[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2014 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms by which Xist RNA associates with the X chromosome to mediate alterations in chromatin structure remain mysterious. Recent genome-wide Xist RNA distribution studies suggest that this long noncoding RNA uses 3-dimensional chromosome contacts to move to its sites of action.
Preview · Article · Jan 2014 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells are pluripotent cells that can self-renew indefinitely or be induced to differentiate into multiple cell lineages, and thus have the potential to be used in regenerative medicine. Pluripotency transcription factors (TFs), such as Oct4, Sox2, and Nanog, function in a regulatory circuit that silences the expression of key TFs required for differentiation and activates the expression of genes important for maintenance of pluripotency. In addition, proteins that remodel chromatin structure also play important roles in determining the ES cell-specific gene expression pattern. Here we review recent studies demonstrating the roles of enzymes that carry out one facet of chromatin regulation, nucleosome remodeling, in control of ES cell self-renewal and differentiation.
Preview · Article · Oct 2010 · Current opinion in genetics & development
[Show abstract][Hide abstract] ABSTRACT: One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · May 2010 · Molecular & Cellular Proteomics
[Show abstract][Hide abstract] ABSTRACT: In an RNA interference screen interrogating regulators of mouse embryonic stem (ES) cell chromatin structure, we previously identified 62 genes required for ES cell viability. Among these 62 genes were Smc2 and -4, which are core components of the two mammalian condensin complexes. In this study, we show that for Smc2 and -4, as well as an additional 49 of the 62 genes, knockdown (KD) in somatic cells had minimal effects on proliferation or viability. Upon KD, Smc2 and -4 exhibited two phenotypes that were unique to ES cells and unique among the ES cell-lethal targets: metaphase arrest and greatly enlarged interphase nuclei. Nuclear enlargement in condensin KD ES cells was caused by a defect in chromatin compaction rather than changes in DNA content. The altered compaction coincided with alterations in the abundance of several epigenetic modifications. These data reveal a unique role for condensin complexes in interphase chromatin compaction in ES cells.
Full-text · Article · Feb 2010 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Polycomb Repressive Complex 2 (PRC2) modifies chromatin to silence many embryonic patterning genes, restricting their expression to the appropriate cell populations. Two reports in Cell by Peng et al. (2009) and Shen et al. (2009) identify Jarid2/Jumonji, a new component of PRC2, which inhibits PRC2 enzymatic activity to fine-tune silencing.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Preview · Article · May 2009 · BMC Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Histone modifications affect chromatin dynamics on several levels by serving as binding sites for regulatory proteins. In many cell types, including embryonic stem cells (ESCs), a subset of genes is marked with histone modifications thought to be both activating and repressing: H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3), respectively. As a result, genes bearing this "bivalent" mark are transcribed at low levels, but are primed for activation, should the cell receive the appropriate cues during differentiation. Recently, we found that the Tip60-p400 acetyltransferase and histone exchange complex is necessary to maintain normal self-renewal in mouse ESCs. While Tip60-p400 has histone acetyltransferase activity, which is generally associated with transcriptional activation, it acts predominantly as a repressor of genes expressed during differentiation. Surprisingly, in ESCs Tip60-p400 localizes to the promoters of genes marked by H3K4me3, which include both highly expressed genes and "bivalent" genes expressed at low levels. Tip60-p400 acetylates histones at these targets, including the promoters for developmental regulators it helps to silence in ESCs. This suggests that the effect of chromatin modifications on transcription is not always simply positive or negative. Rather, we propose that the impact of specific modifications at each promoter is determined by the chromatin context in which they are found.
[Show abstract][Hide abstract] ABSTRACT: X-chromosome inactivation occurs randomly for one of the two X chromosomes in female cells during development. Inactivation occurs when RNA transcribed from the Xist gene on the X chromosome from which it is expressed spreads to coat the whole X chromosome. In the first issue of Epigenetics and Chromatin, Nesterova and colleagues investigate the role of the RNA interference pathway enzyme Dicer in DNA methylation of the Xist promoter.
[Show abstract][Hide abstract] ABSTRACT: Organized by Jessica Tyler (University of Colorado School of Medicine), Michael Carey (UCLA), and Victor Corces (Emory University), the FASEB conference brought together molecular, cellular, and developmental biologists with a shared interest in transcriptional regulation. Reflecting the different areas of expertise among the conference participants, the research presented utilized an impressive arsenal of genetic, biochemical, biophysical, and computational methods. Only by combining these techniques can one begin to understand and appreciate the coordination and complexity required to appropriately regulate transcription. Indeed, it takes an extensive array of factors to control expression of any given gene. Moreover, it is becoming clear that, despite obvious shared features of active and of inactive genes, many genes likely possess their own distinct regulatory signature. That is, their expression relies upon distinct sets of coregulators and/or subtle differences in chromatin state. Major themes discussed at the meeting are outlined in the sections below.
[Show abstract][Hide abstract] ABSTRACT: 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.