Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons.

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.

Myst2/Kat7 histone acetyltransferase interaction proteomics reveals tumour-suppressor Niam as a novel binding partner in embryonic stem cells.

MYST histone acetyltransferases have crucial functions in transcription, replication and DNA repair and are hence implicated in development and cancer. Here we characterise Myst2/Kat7/Hbo1 protein interactions in mouse embryonic stem cells by affinity purification coupled to mass spectrometry. This study confirms that in embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2-associated protein, the tumour suppressor protein Niam (Nuclear Interactor of ARF and Mdm2). Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and Niam in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism early development and survival as well as cancer. Data are available via ProteomeXchange with identifier PXD005987.

Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells.

Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.

Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and ß-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.

ZFP57 and the Targeted Maintenance of Postfertilization Genomic Imprints.

Epigenetic modifications play an important role in modulating genome function. In mammals, inappropriate epigenetic states can cause embryonic lethality and various acquired and inherited diseases; hence, it is important to understand how such states are formed and maintained in particular genomic contexts. Genomic imprinting is a process in which epigenetic states provide a sustained memory of parental origin and cause gene expression/repression from only one of the two parental chromosomes. Genomic imprinting is therefore a valuable model to decipher the principles and processes associated with the targeting and maintenance of epigenetic states in general. Krüppel-associated box zinc finger proteins (KRAB-ZFPs) are proteins that have the potential to mediate this. ZFP57, one of the best characterized proteins in this family, has been shown to target and maintain epigenetic states at imprinting control regions after fertilization. Its role in imprinting through the use of ZFP57 mutants in mouse and the wider implications of KRAB-ZFPs for the targeted maintenance of epigenetic states are discussed here.

Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9.

Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of ß-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.

Efficient knockin mouse generation by ssDNA oligonucleotides and zinc-finger nuclease assisted homologous recombination in zygotes.

The generation of specific mutant animal models is critical for functional analysis of human genes. The conventional gene targeting approach in embryonic stem cells (ESCs) by homologous recombination is however laborious, slow, expensive, and limited to species with functional ESCs. It is therefore a long-sought goal to develop an efficient and simple alternative gene targeting strategy. Here we demonstrate that, by combining an efficient ZFN pair and ssODN, a restriction site and a loxP site were successfully introduced into a specific genomic locus. A targeting efficiency up to 22.22% was achieved by coinciding the insertion site and the ZFN cleavage site isogenic and keeping the length of the homology arms equal and isogenic to the endogenous target locus. Furthermore, we determine that ZFN and ssODN-assisted HR is ssODN homology arm length dependent. We further show that mutant alleles generated by ZFN and ssODN-assisted HR can be transmitted through the germline successfully. This study establishes an efficient gene targeting strategy by ZFN and ssODN-assisted HR in mouse zygotes, and provides a potential avenue for genome engineering in animal species without functional ES cell lines.

Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.

Bi-allelic gene targeting in mouse embryonic stem cells.

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.

Gene body methylation of the dimethylarginine dimethylamino-hydrolase 2 (Ddah2) gene is an epigenetic biomarker for neural stem cell differentiation.

DNA methylation is an important epigenetic mark that is involved in the regulation of many cellular processes such as gene expression, genomic imprinting and silencing of repetitive elements. Because of their ability to cause and capture phenotypic plasticity, epigenetic marks such as DNA methylation represent potential biomarkers to distinguish between different types of tissues and stages of differentiation. Here, we have identified differential DNA methylation in the gene body of the nitric oxide inhibitor Ddah2 that discriminates embryonic stem cells from neural stem cells and is positively correlated with differential gene expression.

ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a.

ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectoderm-derived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.