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.

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.

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1's function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

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.

SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells.

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor.

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.

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.

X-inactivation: Xist RNA uses chromosome contacts to coat the X.

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.

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.

Derivation conditions impact X-inactivation status in female human induced pluripotent stem cells.

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.

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.

Control of embryonic stem cell identity by nucleosome remodeling enzymes.

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.

The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation.

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.

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.

Condensin complexes regulate mitotic progression and interphase chromatin structure in embryonic stem cells.

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.

Fine-tuning silencing.

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.

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.