Publications by authors named "Luca Pinello"

76 Publications

ZNF410 represses fetal globin by singular control of CHD4.

Nat Genet 2021 Apr 15. Epub 2021 Apr 15.

Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA, USA.

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-021-00843-wDOI Listing
April 2021

Author Correction: Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 Apr;53(4):586

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-021-00834-xDOI Listing
April 2021

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 04 1;53(4):511-520. Epub 2021 Mar 1.

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

BCL11A, the major regulator of fetal hemoglobin (HbF, αγ) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, αβ). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-021-00798-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038971PMC
April 2021

PrimeDesign software for rapid and simplified design of prime editing guide RNAs.

Nat Commun 2021 02 15;12(1):1034. Epub 2021 Feb 15.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21337-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884779PMC
February 2021

A Code of Ethics for Gene Drive Research.

CRISPR J 2021 Feb 10;4(1):19-24. Epub 2021 Feb 10.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/crispr.2020.0096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898401PMC
February 2021

Motif-Raptor: A Cell Type-Specific and Transcription Factor Centric Approach for Post-GWAS Prioritization of Causal Regulators.

Bioinformatics 2021 Feb 3. Epub 2021 Feb 3.

Department of Pathology, Massachusetts General Hospital, Charlestown, MA, USA.

Motivation: Genome-wide association studies (GWAS) have identified thousands of common trait-associated genetic variants but interpretation of their function remains challenging. These genetic variants can overlap the binding sites of transcription factors (TFs) and therefore could alter gene expression. However, we currently lack a systematic understanding on how this mechanism contributes to phenotype.

Results: We present Motif-Raptor, a TF-centric computational tool that integrates sequence-based predictive models, chromatin accessibility, gene expression datasets and GWAS summary statistics to systematically investigate how TF function is affected by genetic variants. Given trait associated non-coding variants, Motif-Raptor can recover relevant cell types and critical TFs to drive hypotheses regarding their mechanism of action. We tested Motif-Raptor on complex traits such as rheumatoid arthritis and red blood cell count and demonstrated its ability to prioritize relevant cell types, potential regulatory TFs and non-coding SNPs which have been previously characterized and validated.

Availability: Motif-Raptor is freely available as a Python package at: https://github.com/pinellolab/MotifRaptor.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btab072DOI Listing
February 2021

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Cell Stem Cell 2021 Jan 25. Epub 2021 Jan 25.

Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2020.12.015DOI Listing
January 2021

Aging-Associated Alterations in Mammary Epithelia and Stroma Revealed by Single-Cell RNA Sequencing.

Cell Rep 2020 Dec;33(13):108566

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Aging is closely associated with increased susceptibility to breast cancer, yet there have been limited systematic studies of aging-induced alterations in the mammary gland. Here, we leverage high-throughput single-cell RNA sequencing to generate a detailed transcriptomic atlas of young and aged murine mammary tissues. By analyzing epithelial, stromal, and immune cells, we identify age-dependent alterations in cell proportions and gene expression, providing evidence that suggests alveolar maturation and physiological decline. The analysis also uncovers potential pro-tumorigenic mechanisms coupled to the age-associated loss of tumor suppressor function and change in microenvironment. In addition, we identify a rare, age-dependent luminal population co-expressing hormone-sensing and secretory-alveolar lineage markers, as well as two macrophage populations expressing distinct gene signatures, underscoring the complex heterogeneity of the mammary epithelia and stroma. Collectively, this rich single-cell atlas reveals the effects of aging on mammary physiology and can serve as a useful resource for understanding aging-associated cancer risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898263PMC
December 2020

Robust and Scalable Learning of Complex Intrinsic Dataset Geometry via ElPiGraph.

Entropy (Basel) 2020 Mar 4;22(3). Epub 2020 Mar 4.

Institut Curie, PSL Research University, 75005 Paris, France.

Multidimensional datapoint clouds representing large datasets are frequently characterized by non-trivial low-dimensional geometry and topology which can be recovered by unsupervised machine learning approaches, in particular, by principal graphs. Principal graphs approximate the multivariate data by a graph injected into the data space with some constraints imposed on the node mapping. Here we present ElPiGraph, a scalable and robust method for constructing principal graphs. ElPiGraph exploits and further develops the concept of elastic energy, the topological graph grammar approach, and a gradient descent-like optimization of the graph topology. The method is able to withstand high levels of noise and is capable of approximating data point clouds via principal graph ensembles. This strategy can be used to estimate the statistical significance of complex data features and to summarize them into a single consensus principal graph. ElPiGraph deals efficiently with large datasets in various fields such as biology, where it can be used for example with single-cell transcriptomic or epigenomic datasets to infer gene expression dynamics and recover differentiation landscapes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/e22030296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516753PMC
March 2020

Epigenetic Alterations in Keratinocyte Carcinoma.

J Invest Dermatol 2020 Nov 16. Epub 2020 Nov 16.

Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care Institute, Boston, Massachusetts, USA.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are both derived from epidermal keratinocytes but are phenotypically diverse. To improve the understanding of keratinocyte carcinogenesis, it is critical to understand epigenetic alterations, especially those that govern gene expression. We examined changes to the enhancer-associated histone acetylation mark H3K27ac by mapping matched tumor-normal pairs from 11 patients (five with BCC and six with SCC) undergoing Mohs surgery. Our analysis uncovered cancer-specific enhancers on the basis of differential H3K27ac peaks between matched tumor-normal pairs. We also uncovered biological pathways potentially altered in keratinocyte carcinoma, including enriched epidermal development and Wnt signaling pathways enriched in BCCs and enriched immune response and cell activation pathways in SCCs. We also observed enrichment of transcription factors that implicated SMAD and JDP2 in BCC pathogenesis and FOXP1 in SCC pathogenesis. On the basis of these findings, we prioritized three loci with putative regulation events (FGFR2 enhancer in BCC, intragenic regulation of FOXP1 in SCC, and WNT5A promoter in both subtypes) and validated our findings with published gene expression data. Our findings highlight unique and shared epigenetic alterations in histone modifications and potential regulators for BCCs and SCCs that likely impact the divergent oncogenic pathways, paving the way for targeted drug discoveries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2020.10.018DOI Listing
November 2020

High throughput single-cell detection of multiplex CRISPR-edited gene modifications.

Genome Biol 2020 10 20;21(1):266. Epub 2020 Oct 20.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-020-02174-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574538PMC
October 2020

Parallel Single-Cell RNA-Seq and Genetic Recording Reveals Lineage Decisions in Developing Embryoid Bodies.

Cell Rep 2020 10;33(1):108222

Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Early developmental specification can be modeled by differentiating embryonic stem cells (ESCs) to embryoid bodies (EBs), a heterogeneous mixture of three germ layers. Here, we combine single-cell transcriptomics and genetic recording to characterize EB differentiation. We map transcriptional states along a time course and model cell fate trajectories and branchpoints as cells progress to distinct germ layers. To validate this inferential model, we propose an innovative inducible genetic recording technique that leverages recombination to generate cell-specific, timestamp barcodes in a narrow temporal window. We validate trajectory architecture and key branchpoints, including early specification of a primordial germ cell (PGC)-like lineage from preimplantation epiblast-like cells. We further identify a temporally defined role of DNA methylation in this PGC-epiblast decision. Our study provides a high-resolution lineage map for an organoid model of embryogenesis, insights into epigenetic determinants of fate specification, and a strategy for lineage mapping of rapid differentiation processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646252PMC
October 2020

A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies and regulatory elements of in murine ESCs.

J Biol Chem 2020 11 29;295(47):15797-15809. Epub 2020 Sep 29.

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia. Electronic address:

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in or in Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed and regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb -region (REs; CREs) of the gene locus, as well as genome-wide 2,613 high-confidence REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling gene expression. A subset of active CREs and TREs physically interact with the promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case ) in their native context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681025PMC
November 2020

Technologies and Computational Analysis Strategies for CRISPR Applications.

Mol Cell 2020 07;79(1):11-29

Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA; Department of Pathology, Harvard Medical School, Boston, MA, USA; The Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address:

The CRISPR-Cas system offers a programmable platform for eukaryotic genome and epigenome editing. The ability to perform targeted genetic and epigenetic perturbations enables researchers to perform a variety of tasks, ranging from investigating questions in basic biology to potentially developing novel therapeutics for the treatment of disease. While CRISPR systems have been engineered to target DNA and RNA with increased precision, efficiency, and flexibility, assays to identify off-target editing are becoming more comprehensive and sensitive. Furthermore, techniques to perform high-throughput genome and epigenome editing can be paired with a variety of readouts and are uncovering important cellular functions and mechanisms. These technological advances drive and are driven by accompanying computational approaches. Here, we briefly present available CRISPR technologies and review key computational advances and considerations for various CRISPR applications. In particular, we focus on the analysis of on- and off-target editing and CRISPR pooled screen data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497852PMC
July 2020

Publisher Correction: Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Nat Biotechnol 2020 Jul;38(7):901

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-020-0587-zDOI Listing
July 2020

Therapeutic base editing of human hematopoietic stem cells.

Nat Med 2020 04 16;26(4):535-541. Epub 2020 Mar 16.

Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA, USA.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34 hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-0790-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869435PMC
April 2020

Eleven grand challenges in single-cell data science.

Genome Biol 2020 02 7;21(1):31. Epub 2020 Feb 7.

Life Sciences and Health, Centrum Wiskunde & Informatica, Amsterdam, The Netherlands.

The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-020-1926-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007675PMC
February 2020

CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome editing.

Bioinformatics 2020 04;36(7):2001-2008

Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.

Motivation: Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects.

Results: To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project).

Availability And Implementation: The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btz867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141852PMC
April 2020

Assessment of computational methods for the analysis of single-cell ATAC-seq data.

Genome Biol 2019 11 18;20(1):241. Epub 2019 Nov 18.

Molecular Pathology Unit, Massachusetts General Hospital Research Institute, Charlestown, MA, 02129, USA.

Background: Recent innovations in single-cell Assay for Transposase Accessible Chromatin using sequencing (scATAC-seq) enable profiling of the epigenetic landscape of thousands of individual cells. scATAC-seq data analysis presents unique methodological challenges. scATAC-seq experiments sample DNA, which, due to low copy numbers (diploid in humans), lead to inherent data sparsity (1-10% of peaks detected per cell) compared to transcriptomic (scRNA-seq) data (10-45% of expressed genes detected per cell). Such challenges in data generation emphasize the need for informative features to assess cell heterogeneity at the chromatin level.

Results: We present a benchmarking framework that is applied to 10 computational methods for scATAC-seq on 13 synthetic and real datasets from different assays, profiling cell types from diverse tissues and organisms. Methods for processing and featurizing scATAC-seq data were compared by their ability to discriminate cell types when combined with common unsupervised clustering approaches. We rank evaluated methods and discuss computational challenges associated with scATAC-seq analysis including inherently sparse data, determination of features, peak calling, the effects of sequencing coverage and noise, and clustering performance. Running times and memory requirements are also discussed.

Conclusions: This reference summary of scATAC-seq methods offers recommendations for best practices with consideration for both the non-expert user and the methods developer. Despite variation across methods and datasets, SnapATAC, Cusanovich2018, and cisTopic outperform other methods in separating cell populations of different coverages and noise levels in both synthetic and real datasets. Notably, SnapATAC is the only method able to analyze a large dataset (> 80,000 cells).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-019-1854-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859644PMC
November 2019

LSD1 suppresses invasion, migration and metastasis of luminal breast cancer cells via activation of GATA3 and repression of TRIM37 expression.

Oncogene 2019 10 13;38(44):7017-7034. Epub 2019 Aug 13.

Division of Genetics, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA.

LSD1 (KDM1A) is a histone demethylase that plays both oncogenic and tumor suppressor roles in breast cancer. However, the exact contexts under which it plays these opposite functions remain largely elusive. By characterizing its role in luminal breast epithelial cells, here we show that inhibition of LSD1 by both genetic and pharmacological approaches increases their invasion and migration, whereas its inhibition by genetic approach, but not by pharmacological approach, impairs their proliferation/survival. Induced loss of LSD1 in luminal cells in a mouse model of luminal breast cancer, MMTV-PyMT, leads to a profound increase in lung metastasis. Mechanistically, LSD1 interacts with GATA3, a key luminal-specific transcription factor (TF), and their common target genes are highly related to breast cancer. LSD1 positively regulates GATA3 expression. It also represses expression of TRIM37, a breast epithelial oncogene encoding a histone H2A ubiquitin ligase, and ELF5, a key TF gene for luminal progenitors and alveolar luminal cells. LSD1-loss also leads to reduced expression of several cell-cell adhesion genes (e.g., CDH1, VCL, CTNNA1), possibly via TRIM37-upregulation and subsequently TRIM37-mediated repression. Collectively, our data suggest LSD1 largely plays a tumor suppressor role in luminal breast cancer and the oncogenic program associated with LSD1-inhibition may be suppressed via TRIM37-inhibition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-019-0923-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823153PMC
October 2019

Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.

Nat Genet 2019 07 28;51(7):1149-1159. Epub 2019 Jun 28.

Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Harvard Medical School, Boston, MA, USA.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-019-0453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650275PMC
July 2019

Impact of Genetic Variation on CRISPR-Cas Targeting.

CRISPR J 2018 04;1(2):159-170

1 Molecular Pathology Unit, Massachusetts General Hospital , Charlestown, Massachusetts.

The CRISPR-CRISPR-associated (Cas) nuclease system offers the ability to perform unprecedented functional genetic experiments and the promise of therapy for a variety of genetic disorders. The understanding of factors contributing to CRISPR targeting efficacy and specificity continues to evolve. As CRISPR systems rely on Watson-Crick base pairing to ultimately mediate genomic cleavage, it logically follows that genetic variation would affect CRISPR targeting by increasing or decreasing sequence homology at on-target and off-target sites or by altering protospacer adjacent motifs. Numerous efforts have been made to document the extent of human genetic variation, which can serve as resources to understand and mitigate the effect of genetic variation on CRISPR targeting. Here, we review efforts to elucidate the effect of human genetic variation on CRISPR targeting at on-target and off-target sites with considerations for laboratory experiments and clinical translation of CRISPR-based therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/crispr.2017.0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319324PMC
April 2018

Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM.

Nat Commun 2019 04 23;10(1):1903. Epub 2019 Apr 23.

Molecular Pathology Unit & Cancer Center, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA, 02114, USA.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09670-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478907PMC
April 2019

TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.

Mol Cell 2019 06 17;74(6):1148-1163.e7. Epub 2019 Apr 17.

Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia. Electronic address:

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671628PMC
June 2019

CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML.

Nat Chem Biol 2019 05 15;15(5):529-539. Epub 2019 Apr 15.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure-activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR-Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-019-0263-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679026PMC
May 2019

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Nat Med 2019 05 25;25(5):776-783. Epub 2019 Mar 25.

Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, αγ). Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0401-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512986PMC
May 2019

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Nat Biotechnol 2019 03 11;37(3):276-282. Epub 2019 Feb 11.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM). To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-018-0011-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401248PMC
March 2019

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia.

Blood 2019 05 31;133(21):2255-2262. Epub 2019 Jan 31.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34 hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2019-01-895094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533605PMC
May 2019