技术资料

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders,some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here,we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program,TALENSeek,(2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol,and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify,construct,and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity. View Publication

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently,the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique,we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen,we further describe strategies for confirming the screening phenotype,as well as genetic perturbation,through analysis of indel rate and transcriptional activation. Beginning with library design,a genome-scale screen can be completed in 9-15 weeks,followed by 4-5 weeks of validation. View Publication

BACKGROUND: Fragile X syndrome (FXS) is a common form of inherited intellectual disability caused by an expansion of CGG repeats located in the 5' untranslated region (UTR) of the FMR1 gene,which leads to hypermethylation and silencing of this locus. Although a dramatic increase in DNA methylation of the FMR1 full mutation allele is well documented,the extent to which these changes affect DNA methylation throughout the rest of the genome has gone unexplored. METHODS: Here we examined genome-wide methylation in both peripheral blood (N = 62) and induced pluripotent stem cells (iPSCs; N = 10) from FXS individuals and controls. RESULTS: We not only found the expected significant DNA methylation differences in the FMR1 promoter and 5' UTR,we also saw that these changes inverse in the FMR1 gene body. Importantly,we found no other differentially methylated loci throughout the remainder of the genome,indicating the aberrant methylation of FMR1 in FXS is locus-specific. CONCLUSIONS: This study provides a comprehensive methylation profile of FXS and helps refine our understanding of the mechanisms behind FMR1 silencing. View Publication

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background,yet the landscapes of in vivo tissues remain largely uncharted. Here,we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages,lineages,and cellular environments. They also reveal global features of the epigenome,related to nuclear architecture,that also vary across cellular phenotypes. Specifically,developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture,and discuss their implications for lineage fidelity,cellular senescence,and reprogramming. ?? 2013 Elsevier Inc. View Publication

How tumor suppressor p53 selectively responds to specific signals,especially in normal cells,is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation,induced by retinoic acid,versus DNA damage,caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and,in pluripotent hESCs,are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases,UTX and JMJD3,to chromatin. In contrast,genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation,a process highly distinct from stress-induced p53 response in hESCs. View Publication

textlessptextgreaterStem cell phenotypes are reflected by post-translational histone modifications,and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs),bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remains to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2,mitotic,and G1 phases of the cell cycle,we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle–dependent fashion. Interestingly,bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore,the histone-modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle–independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in maintenance of pluripotency.textless/ptextgreater View Publication

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved,we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel,mouse embryonic fibroblast feeders,and Matrigel replated on feeders. At the outset,we profiled and quantified their differentiation efficiency,transcriptome,transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome,thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1,FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs,we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly,the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus,we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. View Publication

BACKGROUND: RNAs can be physically classified into poly(A)+ or poly(A)- transcripts according to the presence or absence of a poly(A) tail at their 3' ends. Current deep sequencing approaches largely depend on the enrichment of transcripts with a poly(A) tail,and therefore offer little insight into the nature and expression of transcripts that lack poly(A) tails. RESULTS: We have used deep sequencing to explore the repertoire of both poly(A)+ and poly(A)- RNAs from HeLa cells and H9 human embryonic stem cells (hESCs). Using stringent criteria,we found that while the majority of transcripts are poly(A)+,a significant portion of transcripts are either poly(A)- or bimorphic,being found in both the poly(A)+ and poly(A)- populations. Further analyses revealed that many mRNAs may not contain classical long poly(A) tails and such messages are overrepresented in specific functional categories. In addition,we surprisingly found that a few excised introns accumulate in cells and thus constitute a new class of non-polyadenylated long non-coding RNAs. Finally,we have identified a specific subset of poly(A)- histone mRNAs,including two histone H1 variants,that are expressed in undifferentiated hESCs and are rapidly diminished upon differentiation; further,these same histone genes are induced upon reprogramming of fibroblasts to induced pluripotent stem cells. CONCLUSIONS: We offer a rich source of data that allows a deeper exploration of the poly(A)- landscape of the eukaryotic transcriptome. The approach we present here also applies to the analysis of the poly(A)- transcriptomes of other organisms. View Publication

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis,while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX,indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus,increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. View Publication

Although it has been observed that aggregate size affects cardiac development,an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development,and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100,1000,or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells,with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate,in a defined,serum-free cardiac induction system,that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration,a parameter that can be directly modulated by controlling hESC aggregate size. View Publication

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however,the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF),an orphan nuclear receptor,in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes,GCNF down-regulated 36% of the genes,and up-regulated 64% in undifferentiated hES cells. In addition,GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process. View Publication

Pluripotency is a unique state in which cells can self-renew indefinitely but also retain the ability to differentiate into other cell types upon receipt of extracellular cues. Although it is clear that stem cells have a distinct transcriptional program,little is known about how alterations in post-transcriptional mechanisms,such as mRNA turnover,contribute to the achievement and maintenance of pluripotency. Here we have assessed the rates of decay for the majority of mRNAs expressed in induced pluripotent stem (iPS) cells and the fully differentiated human foreskin fibroblasts (HFFs) they were derived from. Comparison of decay rates in the two cell types led to the discovery of three independent regulatory mechanisms that allow coordinated turnover of specific groups of mRNAs. One mechanism results in increased stability of many histone mRNAs in iPS cells. A second pathway stabilizes a large set of zinc finger protein mRNAs,potentially through reduced levels of miRNAs that target them. Finally,a group of transcripts bearing 3' UTR C-rich sequence elements,many of which encode transcription factors,are significantly less stable in iPS cells. Intriguingly,two poly(C)-binding proteins that recognize this type of element are reciprocally expressed in iPS and HFF cells. Overall,our results highlight the importance of post-transcriptional control in pluripotent cells and identify miRNAs and RNA-binding proteins whose activity may coordinately control expression of a wide range of genes in iPS cells. View Publication