Publications by authors named "Jesse M Engreitz"

34 Publications

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

Nature 2021 07 29;595(7865):107-113. Epub 2021 Apr 29.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63 intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03570-8DOI Listing
July 2021

Genome-wide enhancer maps link risk variants to disease genes.

Nature 2021 05 7;593(7858):238-243. Epub 2021 Apr 7.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease. Many of the underlying causal variants may affect enhancers, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03446-xDOI Listing
May 2021

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

bioRxiv 2021 Feb 25. Epub 2021 Feb 25.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63 intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.02.25.430130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924267PMC
February 2021

Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability.

Nat Neurosci 2021 03 4;24(3):437-448. Epub 2021 Feb 4.

Department of Neurobiology, Harvard Medical School, Boston, MA, USA.

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-020-00786-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933108PMC
March 2021

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.

Proc Natl Acad Sci U S A 2020 12 21;117(52):33404-33413. Epub 2020 Dec 21.

Broad Institute of MIT and Harvard, Cambridge, MA 02142;

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2010738117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776864PMC
December 2020

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Nature 2020 10 14;586(7831):763-768. Epub 2020 Oct 14.

Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer and coronary heart disease-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP). Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2819-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944936PMC
October 2020

Prioritizing disease and trait causal variants at the TNFAIP3 locus using functional and genomic features.

Nat Commun 2020 03 6;11(1):1237. Epub 2020 Mar 6.

Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

Genome-wide association studies have associated thousands of genetic variants with complex traits and diseases, but pinpointing the causal variant(s) among those in tight linkage disequilibrium with each associated variant remains a major challenge. Here, we use seven experimental assays to characterize all common variants at the multiple disease-associated TNFAIP3 locus in five disease-relevant immune cell lines, based on a set of features related to regulatory potential. Trait/disease-associated variants are enriched among SNPs prioritized based on either: (1) residing within CRISPRi-sensitive regulatory regions, or (2) localizing in a chromatin accessible region while displaying allele-specific reporter activity. Of the 15 trait/disease-associated haplotypes at TNFAIP3, 9 have at least one variant meeting one or both of these criteria, 5 of which are further supported by genetic fine-mapping. Our work provides a comprehensive strategy to characterize genetic variation at important disease-associated loci, and aids in the effort to identify trait causal genetic variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15022-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060350PMC
March 2020

Activity-by-contact model of enhancer-promoter regulation from thousands of CRISPR perturbations.

Nat Genet 2019 12 29;51(12):1664-1669. Epub 2019 Nov 29.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-019-0538-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886585PMC
December 2019

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis.

Elife 2019 05 9;8. Epub 2019 May 9.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, United States.

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.44080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534380PMC
May 2019

Discovering metabolic disease gene interactions by correlated effects on cellular morphology.

Mol Metab 2019 06 13;24:108-119. Epub 2019 Mar 13.

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Division of Endocrinology, Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA. Electronic address:

Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation.

Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene.

Results: Over 10 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions.

Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2019.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531784PMC
June 2019

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.

Nature 2018 09 27;561(7721):132-136. Epub 2018 Aug 27.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0453-zDOI Listing
September 2018

Deep-coverage whole genome sequences and blood lipids among 16,324 individuals.

Nat Commun 2018 08 23;9(1):3391. Epub 2018 Aug 23.

School of Medicine, University of Maryland, Baltimore, MD, 21201, USA.

Large-scale deep-coverage whole-genome sequencing (WGS) is now feasible and offers potential advantages for locus discovery. We perform WGS in 16,324 participants from four ancestries at mean depth >29X and analyze genotypes with four quantitative traits-plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, and triglycerides. Common variant association yields known loci except for few variants previously poorly imputed. Rare coding variant association yields known Mendelian dyslipidemia genes but rare non-coding variant association detects no signals. A high 2M-SNP LDL-C polygenic score (top 5th percentile) confers similar effect size to a monogenic mutation (~30 mg/dl higher for each); however, among those with severe hypercholesterolemia, 23% have a high polygenic score and only 2% carry a monogenic mutation. At these sample sizes and for these phenotypes, the incremental value of WGS for discovery is limited but WGS permits simultaneous assessment of monogenic and polygenic models to severe hypercholesterolemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-05747-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107638PMC
August 2018

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

Cell 2018 03 15;173(1):90-103.e19. Epub 2018 Mar 15.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.02.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866246PMC
March 2018

Cohesin Loss Eliminates All Loop Domains.

Cell 2017 Oct;171(2):305-320.e24

The Center for Genome Architecture, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Center for Theoretical Biological Physics, Rice University, Houston, TX 77030, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA; Departments of Computer Science and Computational and Applied Mathematics, Rice University, Houston, TX 77030, USA. Electronic address:

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846482PMC
October 2017

Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.

Nature 2017 08 11;548(7667):343-346. Epub 2017 Aug 11.

Department of Biological Engineering, MIT, Cambridge, Massachusetts 02139, USA.

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature23451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706657PMC
August 2017

A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression.

Cell 2017 Jul;170(3):522-533.e15

Broad Institute of MIT and Harvard University, Cambridge, MA, USA; Cardiology Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA USA; Center for Genomic Medicine, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Electronic address:

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.06.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785707PMC
July 2017

Recurrent and functional regulatory mutations in breast cancer.

Nature 2017 07 28;547(7661):55-60. Epub 2017 Jun 28.

Massachusetts General Hospital Center for Cancer Research, Charlestown, Massachusetts 02129, USA.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature22992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563978PMC
July 2017

Local regulation of gene expression by lncRNA promoters, transcription and splicing.

Nature 2016 11 26;539(7629):452-455. Epub 2016 Oct 26.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature20149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853796PMC
November 2016

Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression.

Nat Rev Mol Cell Biol 2016 12 26;17(12):756-770. Epub 2016 Oct 26.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples - including Xist, which orchestrates X chromosome inactivation - has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrm.2016.126DOI Listing
December 2016

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Nat Med 2016 12 24;22(12):1402-1410. Epub 2016 Oct 24.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8 T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.4200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209798PMC
December 2016

Systematic mapping of functional enhancer-promoter connections with CRISPR interference.

Science 2016 11 29;354(6313):769-773. Epub 2016 Sep 29.

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the seven enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aag2445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438575PMC
November 2016

RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites.

Cell 2014 Sep;159(1):188-199

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2014.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177037PMC
September 2014

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

Cell 2014 Sep 11;159(1):148-162. Epub 2014 Sep 11.

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA. Electronic address:

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2014.08.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180118PMC
September 2014

Neuregulin autocrine signaling promotes self-renewal of breast tumor-initiating cells by triggering HER2/HER3 activation.

Cancer Res 2014 Jan 31;74(1):341-52. Epub 2013 Oct 31.

Authors' Affiliations: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine and Departments of Pathology and Radiation Oncology, Stanford University School of Medicine, Stanford, California; and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Currently, only patients with HER2-positive tumors are candidates for HER2-targeted therapies. However, recent clinical observations suggest that the survival of patients with HER2-low breast cancers, who lack HER2 amplification, may benefit from adjuvant therapy that targets HER2. In this study, we explored a mechanism through which these benefits may be obtained. Prompted by the hypothesis that HER2/HER3 signaling in breast tumor-initiating cells (TIC) promotes self-renewal and survival, we obtained evidence that neuregulin 1 (NRG1) produced by TICs promotes their proliferation and self-renewal in HER2-low tumors, including in triple-negative breast tumors. Pharmacologic inhibition of EGFR, HER2, or both receptors reduced breast TIC survival and self-renewal in vitro and in vivo and increased TIC sensitivity to ionizing radiation. Through a tissue microarray analysis, we found that NRG1 expression and associated HER2 activation occurred in a subset of HER2-low breast cancers. Our results offer an explanation for why HER2 inhibition blocks the growth of HER2-low breast tumors. Moreover, they argue that dual inhibition of EGFR and HER2 may offer a useful therapeutic strategy to target TICs in these tumors. In generating a mechanistic rationale to apply HER2-targeting therapies in patients with HER2-low tumors, this work shows why these therapies could benefit a considerably larger number of patients with breast cancer than they currently reach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-13-1055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917843PMC
January 2014

The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome.

Science 2013 Aug 4;341(6147):1237973. Epub 2013 Jul 4.

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1237973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778663PMC
August 2013

Three-dimensional genome architecture influences partner selection for chromosomal translocations in human disease.

PLoS One 2012 28;7(9):e44196. Epub 2012 Sep 28.

Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, United States of America.

Chromosomal translocations are frequent features of cancer genomes that contribute to disease progression. These rearrangements result from formation and illegitimate repair of DNA double-strand breaks (DSBs), a process that requires spatial colocalization of chromosomal breakpoints. The "contact first" hypothesis suggests that translocation partners colocalize in the nuclei of normal cells, prior to rearrangement. It is unclear, however, the extent to which spatial interactions based on three-dimensional genome architecture contribute to chromosomal rearrangements in human disease. Here we intersect Hi-C maps of three-dimensional chromosome conformation with collections of 1,533 chromosomal translocations from cancer and germline genomes. We show that many translocation-prone pairs of regions genome-wide, including the cancer translocation partners BCR-ABL and MYC-IGH, display elevated Hi-C contact frequencies in normal human cells. Considering tissue specificity, we find that translocation breakpoints reported in human hematologic malignancies have higher Hi-C contact frequencies in lymphoid cells than those reported in sarcomas and epithelial tumors. However, translocations from multiple tissue types show significant correlation with Hi-C contact frequencies, suggesting that both tissue-specific and universal features of chromatin structure contribute to chromosomal alterations. Our results demonstrate that three-dimensional genome architecture shapes the landscape of rearrangements directly observed in human disease and establish Hi-C as a key method for dissecting these effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044196PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460994PMC
March 2013
-->