ABSTRACT
Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.
Subject(s)
Cell Communication/genetics , Disease/genetics , Oligodendroglia/metabolism , Animals , Brain/metabolism , Central Nervous System/metabolism , Demyelinating Diseases/metabolism , Demyelinating Diseases/pathology , Humans , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Multiple Sclerosis/physiopathology , Myelin Sheath/metabolism , Neurons/metabolism , Oligodendroglia/physiology , Risk FactorsABSTRACT
Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
Subject(s)
Chromosomes, Human/genetics , Enhancer Elements, Genetic , Gene Amplification , Oncogenes , Acetylation , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Survival/genetics , Chromatin/metabolism , DNA, Neoplasm/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Genes, Neoplasm , Genetic Loci , Glioblastoma/genetics , Glioblastoma/pathology , Histones/metabolism , Humans , Neuroglia/metabolismABSTRACT
Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
Subject(s)
Chromatin/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Genome, Human , Neurogenesis/genetics , Promoter Regions, Genetic , Adult , Cell Line , Cerebrum/cytology , Cerebrum/growth & development , Cerebrum/metabolism , Chromatin/ultrastructure , Chromosome Mapping , Fetus , Histones/genetics , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Nerve Tissue Proteins/classification , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neurons/cytology , Neurons/metabolism , Temporal Lobe/cytology , Temporal Lobe/growth & development , Temporal Lobe/metabolism , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Opioid use disorder is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. Opioid overdose, the most severe outcome of opioid use disorder, remains the leading cause of accidental death in the United States. We interrogated the effects of opioid overdose on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicted case-control status with high accuracy. We focused on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid use disorder and captures genetic and environmental factors perpetuating the opioid epidemic.
Subject(s)
Opiate Overdose , Opioid-Related Disorders , Analgesics, Opioid/therapeutic use , Epigenesis, Genetic/genetics , Humans , Machine Learning , Opioid-Related Disorders/drug therapy , United StatesABSTRACT
CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+), Chd7(+/-), and Chd7(-/-) ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.
Subject(s)
DNA-Binding Proteins/physiology , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Animals , Binding Sites , DNA-Binding Proteins/metabolism , E1A-Associated p300 Protein/metabolism , Gene Expression Profiling , Histones/metabolism , Mice , Mice, Knockout , Protein BindingABSTRACT
Aim: Obesity and obesogenic diets might partly accelerate cancer development through epigenetic mechanisms. To determine these early effects, we investigated the impact of three days of a high-fat diet on epigenomic and transcriptomic changes in Apc Min/+ murine intestinal epithelia. Method: ChIP-Seq and RNA-Seq were performed on small intestinal epithelia of WT and Apc Min/+ male mice fed high-fat diet (HFD) or low-fat diet (LFD) for three days to identify genomic regions associated with differential H3K27ac levels as a marker of variant enhancer loci (VELs) as well as differentially expressed genes (DEGs). Results: Regarding epigenetic and transcriptomic changes, diet type (LFD vs. HFD) showed a significant impact, and genotype (WT vs.Apc Min/+) showed a small impact. Compared to LFD, HFD resulted in 1306 gained VELs, 230 lost VELs, 133 upregulated genes, and 127 downregulated genes in WT mice, with 1056 gained VELs, 371 lost VELs, 222 upregulated genes, and 182 downregulated genes in Apc Min/+ mice. Compared to the WT genotype, the Apc Min/+ genotype resulted in zero changed VELs for either diet type group, 21 DEGs for LFD, and 48 DEGs for HFD. Most gained VELs, and upregulated genes were associated with lipid metabolic processes. Gained VELs were also associated with Wnt signaling. Downregulated genes were associated with antigen presentation and processing. Conclusion: Three days of HFD-induced epigenomic and transcriptomic changes involving metabolic and immunologic pathways that may promote tumor growth in the genetically predisposed murine intestine without affecting key cancer signaling pathways.
ABSTRACT
The metastasis-invasion cascade describes the series of steps required for a cancer cell to successfully spread from its primary tumor and ultimately grow within a secondary organ. Despite metastasis being a dynamic, multistep process, most omics studies to date have focused on comparing primary tumors to the metastatic deposits that define end-stage disease. This static approach means we lack information about the genomic and epigenomic changes that occur during the majority of tumor progression. One particularly understudied phase of tumor progression is metastatic colonization, during which cells must adapt to the new microenvironment of the secondary organ. Through temporal profiling of chromatin accessibility and gene expression in vivo, we identify dynamic changes in the epigenome that occur as osteosarcoma tumors form and grow within the lung microenvironment. Furthermore, we show through paired in vivo and in vitro CRISPR drop-out screens and pharmacological validation that the upstream transcription factors represent a class of metastasis-specific dependency genes. While current models depict lung colonization as a discrete step within the metastatic cascade, our study shows it is a defined trajectory through multiple epigenetic states, revealing new therapeutic opportunities undetectable with standard approaches.
Subject(s)
Bone Neoplasms , Osteosarcoma , Humans , Chromatin/genetics , Osteosarcoma/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Epigenome , Bone Neoplasms/genetics , Tumor MicroenvironmentABSTRACT
Progressive pseudorheumatoid dysplasia (PPRD) is a genetic, non-inflammatory arthropathy caused by recessive loss of function mutations in WISP3 (Wnt1-inducible signaling pathway protein 3; MIM 603400), encoding for a signaling protein. The disease is clinically silent at birth and in infancy. It manifests between the age of 3 and 6 years with joint pain and progressive joint stiffness. Affected children are referred to pediatric rheumatologists and orthopedic surgeons; however, signs of inflammation are absent and anti-inflammatory treatment is of little help. Bony enlargement at the interphalangeal joints progresses leading to camptodactyly. Spine involvement develops in late childhood and adolescence leading to short trunk with thoracolumbar kyphosis. Adult height is usually below the 3rd percentile. Radiographic signs are relatively mild. Platyspondyly develops in late childhood and can be the first clue to the diagnosis. Enlargement of the phalangeal metaphyses develops subtly and is usually recognizable by 10 years. The femoral heads are large and the acetabulum forms a distinct "lip" overriding the femoral head. There is a progressive narrowing of all articular spaces as articular cartilage is lost. Medical management of PPRD remains symptomatic and relies on pain medication. Hip joint replacement surgery in early adulthood is effective in reducing pain and maintaining mobility and can be recommended. Subsequent knee joint replacement is a further option. Mutation analysis of WISP3 allowed the confirmation of the diagnosis in 63 out of 64 typical cases in our series. Intronic mutations in WISP3 leading to splicing aberrations can be detected only in cDNA from fibroblasts and therefore a skin biopsy is indicated when genomic analysis fails to reveal mutations in individuals with otherwise typical signs and symptoms. In spite of the first symptoms appearing in early childhood, the diagnosis of PPRD is most often made only in the second decade and affected children often receive unnecessary anti-inflammatory and immunosuppressive treatments. Increasing awareness of PPRD appears to be essential to allow for a timely diagnosis.
Subject(s)
Arthropathy, Neurogenic/diagnostic imaging , Arthropathy, Neurogenic/genetics , CCN Intercellular Signaling Proteins/genetics , Mutation/genetics , Adult , Alternative Splicing/genetics , Arthropathy, Neurogenic/ethnology , Arthropathy, Neurogenic/pathology , CCN Intercellular Signaling Proteins/chemistry , Calcinosis/diagnostic imaging , Child , Child, Preschool , DNA, Complementary/genetics , Hand/diagnostic imaging , Humans , Joint Diseases/congenital , Pelvis/diagnostic imaging , Pelvis/pathology , Polymorphism, Single Nucleotide/genetics , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Radiography , Reproducibility of Results , Spine/diagnostic imaging , Spine/pathologyABSTRACT
BACKGROUND: Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. METHODS: LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). RESULTS: Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p < 0.002). CONCLUSIONS: Our results provide additional information on the role of LRP5 mutations and their effects on the development of juvenile-onset primary osteoporosis, and hence the pathogenesis of the disorder. The mutations causing primary osteoporosis reduce the signaling activity of the canonical Wnt signaling pathway and may therefore result in decreased bone formation. The specific mechanism affecting signaling activity remains to be resolved in future studies.
Subject(s)
Low Density Lipoprotein Receptor-Related Protein-5/genetics , Osteoporosis/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway , Animals , Bone Density/genetics , CHO Cells , Cricetinae , Cricetulus , Genes, Reporter , Heterozygote , Humans , Low Density Lipoprotein Receptor-Related Protein-5/metabolism , Mutation, Missense , Osteogenesis Imperfecta/genetics , Phenotype , Real-Time Polymerase Chain Reaction , Receptors, Serotonin/metabolism , Serotonin/metabolism , Transfection , Tryptophan Hydroxylase/metabolismABSTRACT
In multiple types of cancer, an increased frequency in myeloid-derived suppressor cells (MDSC) is associated with worse outcomes and poor therapeutic response. In the glioblastoma (GBM) microenvironment, monocytic (m) MDSCs represent the predominant subset. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared with granulocytic (g) MDSCs has yet to be determined. Here we performed the first broad epigenetic profiling of MDSC subsets to define underlying cell-intrinsic differences in behavior and found that enhanced gene accessibility of cell adhesion programs in mMDSCs is linked to their tumor-accelerating ability in GBM models upon adoptive transfer. Mouse and human mMDSCs expressed higher levels of integrin ß1 and dipeptidyl peptidase-4 (DPP-4) compared with gMDSCs as part of an enhanced cell adhesion signature. Integrin ß1 blockade abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment, whereas treatment with a DPP-4 inhibitor extended survival in preclinical GBM models. Targeting DPP-4 in mMDSCs reduced pERK signaling and their migration towards tumor cells. These findings uncover a fundamental difference in the molecular basis of MDSC subsets and suggest that integrin ß1 and DPP-4 represent putative immunotherapy targets to attenuate myeloid cell-driven immune suppression in GBM. SIGNIFICANCE: Epigenetic profiling uncovers cell adhesion programming as a regulator of the tumor-promoting functions of monocytic myeloid-derived suppressor cells in glioblastoma, identifying therapeutic targets that modulate the immune response and suppress tumor growth.
Subject(s)
Cell Adhesion , Glioblastoma , Myeloid-Derived Suppressor Cells , Animals , Humans , Mice , Glioblastoma/metabolism , Glioblastoma/pathology , Integrin beta1/metabolism , Myeloid-Derived Suppressor Cells/pathology , Tumor MicroenvironmentABSTRACT
We developed a strategy to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation-microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.
Subject(s)
Chromatin Immunoprecipitation/methods , Chromosome Mapping/methods , Epitope Mapping/methods , Genetic Vectors/genetics , Oligonucleotide Array Sequence Analysis/methods , Proteins/genetics , Proteins/metabolism , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Proteins/immunology , Staining and LabelingABSTRACT
The six mammalian CCN genes (Cyr61, CTGF, Nov, WISP1, WISP2, WISP3) encode a family of secreted, cysteine-rich, multimodular proteins having roles in cell proliferation, adhesion, migration, and differentiation during embryogenesis, wound healing, and angiogenesis. We used bioinformatics to identify 9 CCN genes in zebrafish (zCCNs), 6 of which have not been previously described. When compared with mammalian CCN family members, 3 were paralogs of Cyr61, 2 of CTGF, 2 of WISP1, 1 of WISP2, and 1 of WISP3. No paralog of Nov was found. In situ hybridization was performed to characterize the sites of expression of the zCCNs during early zebrafish development. zCCNs demonstrated both unique and overlapping patterns of expression, suggesting potential division of labor between orthologous genes and providing an alternate approach to gene function studies that will complement studies in mammalian models.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , CCN Intercellular Signaling Proteins/genetics , Connective Tissue Growth Factor/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Base Sequence , Cell Differentiation/genetics , Genes , Molecular Sequence Data , Zebrafish/metabolismABSTRACT
Mammalian cells respond to insufficient oxygen through transcriptional regulators called hypoxia-inducible factors (HIFs). Although transiently protective, prolonged HIF activity drives distinct pathological responses in different tissues. Using a model of chronic HIF1a accumulation in pluripotent-stem-cell-derived oligodendrocyte progenitors (OPCs), we demonstrate that HIF1a activates non-canonical targets to impair generation of oligodendrocytes from OPCs. HIF1a activated a unique set of genes in OPCs through interaction with the OPC-specific transcription factor OLIG2. Non-canonical targets, including Ascl2 and Dlx3, were sufficient to block differentiation through suppression of the oligodendrocyte regulator Sox10. Chemical screening revealed that inhibition of MEK/ERK signaling overcame the HIF1a-mediated block in oligodendrocyte generation by restoring Sox10 expression without affecting canonical HIF1a activity. MEK/ERK inhibition also drove oligodendrocyte formation in hypoxic regions of human oligocortical spheroids. This work defines mechanisms by which HIF1a impairs oligodendrocyte formation and establishes that cell-type-specific HIF1a targets perturb cell function in response to low oxygen.
Subject(s)
Oligodendrocyte Precursor Cells , Pluripotent Stem Cells , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation , Cells, Cultured , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , OligodendrogliaABSTRACT
We extend the spectrum of phenotypes caused by mutations in the Wnt/Norrin coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) by identifying two novel types of mutation in related individuals whose presenting features were profound muscle hypotonia, mild mental retardation, blindness, and growth retardation. One mutation removes 6 out of 9 consecutive leucine residues in the LRP5 signal peptide (c.43_60del or p.Leu15_Leu20del), which impairs polypeptide entry into the endoplasmic reticulum (ER), trafficking to the cell membrane, and signal transduction. The second mutation resulted from nonhomologous recombination between Alu repeat sequences, which deleted exons 14-16 and would produce a nonfunctional, truncated, and frameshifted polypeptide, if expressed [chr11:g.(13871447_1387511)_(13879636_13879700)del (NW_925106.1) or p.Pro1010GlnfsX38]. We confirmed that the length of the LRP5 signal peptide poly-leucine repeat is polymorphic in the general population, and, importantly, we were able to demonstrate in independent in vitro assays that different allele sizes affect receptor processing and signal transduction. Consequently, this polymorphism may have physiologic effects in vivo. This latter finding is relevant since through a genomewide search we identified nearly 400 human proteins that contain poly-leucine repeats within their signal peptide. We chose 18 of these proteins and genotyped the underlying trinucleotide repeat in healthy Caucasian individuals. More than one length allele was observed in one-half of the proteins. We therefore propose that natural variation in poly-leucine-stretches within signal peptides constitutes a currently unrecognized source of variability in protein translation and expression.
Subject(s)
Abnormalities, Multiple/genetics , LDL-Receptor Related Proteins/genetics , Mutation , Osteoporosis/pathology , Protein Sorting Signals/genetics , Trinucleotide Repeats/genetics , Abnormalities, Multiple/pathology , Amino Acid Sequence , Base Sequence , Blotting, Western , Cell Line , Eye Diseases/pathology , Family Health , Female , Gene Frequency , Genotype , Humans , Leucine/genetics , Low Density Lipoprotein Receptor-Related Protein-5 , Luciferases/genetics , Luciferases/metabolism , Male , Pedigree , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Syndrome , TurkeyABSTRACT
Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent. The indels affect short homopolymer tracts of A/T and increase affinity for FOX transcription factors. We further demonstrate that signature mismatch-repair (MMR) mutations activate enhancers using a xenograft tumor metastasis model, where mutations are induced naturally via CRISPR/Cas9 inactivation of MLH1 prior to tumor cell injection. Our results suggest that MMR signature mutations activate enhancers in CRC tumor epigenomes to provide a selective advantage.
Subject(s)
Colorectal Neoplasms/genetics , DNA Mismatch Repair/genetics , Enhancer Elements, Genetic/genetics , Epigenome , Mutation/genetics , Acetylation , Animals , Base Sequence , Cell Line, Tumor , Gene Expression Regulation , Histones/metabolism , Humans , INDEL Mutation/genetics , Lysine/metabolism , Mice , Microsatellite Instability , Nucleotide Motifs/genetics , Phenotype , Selection, Genetic , Transcription Factors/metabolismABSTRACT
CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.
Subject(s)
Aldehyde Oxidoreductases/metabolism , CHARGE Syndrome/genetics , DNA Helicases/deficiency , DNA-Binding Proteins/deficiency , Gene Expression Regulation, Developmental , Retinal Dehydrogenase/metabolism , Tretinoin/metabolism , Aldehyde Oxidoreductases/genetics , Animals , CHARGE Syndrome/pathology , Cell Line, Tumor , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Disease Models, Animal , Ear, Inner/embryology , Embryo, Mammalian , Female , Gene Expression Profiling , Gene Knockdown Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Organogenesis/genetics , RNA, Small Interfering/metabolism , Retinal Dehydrogenase/geneticsABSTRACT
This corrects the article DOI: 10.1038/nm.4475.
ABSTRACT
Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.
Subject(s)
Carcinogenesis/genetics , Enhancer Elements, Genetic/genetics , Lung Neoplasms/genetics , Osteosarcoma/genetics , Cell Line, Tumor , Epigenomics , Gene Expression Regulation, Neoplastic , Genome, Human/genetics , Humans , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Neoplasm Metastasis/genetics , Osteosarcoma/pathology , Proteins/antagonists & inhibitors , Proteins/genetics , Selection, Genetic , Thromboplastin/genetics , Transcription Factor AP-1/antagonists & inhibitors , Transcription Factor AP-1/genetics , Tumor Microenvironment/geneticsABSTRACT
In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival.