RESUMO
Cleavage under targets and tagmentation (CUT&Tag) is an antibody-directed in situ chromatin profiling strategy that is rapidly replacing immune precipitation-based methods, such as chromatin immunoprecipitation-sequencing. The efficiency of the method enables chromatin profiling in single cells but is limited by the numbers of cells that can be profiled. Here, we describe a combinatorial barcoding strategy for CUT&Tag that harnesses a nanowell dispenser for simple, high-resolution, high-throughput, single-cell chromatin profiling. In this single-cell combinatorial indexing CUT&Tag (sciCUT&Tag) protocol, lightly cross-linked nuclei are bound to magnetic beads and incubated with primary and secondary antibodies in bulk and then arrayed in a 96-well plate for a first round of cellular indexing by antibody-directed Tn5 tagmentation. The sample is then repooled, mixed and arrayed across 5,184 nanowells at a density of 12-24 nuclei per well for a second round of cellular indexing during PCR amplification of the sequencing-ready library. This protocol can be completed in 1.5 days by a research technician, and we illustrate the optimized protocol by profiling histone modifications associated with developmental gene repression (H3K27me3) as well as transcriptional activation (H3K4me1-2-3) in human peripheral blood mononuclear cells and use single-nucleotide polymorphisms to facilitate collision removal. We have also used sciCUT&Tag for simultaneous profiling of multiple chromatin epitopes in single cells. The reduced cost, improved resolution and scalability of sciCUT&Tag make it an attractive platform to profile chromatin features in single cells.
Assuntos
Histonas , Leucócitos Mononucleares , Humanos , Histonas/genética , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Cromatina/genética , Processamento de Proteína Pós-Traducional , Código das Histonas , Análise de Célula Única/métodosRESUMO
For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.
Assuntos
Cromatina , Epigenômica , Animais , Camundongos , Inclusão em Parafina , Estudos Retrospectivos , Cromatina/genética , FormaldeídoRESUMO
Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Histonas/metabolismo , Proteínas Nucleares/metabolismo , HIV-1/fisiologia , Fatores de Transcrição/metabolismo , Latência Viral/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Soropositividade para HIV/genética , Provírus/genética , Linfócitos T CD4-Positivos , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
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
Chromosomal translocations involving the Lysine-Methyl-Tansferase-2A ( KMT2A ) locus generate potent oncogenes that cause highly aggressive acute leukemias 1 . KMT2A and the most frequent translocation partners encode proteins that interact with DNA to regulate developmental gene expression 2 . KMT2A-oncogenic fusion proteins (oncoproteins) contribute to the epigenetic mechanisms that allow KMT2A -rearranged leukemias to evade targeted therapies. By profiling the oncoprotein-target sites of 34 KMT2A -rearranged leukemia samples, we find that the genomic enrichment of oncoprotein binding is highly variable between samples. At high levels of expression, the oncoproteins preferentially activate either the lymphoid or myeloid lineage program depending on the fusion partner. These fusion-partner-dependent binding sites correspond to the frequencies of each mutation in acute lymphoid leukemia versus acute myeloid leukemia. By profiling a sample that underwent a lymphoid-to-myeloid lineage switching event in response to lymphoid-directed treatment, we find the global oncoprotein levels are reduced and the oncoprotein-target gene network changes. At lower levels of expression, the oncoprotein shifts to a non-canonical regulatory program that favors the myeloid lineage, and in a subset of resistant patients, the Menin inhibitor Revumenib induces a similar response. The dynamic shifts in KMT2A oncoproteins we describe likely contribute to epigenetic resistance of KMT2A -rearranged leukemias to targeted therapies.
RESUMO
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
The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.
Assuntos
Neoplasias Colorretais , Proteínas F-Box , Proteína 7 com Repetições F-Box-WD/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/patologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , Mutação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/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
Stem cells that indirectly generate differentiated cells through intermediate progenitors drives vertebrate brain evolution. Due to a lack of lineage information, how stem cell functionality, including the competency to generate intermediate progenitors, becomes extinguished during progenitor commitment remains unclear. Type II neuroblasts in fly larval brains divide asymmetrically to generate a neuroblast and a progeny that commits to an intermediate progenitor (INP) identity. We identified Tailless (Tll) as a master regulator of type II neuroblast functional identity, including the competency to generate INPs. Successive expression of transcriptional repressors functions through Hdac3 to silence tll during INP commitment. Reducing repressor activity allows re-activation of Notch in INPs to ectopically induce tll expression driving supernumerary neuroblast formation. Knocking-down hdac3 function prevents downregulation of tll during INP commitment. We propose that continual inactivation of stem cell identity genes allows intermediate progenitors to stably commit to generating diverse differentiated cells during indirect neurogenesis.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Células-Tronco Neurais/metabolismo , Neurogênese , Fatores de Transcrição/genética , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Linhagem da Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases , Larva/genética , Larva/metabolismo , Fenótipo , Receptores Notch , Proteínas Repressoras , Fatores de Transcrição/metabolismoRESUMO
We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.
Assuntos
Cromatina/genética , Mapeamento Cromossômico/métodos , Epigenômica/métodos , Sequência de Bases , DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Transposases/genética , Transposases/metabolismoRESUMO
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
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
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
How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts. Within 2 hr of immature INP birth, downregulation of repressor activities alleviates Rpd3-mediated repression on the erm enhancer, enabling acetylation of multiple histone proteins and activating Erm expression. Erm restricts the developmental potential in immature INPs by repressing genes encoding neuroblast transcriptional activators. We propose that poising the fast-activating enhancers of master regulators of differentiation through continual histone deacetylation in stem cells enables self-renewal and rapid restriction of developmental potential following asymmetric division.
Assuntos
Divisão Celular Assimétrica , Autorrenovação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Histona Desacetilase 1/metabolismo , Acetilação , Animais , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular/genética , Sequência Consenso/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos/genética , Retroalimentação Fisiológica , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.
Assuntos
Encéfalo/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/genética , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Linhagem da Célula/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Epigênese Genética , Loci Gênicos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Células-Tronco Neurais/citologia , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
During malignant transformation the cells of origin give rise to cancer stem cells which possess the capacity to undergo limitless rounds of self-renewing division, regenerating themselves while producing more tumor cells. Within normal tissues, a limitless self-renewal capacity is unique to the stem cells, which divide asymmetrically to produce more restricted progenitors. Accumulating evidence suggests that misregulation of the self-renewal machinery in stem cell progeny can lead to tumorigenesis, but how it influences the properties of the resulting tumors remains unclear. Studies of the type II neural stem cell (neuroblast) lineages in the Drosophila larval brain have identified a regulatory cascade that promotes commitment to a progenitor cell identity by restricting their response to the self-renewal machinery. Brain tumor (Brat) and Numb initiate this cascade by asymmetrically extinguishing the activity of the self-renewal factors. Subsequently, Earmuff (Erm) and the SWI/SNF complex stably restrict the competence of the progenitor cell to respond to reactivation of self-renewal mechanisms. Together, this cascade programs the progenitor cell to undergo limited rounds of division, generating exclusive differentiated progeny. Here we review how defects in this cascade lead to tumor initiation and how inhibiting the self-renewal mechanisms may be an effective strategy to block CSC expansion.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neurais/citologia , Animais , Neoplasias Encefálicas/metabolismo , Modelos Animais de Doenças , Drosophila , Humanos , Larva , Células-Tronco Neoplásicas/metabolismoRESUMO
Despite expressing stem cell self-renewal factors, intermediate progenitor cells possess restricted developmental potential, which allows them to give rise exclusively to differentiated progeny rather than stem cell progeny. Failure to restrict the developmental potential can allow intermediate progenitor cells to revert into aberrant stem cells that might contribute to tumorigenesis. Insight into stable restriction of the developmental potential in intermediate progenitor cells could improve our understanding of the development and growth of tumors, but the mechanisms involved remain largely unknown. Intermediate neural progenitors (INPs), generated by type II neural stem cells (neuroblasts) in fly larval brains, provide an in vivo model for investigating the mechanisms that stably restrict the developmental potential of intermediate progenitor cells. Here, we report that the transcriptional repressor protein Earmuff (Erm) functions temporally after Brain tumor (Brat) and Numb to restrict the developmental potential of uncommitted (immature) INPs. Consistently, endogenous Erm is detected in immature INPs but undetectable in INPs. Erm-dependent restriction of the developmental potential in immature INPs leads to attenuated competence to respond to all known neuroblast self-renewal factors in INPs. We also identified that the BAP chromatin-remodeling complex probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data led us to conclude that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. We propose that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.