RESUMO
Oncogenic fusions involving transcription factors are present in the majority of pediatric leukemias; however, the context-specific mechanisms they employ to drive cancer remain poorly understood. CBFA2T3-GLIS2 (C/G) fusions occur in treatment-refractory acute myeloid leukemias and are restricted to young children. To understand how the C/G fusion drives oncogenesis we applied CUT&RUN chromatin profiling to an umbilical cord blood/endothelial cell (EC) co-culture model of C/G AML that recapitulates the biology of this malignancy. We find C/G fusion binding is mediated by its zinc finger domains. Integration of fusion binding sites in C/G- transduced cells with Polycomb Repressive Complex 2 (PRC2) sites in control cord blood cells identifies MYCN, ZFPM1, ZBTB16 and LMO2 as direct C/G targets. Transcriptomic analysis of a large pediatric AML cohort shows that these genes are upregulated in C/G patient samples. Single cell RNA-sequencing of umbilical cord blood identifies a population of megakaryocyte precursors that already express many of these genes despite lacking the fusion. By integrating CUT&RUN data with CRISPR dependency screens we identify BRG1/SMARCA4 as a vulnerability in C/G AML. BRG1 profiling in C/G patient-derived cell lines shows that the CBFA2T3 locus is a binding site, and treatment with clinically-available BRG1 inhibitors reduces fusion levels and downstream C/G targets including N-MYC, resulting in C/G leukemia cell death and extending survival in a murine xenograft model.
RESUMO
Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or replication-independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.
Assuntos
Proteínas de Drosophila , Histonas , Animais , Lisina , Cromatina , Drosophila , Proteínas do Grupo PolycombRESUMO
Chromatin profiling at locus resolution uncovers gene regulatory features that define cell types and developmental trajectories, but it remains challenging to map and compare different chromatin-associated proteins in the same sample. Here we describe Multiple Target Identification by Tagmentation (MulTI-Tag), an antibody barcoding approach for profiling multiple chromatin features simultaneously in single cells. We optimized MulTI-Tag to retain high sensitivity and specificity, and we demonstrate detection of up to three histone modifications in the same cell: H3K27me3, H3K4me1/2 and H3K36me3. We apply MulTI-Tag to resolve distinct cell types and developmental trajectories; to distinguish unique, coordinated patterns of active and repressive element regulatory usage associated with differentiation outcomes; and to uncover associations between histone marks. Multifactorial epigenetic profiling holds promise for comprehensively characterizing cell-specific gene regulatory landscapes in development and disease.
Assuntos
Cromatina , Histonas , Histonas/genética , Histonas/metabolismo , Cromatina/genética , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Epigênese Genética/genéticaRESUMO
Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. SIGNIFICANCE: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517.
Assuntos
DNA Tumoral Circulante , Neoplasias da Próstata , Masculino , Humanos , DNA Tumoral Circulante/genética , Nucleossomos/genética , Medicina de Precisão , Neoplasias da Próstata/patologia , Regulação Neoplásica da Expressão Gênica , FenótipoRESUMO
Cleavage Under Targets and Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers, and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.
Assuntos
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , RNA Polimerase II/genética , Sequências Reguladoras de Ácido Nucleico , Transposases/metabolismoRESUMO
Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.
Assuntos
Cromatina/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia/genética , Leucemia/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/metabolismo , Antineoplásicos/farmacologia , Automação Laboratorial , Benzamidas/farmacologia , Benzimidazóis/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas , Humanos , Leucemia/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirimidinas/farmacologia , Análise de Célula Única/métodos , Fatores de Elongação da Transcrição/genéticaRESUMO
Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.
Assuntos
Androgênios/farmacologia , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Di-Hidrotestosterona/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Transdução de Sinais/genéticaRESUMO
Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. We show that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNAreplication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, we demonstrate that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. We propose that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.
Assuntos
Cromatina , Regulação Neoplásica da Expressão Gênica/genética , Histonas , Mutação/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Drosophila/genética , Glioma/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Larva/genética , Larva/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Pré-Escolar , Estudos de Coortes , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Nuclear Ligada ao X/metabolismoRESUMO
BACKGROUND: CUT&RUN is an efficient epigenome profiling method that identifies sites of DNA binding protein enrichment genome-wide with high signal to noise and low sequencing requirements. Currently, the analysis of CUT&RUN data is complicated by its exceptionally low background, which renders programs designed for analysis of ChIP-seq data vulnerable to oversensitivity in identifying sites of protein binding. RESULTS: Here we introduce Sparse Enrichment Analysis for CUT&RUN (SEACR), an analysis strategy that uses the global distribution of background signal to calibrate a simple threshold for peak calling. SEACR discriminates between true and false-positive peaks with near-perfect specificity from "gold standard" CUT&RUN datasets and efficiently identifies enriched regions for several different protein targets. We also introduce a web server ( http://seacr.fredhutch.org ) for plug-and-play analysis with SEACR that facilitates maximum accessibility across users of all skill levels. CONCLUSIONS: SEACR is a highly selective peak caller that definitively validates the accuracy of CUT&RUN for datasets with known true negatives. Its ease of use and performance in comparison with existing peak calling strategies make it an ideal choice for analyzing CUT&RUN data.
Assuntos
Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/análise , Epigenômica/métodos , Algoritmos , Animais , Sítios de Ligação/genética , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/genética , Genoma , Humanos , Ligação Proteica/genética , Análise de Sequência de DNARESUMO
Although the in vitro structural and in vivo spatial characteristics of transcription factor (TF) binding are well defined, TF interactions with chromatin and other companion TFs during development are poorly understood. To analyze such interactions in vivo, we profiled several TFs across a time course of human embryonic stem cell differentiation and studied their interactions with nucleosomes and co-occurring TFs by enhanced chromatin occupancy (EChO), a computational strategy for classifying TF interactions with chromatin. EChO shows that multiple individual TFs can employ either direct DNA binding or "pioneer" nucleosome binding at different enhancer targets. Nucleosome binding is not exclusively confined to inaccessible chromatin but rather correlated with local binding of other TFs and degeneracy at key bases in the pioneer factor target motif responsible for direct DNA binding. Our strategy reveals a dynamic exchange of TFs at enhancers across developmental time that is aided by pioneer nucleosome binding.
Assuntos
Diferenciação Celular/genética , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Humanos , Nucleossomos/genéticaRESUMO
Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.
Assuntos
Cromatina/metabolismo , Técnicas Imunológicas/métodos , Biologia Molecular/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Epigênese Genética , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismoRESUMO
Sophisticated gene-regulatory mechanisms probably evolved in prokaryotes billions of years before the emergence of modern eukaryotes, which inherited the same basic enzymatic machineries. However, the epigenomic landscapes of eukaryotes are dominated by nucleosomes, which have acquired roles in genome packaging, mitotic condensation and silencing parasitic genomic elements. Although the molecular mechanisms by which nucleosomes are displaced and modified have been described, just how transcription factors, histone variants and modifications and chromatin regulators act on nucleosomes to regulate transcription is the subject of considerable ongoing study. We explore the extent to which these transcriptional regulatory components function in the context of the evolutionarily ancient role of chromatin as a barrier to processes acting on DNA and how chromatin proteins have diversified to carry out evolutionarily recent functions that accompanied the emergence of differentiation and development in multicellular eukaryotes.
Assuntos
Montagem e Desmontagem da Cromatina , DNA/genética , Genoma , Nucleossomos/genética , Transcrição Gênica , Animais , Evolução Biológica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/história , Proteínas Cromossômicas não Histona/metabolismo , DNA/história , DNA/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Genômica/métodos , Histonas/genética , Histonas/história , Histonas/metabolismo , História do Século XXI , História Antiga , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Células Procarióticas/citologia , Células Procarióticas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/história , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Our understanding of eukaryotic gene regulation is limited by the complexity of protein-DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease cleavage method that profiles DNA-binding proteins, histones and chromatin-modifying proteins in situ with exceptional sensitivity and resolution. RESULTS: Here, we describe an automated CUT&RUN platform and apply it to characterize the chromatin landscapes of human cells. We find that automated CUT&RUN profiles of histone modifications crisply demarcate active and repressed chromatin regions, and we develop a continuous metric to identify cell-type-specific promoter and enhancer activities. We test the ability of automated CUT&RUN to profile frozen tumor samples and find that our method readily distinguishes two pediatric glioma xenografts by their subtype-specific gene expression programs. CONCLUSIONS: The easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples.
Assuntos
Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica/métodos , Sítios de Ligação , Cromatina/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Ensaios de Triagem em Larga Escala/métodos , Código das Histonas/genética , Histonas/genética , Humanos , Hibridização In Situ/métodos , Células K562 , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Software , Fatores de Transcrição/genéticaRESUMO
Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.
Assuntos
Clonagem Molecular/métodos , Drosophila melanogaster/genética , Técnicas de Transferência de Genes , Histonas/genética , Família Multigênica , Sequências de Repetição em Tandem/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Drosophila melanogaster/embriologia , Feminino , Genoma de Inseto , Masculino , Modelos Animais , Reprodutibilidade dos Testes , TransgenesRESUMO
BACKGROUND: High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis. RESULTS: The data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for "enhancer RNAs" (eRNAs) in defining developmental transcription programs. CONCLUSIONS: High-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.
Assuntos
Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , RNA/genética , Sítio de Iniciação de Transcrição , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Nucleossomos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a 'histone replacement' system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression.
Assuntos
Regulação da Expressão Gênica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Substituição de Aminoácidos , Animais , Drosophila , Perfilação da Expressão Gênica , Histonas/genética , Metilação , Mutação de Sentido IncorretoRESUMO
Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.
Assuntos
Cromatina/genética , Histonas/metabolismo , Família Multigênica/genética , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Replicação do DNA/genética , Drosophila , Epigênese Genética/genética , Histona-Lisina N-Metiltransferase/metabolismo , MetilaçãoRESUMO
Poly(A) RNA-binding proteins (Pabs) bind with high affinity and specificity to polyadenosine RNA. Textbook models show a nuclear Pab, PABPN1, and a cytoplasmic Pab, PABPC, where the nuclear PABPN1 modulates poly(A) tail length and the cytoplasmic PABPC stabilizes poly(A) RNA in the cytoplasm and also enhances translation. While these conventional roles are critically important, the Pab family has expanded recently both in number and in function. A number of novel roles have emerged for both PAPBPN1 and PABPC that contribute to the fine-tuning of gene expression. Furthermore, as the characterization of the nucleic acid binding properties of RNA-binding proteins advances, additional proteins that show high affinity and specificity for polyadenosine RNA are being discovered. With this expansion of the Pab family comes a concomitant increase in the potential for Pabs to modulate gene expression. Further complication comes from an expansion of the potential binding sites for Pab proteins as revealed by an analysis of templated polyadenosine stretches present within the transcriptome. Thus, Pabs could influence mRNA fate and function not only by binding to the nontemplated poly(A) tail but also to internal stretches of adenosine. Understanding the diverse functions of Pab proteins is not only critical to understand how gene expression is regulated but also to understand the molecular basis for tissue-specific diseases that occur when Pab proteins are altered. Here we describe both conventional and recently emerged functions for PABPN1 and PABPC and then introduce and discuss three new Pab family members, ZC3H14, hnRNP-Q1, and LARP4.