Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.

Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program.

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.

Activators and repressors: A balancing act for X-inactivation.

In early female embryos X-chromosome inactivation occurs concomitant with up regulation of the non-coding RNA, Xist, on the future inactive X-chromosome. Up regulation of Xist and coating of the future inactive X is sufficient to induce silencing. Therefore unlocking the mechanisms of X-chromosome inactivation requires thorough understanding of the transcriptional regulators, both activators and repressors, which control Xist. Mouse pluripotent embryonic stem cells, which have two active X chromosomes, provide a tractable ex vivo model system for studying X-chromosome inactivation, since this process is triggered by differentiation signals in these cultured cells. Yet there are significant discrepancies found between ex vivo analyses in mouse embryonic stem cells and in vivo studies of early embryos. In this review we elaborate on potential models of how Xist is up regulated on a single X chromosome in female cells and how ex vivo and in vivo analyses enlighten our understanding of the activators and repressors that control this non-coding RNA gene.

The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development.

Imprinted genes play important roles in placenta development and function. Parthenogenetic embryos, deficient in paternally expressed imprinted genes, lack extra-embryonic tissues of the trophoblast lineage. Parthenogenetic trophoblast stem cells (TSCs) are extremely difficult to derive, suggesting that an imprinted gene(s) is necessary for TSC establishment or maintenance. In a candidate study, we were able to narrow the list to one known paternally expressed gene, Sfmbt2. We show that mouse embryos inheriting a paternal Sfmbt2 gene trap null allele have severely reduced placentae and die before E12.5 due to reduction of all trophoblast cell types. We infected early embryos with lentivirus vectors expressing anti-Sfmbt2 shRNAs and found that TSC derivation was significantly reduced. Together, these observations support the hypothesis that loss of SFMBT2 results in defects in maintenance of trophoblast cell types necessary for development of the extra-embryonic tissues, the placenta in particular.

Quantitative genetic-interaction mapping in mammalian cells.

Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ∼11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape.

Transcriptional regulation: it takes a village.

A FASEB conference on "Transcriptional Regulation during Cell Growth, Differentiation and Development" met in June, 2008, just outside of Aspen in Snowmass Village, Colorado. The meeting covered a broad range of topics, including the structure of transcription factors (TFs), Preinitiation Complex (PIC) assembly, RNA polymerase II (Pol II) pausing, genome-wide patterns of histone modifications, and the role of TFs in development.

Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.

Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells.

The monosaccharide addition of an N-acetylglucosamine to serine and threonine residues of nuclear and cytosolic proteins (O-GlcNAc) is a posttranslational modification emerging as a general regulator of many cellular processes, including signal transduction, cell division, and transcription. The sole mouse O-GlcNAc transferase (OGT) is essential for embryonic development. To understand the role of OGT in mouse development better, we mapped sites of O-GlcNAcylation of nuclear proteins in mouse embryonic stem cells (ESCs). Here, we unambiguously identify over 60 nuclear proteins as O-GlcNAcylated, several of which are crucial for mouse ESC cell maintenance. Furthermore, we extend the connection between OGT and Polycomb group genes from flies to mammals, showing Polycomb repressive complex 2 is necessary to maintain normal levels of OGT and for the correct cellular distribution of O-GlcNAc. Together, these results provide insight into how OGT may regulate transcription in early development, possibly by modifying proteins important to maintain the ESC transcriptional repertoire.

The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation.

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage. Here, we demonstrate that the inactive X-chromosome in Eed(-/-) trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed(-/-) embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed(-/-) trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

X Chromosome Factor Kdm6a Enhances Cognition Independent of Its Demethylase Function in the Aging XY Male Brain.

Males exhibit shorter life span and more cognitive deficits, in the absence of dementia, in aging human populations. In mammals, the X chromosome is enriched for neural genes and is a major source of biologic sex difference, in part, because males show decreased expression of select X factors (XY). While each sex (XX and XY) harbors one active X due to X chromosome inactivation in females, some genes, such as Kdm6a, transcriptionally escape silencing in females-resulting in lower transcript levels in males. Kdm6a is a known histone demethylase (H3K27me2/3) with multiple functional domains that is linked with synaptic plasticity and cognition. Whether elevating Kdm6a could benefit the aged male brain and whether this requires its demethylase function remains unknown. We used lentiviral-mediated overexpression of the X factor in the hippocampus of aging male mice and tested their cognition and behavior in the Morris water-maze. We found that acutely increasing Kdm6a-in a form without demethylase function-selectively improved learning and memory, in the aging XY brain, without altering total activity or anxiety-like measures. Further understanding the demethylase-independent downstream mechanisms of Kdm6a may lead to novel therapies for treating age-induced cognitive deficits in both sexes.

Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed.

A number of mammalian genes are expressed from only one of the two homologous chromosomes, selected at random in each cell. These include genes subject to X-inactivation, olfactory receptor (OR) genes, and several classes of immune system genes. The means by which monoallelic expression is established are only beginning to be understood. Using a cytological assay, we show that the two homologous alleles of autosomal random monoallelic loci differ from each other in embryonic stem (ES) cells, before establishment of monoallelic expression. The Polycomb Group gene Eed is required to establish this distinctive behavior. In addition, we found that when Eed mutant ES cells are differentiated, they fail to establish asynchronous replication timing at OR loci. These results suggest a common mechanism for random monoallelic expression on autosomes and the X chromosome, and implicate Eed in establishing differences between homologous OR loci before and after differentiation.

High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: determination of abundant histone isoforms and post-translational modifications.

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications.

X-inactivation: it takes two to count.

Mammals balance X-linked gene dosage by silencing one X chromosome in female cells, a process that begins with counting the number of X chromosomes. Recent reports suggest homologous X chromosome pairing may be a prerequisite for silencing, providing a basis for counting by ensuring that silencing only occurs in cells with two X chromosomes.

Epigenetic gene regulation by noncoding RNAs.

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.

A second X chromosome contributes to resilience in a mouse model of Alzheimer's disease.

A major sex difference in Alzheimer's disease (AD) is that men with the disease die earlier than do women. In aging and preclinical AD, men also show more cognitive deficits. Here, we show that the X chromosome affects AD-related vulnerability in mice expressing the human amyloid precursor protein (hAPP), a model of AD. XY-hAPP mice genetically modified to develop testicles or ovaries showed worse mortality and deficits than did XX-hAPP mice with either gonad, indicating a sex chromosome effect. To dissect whether the absence of a second X chromosome or the presence of a Y chromosome conferred a disadvantage on male mice, we varied sex chromosome dosage. With or without a Y chromosome, hAPP mice with one X chromosome showed worse mortality and deficits than did those with two X chromosomes. Thus, adding a second X chromosome conferred resilience to XY males and XO females. In addition, the Y chromosome, its sex-determining region Y gene (Sry), or testicular development modified mortality in hAPP mice with one X chromosome such that XY males with testicles survived longer than did XY or XO females with ovaries. Furthermore, a second X chromosome conferred resilience potentially through the candidate gene Kdm6a, which does not undergo X-linked inactivation. In humans, genetic variation in KDM6A was linked to higher brain expression and associated with less cognitive decline in aging and preclinical AD, suggesting its relevance to human brain health. Our study suggests a potential role for sex chromosomes in modulating disease vulnerability related to AD.

The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels.

BACKGROUND: The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male Drosophila cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the maleless (mle) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted. To understand the molecular basis of Zn72D function, we identified proteins that interact with Zn72D. RESULTS: Among several proteins that associate with Zn72D, we found the DEAD box helicase Belle (Bel). Simultaneous knockdown of Zn72D and bel restored MSL complex localization to the X chromosome and dosage compensation. MLE protein was restored to 70% of wild-type levels, although the level of productively spliced mle transcript was still four-fold lower than in wild-type cells. The increase in production of MLE protein relative to the amount of correctly spliced mle mRNA could not be attributed to an alteration in MLE stability. CONCLUSION: These data indicate that Zn72D and Bel work together to control mle splicing and protein levels. Thus Zn72D and Bel may be factors that coordinate splicing and translational regulation.

A thermodynamic switch for chromosome colocalization.

A general model for the early recognition and colocalization of homologous DNA sequences is proposed. We show, on thermodynamic grounds, how the distance between two homologous DNA sequences is spontaneously regulated by the concentration and affinity of diffusible mediators binding them, which act as a switch between two phases corresponding to independence or colocalization of pairing regions.

Recognition and modification of seX chromosomes.

Flies, worms and mammals employ dosage compensation complexes that alter chromatin or chromosome structure to equalize X-linked gene expression between the sexes. Recent work has improved our understanding of how dosage compensation complexes achieve X chromosome-wide association and has provided significant insight into the epigenetic modifications directed by these complexes to modulate gene expression. In flies, the prevailing view that dosage compensation complexes assemble on the X chromosome at approximately 35 chromatin-entry sites and then spread in cis to cover the chromosome has been re-evaluated in light of the evidence that these chromatin-entry sites are not required for localization of the complex. By contrast, identification of discrete recruitment elements indicates that nucleation at and spread from a limited number of sites directs dosage compensation complex localization on the worm X-chromosome. Studies in flies and mammals have extended our understanding of how ribonucleoprotein complexes are used to modify X chromatin, for either activation or repression of transcription. Finally, evidence from mammals suggests that the chromatin modifications that mediate dosage compensation are very dynamic, because they are established, reversed and re-established early in development.

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome.

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.