de Compensação de Dose , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/genética , Hibridização in Situ Fluorescente , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Sítio de Iniciação de TranscriçãoRESUMO
Trithorax and polycomb group proteins are generally thought to antagonize one another. The trithorax family member MLL (myeloid/lymphoid or mixed-lineage leukemia) is presumed to activate Hox expression, counteracting polycomb-mediated repression. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, whose PWWP domain binds to H3K36me3, interacts with MLL and tethers MLL fusion proteins to HOXA9 in leukaemias. Here we show, unexpectedly, that Psip1/p75 regulates homeotic genes by recruiting not only MLL complexes, but also the polycomb group protein Bmi1. In Psip1(-/-) cells binding of Mll1/2, Bmi1 and the co-repressor Ctbp1 at Hox loci are all abrogated and Hoxa and Hoxd mRNA expression increased. Our data not only reveal a potential mechanism of action for Psip1 in the regulation of Hox genes but also suggest an unexpected interplay between proteins usually considered as transcriptional activators and repressors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Genes Homeobox , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Oxirredutases do Álcool/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.
Assuntos
Ácidos Indolacéticos , Ubiquitina-Proteína Ligases , Animais , Cromossomos/metabolismo , Ácidos Indolacéticos/metabolismo , Ligantes , Mamíferos/metabolismo , Camundongos , Proteólise , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity, with an abundant chromatin-associated factor, is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe.
RESUMO
Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein ( PSIP1/LEDGF) is a transcriptional coactivator, possesses a H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). Furthermore, we performed stable isotope labelling with amino acids in cell culture (SILAC) for a longer isoform of PSIP1 (p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts (MEFS). Results: Proteomic analysis of H3K36me3 chromatin show association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP1/p75. We validated the association of PSIP1/p75 with gamma H2A.X, an early marker of DNA damage and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP1/p75 in promoting HDR in mammals, our data supports the wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting several DNA repair proteins to transcribed gene bodies.
RESUMO
Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.