Publications by authors named "Sherman M Weissman"

68 Publications

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture.

Cell Discov 2021 Jun 29;7(1):47. Epub 2021 Jun 29.

Department of Genetics, Yale School of Medicine, New Haven, USA.

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2 fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2 fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.
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http://dx.doi.org/10.1038/s41421-021-00266-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238952PMC
June 2021

Colorectal Cancer Stem Cell States Uncovered by Simultaneous Single-Cell Analysis of Transcriptome and Telomeres.

Adv Sci (Weinh) 2021 04 8;8(8):2004320. Epub 2021 Feb 8.

State Key Laboratory of Medicinal Chemical Biology Nankai University Tianjin 300350 China.

Cancer stem cells (CSCs) presumably contribute to tumor progression and drug resistance, yet their definitive features have remained elusive. Here, simultaneous measurement of telomere length and transcriptome in the same cells enables systematic assessment of CSCs in primary colorectal cancer (CRC). The in-depth transcriptome profiled by SMART-seq2 is independently validated by high-throughput scRNA-seq using 10 × Genomics. It is found that rare CSCs exist in dormant state and display plasticity toward cancer epithelial cells (EPCs) that essentially are presumptive tumor-initiating cells (TICs), while both retaining the prominent signaling pathways including WNT, TGF-, and HIPPO/YAP. Moreover, CSCs exhibit chromosome copy number variation (CNV) pattern resembling cancer EPCs but distinct from normal stem cells, suggesting the phylogenetic relationship between CSCs and cancer EPCs. Notably, CSCs maintain shorter telomeres and possess minimal telomerase activity consistent with their nonproliferative nature, unlike cancer EPCs. Additionally, the specific signature of CSCs particularly , , , , and correlates with the prognosis of CRC. These findings characterize the heterogeneity of CSCs and their linkage to cancer EPCs/TICs, some of which are conventionally regarded as CSCs.
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http://dx.doi.org/10.1002/advs.202004320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061397PMC
April 2021

Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons.

Mol Cell 2020 07 10;79(1):84-98.e9. Epub 2020 Jun 10.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
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http://dx.doi.org/10.1016/j.molcel.2020.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375197PMC
July 2020

An Overview of Advances in Cell-Based Cancer Immunotherapies Based on the Multiple Immune-Cancer Cell Interactions.

Methods Mol Biol 2020 ;2097:139-171

Department of Pathology, Yale School of Medicine, New Haven, CT, USA.

Tumors have a complex ecosystem in which behavior and fate are determined by the interaction of diverse cancerous and noncancerous cells at local and systemic levels. A number of studies indicate that various immune cells participate in tumor development (Fig. 1). In this review, we will discuss interactions among T lymphocytes (T cells), B cells, natural killer (NK) cells, dendritic cells (DCs), tumor-associated macrophages (TAMs), neutrophils, and myeloid-derived suppressor cells (MDSCs). In addition, we will touch upon attempts to either use or block subsets of immune cells to target cancer.
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http://dx.doi.org/10.1007/978-1-0716-0203-4_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062409PMC
January 2021

A versatile flow-based assay for immunocyte-mediated cytotoxicity.

J Immunol Methods 2019 11 13;474:112668. Epub 2019 Sep 13.

Department of Pathology, Yale School of Medicine, New Haven, CT 06525, USA; Yale Comprehensive Cancer Center, Yale School of Medicine, New Haven, CT 06525, USA. Electronic address:

Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.
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http://dx.doi.org/10.1016/j.jim.2019.112668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891822PMC
November 2019

Telomere Maintenance-Associated PML Is a Potential Specific Therapeutic Target of Human Colorectal Cancer.

Transl Oncol 2019 Sep 15;12(9):1164-1176. Epub 2019 Jun 15.

State Key Laboratory of Medicinal Chemical Biology, 2011 Collaborative Innovation Center for Biotherapy; Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China. Electronic address:

Telomere length maintenance is essential for cell proliferation, which is particularly prominent in cancer. We validate that the primary colorectal tumors exhibit heterogeneous telomere lengths but mostly (90%) short telomeres relative to normal tissues. Intriguingly, relatively short telomeres are associated with tumor malignancy as indicated by poorly differentiated state, and these tumors contain more cancer stem-like cells (CSLCs) identified by several commonly used markers CD44, EPHB2 or LGR5. Moreover, promyelocytic leukemia (PML) and ALT-associated PML nuclear bodies (APBs) are frequently found in tumors with short telomeres and high proliferation. In contrast, distant normal tissues rarely or only minimally express PML. Inhibition of PML and APBs by an ATR inhibitor decreases proliferation of CSLCs and organoids, suggesting a potential therapeutic target to progressive colorectal tumors. Together, telomere maintenance underling tumor progression is connected with CSLCs.
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http://dx.doi.org/10.1016/j.tranon.2019.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580093PMC
September 2019

hESC-Derived Thalamic Organoids Form Reciprocal Projections When Fused with Cortical Organoids.

Cell Stem Cell 2019 03 21;24(3):487-497.e7. Epub 2019 Feb 21.

Department of Genetics, Yale Stem Cell Center, Yale Child Study Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. These efforts have largely focused on generating telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid models for the human thalamus has not been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing revealed a formation of distinct thalamic lineages, which diverge from telencephalic fate. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex. Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.
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http://dx.doi.org/10.1016/j.stem.2018.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853597PMC
March 2019

Correction to: MISC: missing imputation for single-cell RNA sequencing data.

BMC Syst Biol 2019 Jan 22;13(1):13. Epub 2019 Jan 22.

Joint Bioinformatics Program, University of Arkansas Little Rock George Washington Donaghey College of Engineering & IT and University of Arkansas for Medical Sciences, Little Rock, AR, 72204, USA.

It was highlighted that the original article [1] contained a typesetting error in the last name of Allon Canaan. This was incorrectly captured as Allon Canaann in the original article which has since been updated.
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http://dx.doi.org/10.1186/s12918-019-0681-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343234PMC
January 2019

Local and global chromatin interactions are altered by large genomic deletions associated with human brain development.

Nat Commun 2018 12 17;9(1):5356. Epub 2018 Dec 17.

Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, 94304, CA, USA.

Large copy number variants (CNVs) in the human genome are strongly associated with common neurodevelopmental, neuropsychiatric disorders such as schizophrenia and autism. Here we report on the epigenomic effects of the prominent large deletion CNVs on chromosome 22q11.2 and on chromosome 1q21.1. We use Hi-C analysis of long-range chromosome interactions, including haplotype-specific Hi-C analysis, ChIP-Seq analysis of regulatory histone marks, and RNA-Seq analysis of gene expression patterns. We observe changes on all the levels of analysis, within the deletion boundaries, in the deletion flanking regions, along chromosome 22q, and genome wide. We detect gene expression changes as well as pronounced and multilayered effects on chromatin states, chromosome folding and on the topological domains of the chromatin, that emanate from the large CNV locus. These findings suggest basic principles of how such large genomic deletions can alter nuclear organization and affect genomic molecular activity.
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http://dx.doi.org/10.1038/s41467-018-07766-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297223PMC
December 2018

MISC: missing imputation for single-cell RNA sequencing data.

BMC Syst Biol 2018 12 14;12(Suppl 7):114. Epub 2018 Dec 14.

Joint Bioinformatics Program, University of Arkansas Little Rock George Washington Donaghey College of Engineering & IT and University of Arkansas for Medical Sciences, Little Rock, AR, 72204, USA.

Background: Single-cell RNA sequencing (scRNA-seq) technology provides an effective way to study cell heterogeneity. However, due to the low capture efficiency and stochastic gene expression, scRNA-seq data often contains a high percentage of missing values. It has been showed that the missing rate can reach approximately 30% even after noise reduction. To accurately recover missing values in scRNA-seq data, we need to know where the missing data is; how much data is missing; and what are the values of these data.

Methods: To solve these three problems, we propose a novel model with a hybrid machine learning method, namely, missing imputation for single-cell RNA-seq (MISC). To solve the first problem, we transformed it to a binary classification problem on the RNA-seq expression matrix. Then, for the second problem, we searched for the intersection of the classification results, zero-inflated model and false negative model results. Finally, we used the regression model to recover the data in the missing elements.

Results: We compared the raw data without imputation, the mean-smooth neighbor cell trajectory, MISC on chronic myeloid leukemia data (CML), the primary somatosensory cortex and the hippocampal CA1 region of mouse brain cells. On the CML data, MISC discovered a trajectory branch from the CP-CML to the BC-CML, which provides direct evidence of evolution from CP to BC stem cells. On the mouse brain data, MISC clearly divides the pyramidal CA1 into different branches, and it is direct evidence of pyramidal CA1 in the subpopulations. In the meantime, with MISC, the oligodendrocyte cells became an independent group with an apparent boundary.

Conclusions: Our results showed that the MISC model improved the cell type classification and could be instrumental to study cellular heterogeneity. Overall, MISC is a robust missing data imputation model for single-cell RNA-seq data.
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http://dx.doi.org/10.1186/s12918-018-0638-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293493PMC
December 2018

Ritornello: high fidelity control-free chromatin immunoprecipitation peak calling.

Nucleic Acids Res 2017 Dec;45(21):e173

Department of Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. Chromatin immunoprecipitation (ChIP-seq) is the omics technique that enables genome wide localization of transcription factor (TF) binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP-target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach for finding TF-binding sites in ChIP-seq, with roots in digital signal processing that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq. Ritornello is freely available at https://github.com/KlugerLab/Ritornello.
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http://dx.doi.org/10.1093/nar/gkx799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716106PMC
December 2017

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration.

Cell Stem Cell 2017 09 27;21(3):383-398.e7. Epub 2017 Jul 27.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.
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http://dx.doi.org/10.1016/j.stem.2017.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720381PMC
September 2017

Detecting heterogeneity in single-cell RNA-Seq data by non-negative matrix factorization.

PeerJ 2017 19;5:e2888. Epub 2017 Jan 19.

Epidemiology Program, University of Hawaii Cancer Center , Honolulu , HI , United States.

Single-cell RNA-Sequencing (scRNA-Seq) is a fast-evolving technology that enables the understanding of biological processes at an unprecedentedly high resolution. However, well-suited bioinformatics tools to analyze the data generated from this new technology are still lacking. Here we investigate the performance of non-negative matrix factorization (NMF) method to analyze a wide variety of scRNA-Seq datasets, ranging from mouse hematopoietic stem cells to human glioblastoma data. In comparison to other unsupervised clustering methods including K-means and hierarchical clustering, NMF has higher accuracy in separating similar groups in various datasets. We ranked genes by their importance scores (-scores) in separating these groups, and discovered that NMF uniquely identifies genes expressed at intermediate levels as top-ranked genes. Finally, we show that in conjugation with the modularity detection method FEM, NMF reveals meaningful protein-protein interaction modules. In summary, we propose that NMF is a desirable method to analyze heterogeneous single-cell RNA-Seq data. The NMF based subpopulation detection package is available at: https://github.com/lanagarmire/NMFEM.
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http://dx.doi.org/10.7717/peerj.2888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5251935PMC
January 2017

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

Nucleic Acids Res 2017 Jun;45(10):e77

Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.
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http://dx.doi.org/10.1093/nar/gkx026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605247PMC
June 2017

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

Stem Cell Reports 2016 07 30;7(1):43-54. Epub 2016 Jun 30.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT 06520, USA. Electronic address:

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945581PMC
July 2016

A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions.

Nat Commun 2016 Mar 30;7:11178. Epub 2016 Mar 30.

Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.
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http://dx.doi.org/10.1038/ncomms11178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820989PMC
March 2016

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

Cancer Res 2016 Mar 3;76(6):1305-12. Epub 2016 Mar 3.

Hangzhou Cancer Institution, Hangzhou Cancer Hospital, Hangzhou, Zhejiang Province, P. R. China.

Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1907DOI Listing
March 2016

A comparative encyclopedia of DNA elements in the mouse genome.

Nature 2014 Nov;515(7527):355-64

Bioinformatics and Genomics, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003 Barcelona, Catalonia, Spain.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.
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http://dx.doi.org/10.1038/nature13992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266106PMC
November 2014

Co-detection and sequencing of genes and transcripts from the same single cells facilitated by a microfluidics platform.

Sci Rep 2014 Sep 26;4:6485. Epub 2014 Sep 26.

1] Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA [2] Yale Comprehensive Cancer Center, New Haven, CT 06520, USA.

Despite the recent advance of single-cell gene expression analyses, co-measurement of both genomic and transcriptional signatures at the single-cell level has not been realized. However such analysis is necessary in order to accurately delineate how genetic information is transcribed, expressed, and regulated to give rise to an enormously diverse range of cell phenotypes. Here we report on a microfluidics-facilitated approach that allows for controlled separation of cytoplasmic and nuclear contents of a single cell followed by on-chip amplification of genomic DNA and cytoplasmic mRNA. When coupled with off-chip polymerase chain reaction, gel electrophoresis and Sanger sequencing, a panel of genes and transcripts from the same single cell can be co-detected and sequenced. This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control. It also helps address clinical challenges such as diagnosing intra-tumor heterogeneity and dissecting complex cellular immune responses.
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http://dx.doi.org/10.1038/srep06485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175731PMC
September 2014

Long intergenic non-coding RNA HOTAIRM1 regulates cell cycle progression during myeloid maturation in NB4 human promyelocytic leukemia cells.

RNA Biol 2014 24;11(6):777-87. Epub 2014 Apr 24.

Department of Pediatrics; University of Massachusetts Medical School; Worcester, MA USA; Department of Cancer Biology; University of Massachusetts Medical School; Worcester, MA USA.

HOTAIRM1 is a long intergenic non-coding RNA encoded in the human HOXA gene cluster, with gene expression highly specific for maturing myeloid cells. Knockdown of HOTAIRM1 in the NB4 acute promyelocytic leukemia cell line retarded all-trans retinoid acid (ATRA)-induced granulocytic differentiation, resulting in a significantly larger population of immature and proliferating cells that maintained cell cycle progression from G1 to S phases. Correspondingly, HOTAIRM1 knockdown resulted in retained expression of many otherwise ATRA-suppressed cell cycle and DNA replication genes, and abated ATRA induction of cell surface leukocyte activation, defense response, and other maturation-related genes. Resistance to ATRA-induced cell cycle arrest at the G1/S phase transition in knockdown cells was accompanied by retained expression of ITGA4 (CD49d) and decreased induction of ITGAX (CD11c). The coupling of cell cycle progression with temporal dynamics in the expression patterns of these integrin genes suggests a regulated switch to control the transit from the proliferative phase to granulocytic maturation. Furthermore, ITGAX was among a small number of genes showing perturbation in transcript levels upon HOTAIRM1 knockdown even without ATRA treatment, suggesting a direct pathway of regulation. These results indicate that HOTAIRM1 provides a regulatory link in myeloid maturation by modulating integrin-controlled cell cycle progression at the gene expression level.
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http://dx.doi.org/10.4161/rna.28828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156508PMC
July 2015

Extended lifespan and reduced adiposity in mice lacking the FAT10 gene.

Proc Natl Acad Sci U S A 2014 Apr 24;111(14):5313-8. Epub 2014 Mar 24.

Departments of Genetics, Pediatrics, Pathology, and Immunobiology, Yale University School of Medicine, New Haven, CT 06520.

The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation. Although FAT10 is induced by inflammatory mediators and implicated in immunity, the physiological functions of FAT10 are poorly defined. We report the discovery that FAT10 regulates lifespan through pleiotropic actions on metabolism and inflammation. Median and overall lifespan are increased 20% in FAT10ko mice, coincident with elevated metabolic rate, preferential use of fat as fuel, and dramatically reduced adiposity. This phenotype is associated with metabolic reprogramming of skeletal muscle (i.e., increased AMP kinase activity, β-oxidation and -uncoupling, and decreased triglyceride content). Moreover, knockout mice have reduced circulating glucose and insulin levels and enhanced insulin sensitivity in metabolic tissues, consistent with elevated IL-10 in skeletal muscle and serum. These observations suggest novel roles of FAT10 in immune metabolic regulation that impact aging and chronic disease.
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http://dx.doi.org/10.1073/pnas.1323426111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986194PMC
April 2014

Nonstochastic reprogramming from a privileged somatic cell state.

Cell 2014 Feb 30;156(4):649-62. Epub 2014 Jan 30.

Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA; Department of Genetics, Yale University, New Haven, CT 06520, USA; Yale Comprehensive Cancer Center, Yale University, New Haven, CT 06520, USA.

Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:
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http://dx.doi.org/10.1016/j.cell.2014.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318260PMC
February 2014

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration.

Proc Natl Acad Sci U S A 2013 Jul 8;110(30):12361-6. Epub 2013 Jul 8.

Program in Neurodevelopment and Regeneration, Yale University School of Medicine, New Haven, CT 06520, USA.

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.
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http://dx.doi.org/10.1073/pnas.1309725110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725080PMC
July 2013

Robust measurement of telomere length in single cells.

Proc Natl Acad Sci U S A 2013 May 9;110(21):E1906-12. Epub 2013 May 9.

Department of Obstetrics and Gynecology, New York University Langone Medical Center, NY 10016, USA.

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.
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http://dx.doi.org/10.1073/pnas.1306639110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666709PMC
May 2013

Cell engineering with synthetic messenger RNA.

Methods Mol Biol 2013 ;969:3-28

Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.

mRNA has become an important alternative to DNA as a tool for cell reprogramming. To be expressed, exogenous DNA must be transmitted through the cell cytoplasm and placed into the nucleus. In contrast, exogenous mRNA simply has to be delivered into the cytoplasm. This can result in a highly uniform transfection of the whole population of cells, an advantage that has not been observed with DNA transfer. The use of mRNA, instead of DNA, in medical applications increases protocol safety by abolishing the risk of transgene insertion into host genomes. In this chapter, we review the aspects of mRNA structure and function that are important for its "transgenic" behavior, such as the composition of mRNA molecules and complexes with RNA binding proteins, localization of mRNA in cytoplasmic compartments, translation, and the duration of mRNA expression. In immunotherapy, mRNA is employed in reprogramming of antigen presenting cells (vaccination) and cytolytic lymphocytes. Other applications include generation of induced pluripotent stem (iPS) cells, and genome engineering with modularly assembled nucleases. The most investigated applications of mRNA technology are also reviewed here.
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http://dx.doi.org/10.1007/978-1-62703-260-5_1DOI Listing
June 2013

Two methods for full-length RNA sequencing for low quantities of cells and single cells.

Proc Natl Acad Sci U S A 2013 Jan 24;110(2):594-9. Epub 2012 Dec 24.

Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.

The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.
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http://dx.doi.org/10.1073/pnas.1217322109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545756PMC
January 2013

Extensive genetic variation in somatic human tissues.

Proc Natl Acad Sci U S A 2012 Oct 5;109(44):18018-23. Epub 2012 Oct 5.

Department of Genetics, Biomedical Informatics Training Program, Stanford University School of Medicine, Stanford, CA 94305, USA.

Genetic variation between individuals has been extensively investigated, but differences between tissues within individuals are far less understood. It is commonly assumed that all healthy cells that arise from the same zygote possess the same genomic content, with a few known exceptions in the immune system and germ line. However, a growing body of evidence shows that genomic variation exists between differentiated tissues. We investigated the scope of somatic genomic variation between tissues within humans. Analysis of copy number variation by high-resolution array-comparative genomic hybridization in diverse tissues from six unrelated subjects reveals a significant number of intraindividual genomic changes between tissues. Many (79%) of these events affect genes. Our results have important consequences for understanding normal genetic and phenotypic variation within individuals, and they have significant implications for both the etiology of genetic diseases such as cancer and for immortalized cell lines that might be used in research and therapeutics.
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http://dx.doi.org/10.1073/pnas.1213736109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497787PMC
October 2012

A highly integrated and complex PPARGC1A transcription factor binding network in HepG2 cells.

Genome Res 2012 Sep;22(9):1668-79

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.

PPARGC1A is a transcriptional coactivator that binds to and coactivates a variety of transcription factors (TFs) to regulate the expression of target genes. PPARGC1A plays a pivotal role in regulating energy metabolism and has been implicated in several human diseases, most notably type II diabetes. Previous studies have focused on the interplay between PPARGC1A and individual TFs, but little is known about how PPARGC1A combines with all of its partners across the genome to regulate transcriptional dynamics. In this study, we describe a core PPARGC1A transcriptional regulatory network operating in HepG2 cells treated with forskolin. We first mapped the genome-wide binding sites of PPARGC1A using chromatin-IP followed by high-throughput sequencing (ChIP-seq) and uncovered overrepresented DNA sequence motifs corresponding to known and novel PPARGC1A network partners. We then profiled six of these site-specific TF partners using ChIP-seq and examined their network connectivity and combinatorial binding patterns with PPARGC1A. Our analysis revealed extensive overlap of targets including a novel link between PPARGC1A and HSF1, a TF regulating the conserved heat shock response pathway that is misregulated in diabetes. Importantly, we found that different combinations of TFs bound to distinct functional sets of genes, thereby helping to reveal the combinatorial regulatory code for metabolic and other cellular processes. In addition, the different TFs often bound near the promoters and coding regions of each other's genes suggesting an intricate network of interdependent regulation. Overall, our study provides an important framework for understanding the systems-level control of metabolic gene expression in humans.
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http://dx.doi.org/10.1101/gr.127761.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431484PMC
September 2012

Architecture of the human regulatory network derived from ENCODE data.

Nature 2012 Sep;489(7414):91-100

Department of Genetics, Stanford University, 300 Pasteur Dr., M-344 Stanford, CA 94305, USA.

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
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http://dx.doi.org/10.1038/nature11245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154057PMC
September 2012
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