RESUMO
Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.
RESUMO
Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to â¼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.
Assuntos
Síndrome de Angelman , Animais , Camundongos , Síndrome de Angelman/genética , Síndrome de Angelman/terapia , Síndrome de Angelman/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Fenótipo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Precision epigenome editing has gained significant attention as a method to modulate gene expression without altering genetic information. However, a major limiting factor has been that the gene expression changes are often transient, unlike the life-long epigenetic changes that occur frequently in nature. Here, we systematically interrogate the ability of CRISPR/dCas9-based epigenome editors (Epi-dCas9) to engineer persistent epigenetic silencing. We elucidated cis regulatory features that contribute to the differential stability of epigenetic reprogramming, such as the active transcription histone marks H3K36me3 and H3K27ac strongly correlating with resistance to short-term repression and resistance to long-term silencing, respectively. H3K27ac inversely correlates with increased DNA methylation. Interestingly, the dependance on H3K27ac was only observed when a combination of KRAB-dCas9 and targetable DNA methyltransferases (DNMT3A-dCas9 + DNMT3L) was used, but not when KRAB was replaced with the targetable H3K27 histone methyltransferase Ezh2. In addition, programmable Ezh2/DNMT3A + L treatment demonstrated enhanced engineering of localized DNA methylation and was not sensitive to a divergent chromatin state. Our results highlight the importance of local chromatin features for heritability of programmable silencing and the differential response to KRAB- and Ezh2-based epigenetic editing platforms. The information gained in this study provides fundamental insights into understanding contextual cues to more predictably engineer persistent silencing.
Assuntos
Epigenoma , Edição de Genes , Sistemas CRISPR-Cas , Cromatina , Metilação de DNA/genética , Epigênese Genética , Edição de Genes/métodos , Inativação GênicaRESUMO
Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.
Assuntos
Síndrome de Angelman/terapia , Terapia Genética , Deficiência Intelectual/terapia , Convulsões/terapia , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Animais , Antígenos CD34/genética , Ataxia/genética , Ataxia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/terapia , Modelos Animais de Doenças , Eletroencefalografia , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Deficiência Intelectual/genética , Interleucina-2/genética , Lentivirus/genética , Camundongos , Transtornos das Habilidades Motoras/genética , Transtornos das Habilidades Motoras/patologia , Transtornos das Habilidades Motoras/terapia , Convulsões/genéticaRESUMO
Emerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. We compared cross-tissue mRNA levels of 75 HSD genes from 30 families between humans and chimpanzees and found expression patterns consistent with relaxed selection on or neofunctionalization of derived paralogs. In general, ancestral paralogs exhibited greatest expression conservation with chimpanzee orthologs, though exceptions suggest certain derived paralogs may retain or supplant ancestral functions. Concordantly, analysis of long-read isoform sequencing data sets from diverse human tissues and cell lines found that about half of derived paralogs exhibited globally lower expression. To understand mechanisms underlying these differences, we leveraged data from human lymphoblastoid cell lines (LCLs) and found no relationship between paralogous expression divergence and post-transcriptional regulation, sequence divergence, or copy-number variation. Considering cis-regulation, we reanalyzed ENCODE data and recovered hundreds of previously unidentified candidate CREs in HSDs. We also generated large-insert ChIP-sequencing data for active chromatin features in an LCL to better distinguish paralogous regions. Some duplicated CREs were sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogous genes. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.
Assuntos
Duplicação Gênica , Regulação da Expressão Gênica , Genoma Humano , Duplicações Segmentares Genômicas , Animais , Linhagem Celular , Variações do Número de Cópias de DNA , Humanos , Pan troglodytes , Regiões Promotoras GenéticasRESUMO
BACKGROUND: LRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited. METHODS: In silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2'-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system. RESULTS: Across breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2'-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer. CONCLUSIONS: Our study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.
Assuntos
Metilação de DNA , Glicoproteínas de Membrana , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Ilhas de CpG/genética , Decitabina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.
Assuntos
Cromossomos Humanos X/química , Epigênese Genética , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Inativação do Cromossomo X , Alelos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Cromossomos Humanos X/metabolismo , Ilhas de CpG , Edição de Genes , Inativação Gênica , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Ubiquitin E3 ligase 3A (UBE3A) encodes an E3 ubiquitin ligase whose loss from the maternal allele causes the neurodevelopmental disorder Angelman syndrome (AS). Previous studies of UBE3A function have not examined full Ube3a deletion in mouse, the complexity of imprinted gene networks in brain nor the molecular basis of systems-level cognitive dysfunctions in AS. We therefore utilized a systems biology approach to elucidate how UBE3A loss impacts the early postnatal brain in a novel CRISPR/Cas9-engineered rat Angelman model of a complete Ube3a deletion. Strand-specific transcriptome analysis of offspring from maternally or paternally inherited Ube3a deletions revealed the expected parental expression patterns of Ube3a sense and antisense transcripts by postnatal day 2 (P2) in hypothalamus and day 9 (P9) in cortex, compared to wild-type littermates. The dependency of genome-wide effects on parent-of-origin, Ube3a genotype and time (P2 and P9) was investigated through transcriptome (RNA sequencing of cortex and hypothalamus) and methylome (whole-genome bisulfite sequencing of hypothalamus). Weighted gene co-expression and co-methylation network analyses identified co-regulated networks in maternally inherited Ube3a deletion offspring enriched in postnatal developmental processes including Wnt signaling, synaptic regulation, neuronal and glial functions, epigenetic regulation, ubiquitin, circadian entrainment and splicing. Furthermore, we showed that loss of the paternal Ube3a antisense transcript resulted in both unique and overlapping dysregulated gene pathways with maternal loss, predominantly at the level of differential methylation. Together, these results provide a holistic examination of the molecular impacts of UBE3A loss in brain, supporting the existence of interactive epigenetic networks between maternal and paternal transcripts at the Ube3a locus.
Assuntos
Impressão Genômica , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Animais , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Epigênese Genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Hipotálamo/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/genética , Sinapses/metabolismo , Biologia de Sistemas , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização WntRESUMO
Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3'-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3'-end was broadly tolerated by Streptococcus pyogenes Cas9 in an inâ vitro DNA cleavage assay. The 3'-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNPâ K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.
Assuntos
Biotina/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , DNA/química , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/química , Sítios de Ligação , Biotinilação , Proteína 9 Associada à CRISPR/metabolismo , Reagentes de Ligações Cruzadas/química , DNA/metabolismo , Clivagem do DNA , Corantes Fluorescentes/química , Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.
Assuntos
Cromatina/química , Endonucleases/genética , Epigenômica/métodos , Inativação Gênica , Histonas/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Endonucleases/metabolismo , Edição de Genes , Células HCT116 , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
Assuntos
DNA/genética , Enciclopédias como Assunto , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Alelos , Linhagem Celular , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Genômica , Humanos , Células K562 , Especificidade de Órgãos , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Mapas de Interação de Proteínas , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Seleção Genética/genética , Sítio de Iniciação de TranscriçãoRESUMO
Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak.
Assuntos
Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Transcriptoma , Acetilação , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Células HCT116 , Células HEK293 , Humanos , Proteínas E7 de Papillomavirus/genética , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional , Deleção de SequênciaRESUMO
Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.
Assuntos
Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Loci Gênicos , Camundongos , Fatores de Transcrição/administração & dosagem , Dedos de ZincoRESUMO
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.
Assuntos
Cromatina/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Genoma Humano/genética , Sistema Hematopoético/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Imunoprecipitação da Cromatina , Biologia Computacional , Perfilação da Expressão Gênica , Loci Gênicos , Homeostase , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de DNA , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.
Assuntos
Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Estudo de Associação Genômica Ampla , Mutação INDEL , Camundongos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMO
DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.
Assuntos
Algoritmos , Metilação de DNA , Genoma Humano , Análise de Sequência de DNA/métodos , Interpretação Estatística de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Especificidade de ÓrgãosRESUMO
Biologists possess the detailed knowledge critical for extracting biological insight from genome-wide data resources, and yet they are increasingly faced with nontrivial computational analysis challenges posed by genome-scale methodologies. To lower this computational barrier, particularly in the early data exploration phases, we have developed an interactive pattern discovery and visualization approach, Spark, designed with epigenomic data in mind. Here we demonstrate Spark's ability to reveal both known and novel epigenetic signatures, including a previously unappreciated binding association between the YY1 transcription factor and the corepressor CTBP2 in human embryonic stem cells.
Assuntos
Genoma Humano , Ferramenta de Busca , Análise de Sequência de DNA/métodos , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Análise por Conglomerados , Proteínas Correpressoras , Metilação de DNA , Células-Tronco Embrionárias/química , Epigênese Genética , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.
Assuntos
Conyza/genética , Genoma de Cloroplastos/genética , Genoma Mitocondrial/genética , Glicina/análogos & derivados , Resistência a Herbicidas , Herbicidas/farmacologia , Evolução Biológica , Conyza/efeitos dos fármacos , Genômica , Glicina/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Filogenia , Análise de Sequência de DNA , GlifosatoRESUMO
BACKGROUND: The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells. RESULTS: ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival. CONCLUSIONS: Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.
Assuntos
Neoplasias da Mama/metabolismo , Cromatina/metabolismo , Receptor alfa de Estrogênio/genética , Transativadores/fisiologia , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Análise por Conglomerados , Feminino , Fator de Transcrição GATA3/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genoma Humano , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Ligação Proteica , TranscriptomaRESUMO
Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Although the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. Because many questions regarding GATA-2 function in the vascular biology realm remain unanswered, we used ChIP sequencing and loss-of-function strategies to define the GATA-2-instigated genetic network in human endothelial cells. In contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2-occupied sites characteristically contained motifs that bind activator protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Although all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser-73, which stimulates AP-1-dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammatory genes, mechanistic insights underlying GATA-2-AP-1 cooperativity and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.