Christine M. Disteche - US grants

Our aim is to understand the molecular organization and the control of genetic expression of the mouse X and 7 chromosomes. We have isolated and characterized DNA fragments specific to these two mouse chromosomes. We are using these chromosome specific probes to study the mechanism of X inactivation and its spreading in an X;7 chromosomal translacation. We plan to characterize one of the X-specific fragments which is expressed in mouse liver, by nucleotide sequencing of the genomic clone and comparison with its corresponding cDNA. DNA methylation of X-chromosome specific fragments will be compared between male and female mouse DNA to determine if methylation changes occur with X chromosome inactivation. Chromatin structure changes associated with X inactivation will be studied by determining the nuclease sensitivity of specific X-chromosomal loci during inactivation. A restriction enzyme will be chosen for these experiments that distinguishes maternal and paternal X chromosomes on the basis of a DNA restriction site polymorphism (between mouse strains or mouse species). For these experiments, chromatin will be extracted from cloned cell lines that carry either the paternal or maternal X chromosome in an active state, or from extraembryonic membranes which demonstrate preferential paternal X inactivation. To construct a complete correlated genetic and molecular mouse X-chromosome linkage map, mouse X-chromosome fragments already isolated and additional fragments will be localized to the mouse X chromosome by in situ hybridization and the use of recombinant inbred strains of mice. To construct a molecular map of the developmentally important albino locus, a mouse chromosome 7-fragment that we have isolated will be used to isolate additional DNA fragments around that locus. The fragment isolated is located in the overlapping portion of two albino deletions, c3H and c6H. These deletions affect embryogenesis and modify the expresion of several liver enzymes. Chromosome 7-specific DNA fragments will also be used to study the effects of X inactivation on the autosomal portion of the X;7 chromosomal translocation.

X-chromosome inactivation results in the same dosage of gene expression between males and females of mammals. However, not all X-linked genes are subject to X inactivation as recently shown by the finding of several genes that escape X inactivation in human. It is not known whether genes that escape X inactivation in adult do so from the onset of inactivation in embryogenesis. Of special interest is the XIST gene that is expressed only from the inactive X chromosome and may play a role in the onset of X inactivation. In this proposal, we outline experiments to examine in vivo the X-inactivation status of genes in adult and embryo mice. We plan to isolate new mouse genes that escape X inactivation from a human x mouse hybrid cell line that retains only the inactive mouse X chromosome under selective pressure for the neomycin-resistance gene inserted in that chromosome. Mouse-specific transcripts corresponding to genes that escape X inactivation will be isolated from a cDNA library constructed from the hybrid cell line. We will then map, by in situ hybridization to mouse chromosomes, the new genes isolated from the hybrid cell line and existing X-linked genes known to escape X inactivation in human. In addition to locating the genes in mouse, this analysis may reveal the presence of Y homologs. We will determine the inactivation status of the genes in adult mice in vivo, by exploiting a mouse X-autosome translocation where the normal X chromosome is inactive in all cells and the genetic variation between mouse species to evaluate allelic expression at a given locus by a reverse transcriptase polymerase chain reaction assay. We will extend these studies to the mouse embryo by taking advantage of the preferential paternal X- chromosome inactivation in extraembryonic membranes. We will follow the expression of Xist in embryos to see whether the onset of its expression correlates with that X inactivation. The expression of a transgene previously shown to escape X inactivation in adult mouse will be followed during embryogenesis to determine whether there is reactivation of the transgene or whether it escapes inactivation from the onset. Finally, we will look for regions of early replication, in the otherwise late-replicating inactive mouse X chromosome, that may delineate chromosomal regions that contain genes that escape X inactivation.

X inactivation, established during early mammalian development, results in the silencing of all but one X chromosome in cells of female embryos. However, genes that escape inactivation are interspersed on the X chromosome. The unusual expression of some of these genes may explain the association of Turner syndrome with the lack of one X chromosome. Our previous studies have uncovered major differences between human and mouse in terms of location and X-inactivation status of genes. This proposal exploits mouse systems to follow the timing of X inactivation and escape during development, to determine the X-inactivation status of genes in relation to their location and to examine evolution of dosage compensation. Specifically, we plan (l) to construct transgenic mice that contain reporter sequences inserted in the Hprt gene that is subject to X inactivation and the Smcx gene that escapes. Expression of the reporter sequences will be followed during development. We will also continue studies of a transgene that escapes X inactivation. Second, we plan (2) to construct transgenic mice with reporter sequences inserted at different locations from the inactivation center to follow spreading of X inactivation. A parallel approach will be to assay for allele-specific expression of X-linked genes by in situ hybridization. Third, we plan (3) to define the extent of a rearrangement previously detected by mapping of the Clcn4 gene to the X chromosome in one mouse species but to an autosome in another. Expression studies of Clcn4 will be done to determine the consequences of dosage compensation. Finally we plan (4) to determine the X-inactivation status of mouse genes using an X-autosome translocation system developed previously. This study in conjunction with detailed mapping will show whether genes that escape X inactivation are located in specific domains. The experiments planned based on expression and mapping studies of genes that escape X inactivation in comparison to those subject to inactivation, should further elucidate the processes of X-inactivation spreading and maintenance and of evolution of the sex chromosomes.

X-chromosome inactivation results in the same dosage of gene expression between males and females of mammals. However, not all X-linked genes are subject to X inactivation as recently shown by the finding of several genes that escape X inactivation in human. It is not known whether genes that escape X inactivation in adult do so from the onset of inactivation in embryogenesis. Of special interest is the XIST gene that is expressed only from the inactive X chromosome and may play a role in the onset of X inactivation. In this proposal, we outline experiments to examine in vivo the X-inactivation status of genes in adult and embryo mice. We plan to isolate new mouse genes that escape X inactivation from a human x mouse hybrid cell line that retains only the inactive mouse X chromosome under selective pressure for the neomycin-resistance gene inserted in that chromosome. Mouse-specific transcripts corresponding to genes that escape X inactivation will be isolated from a cDNA library constructed from the hybrid cell line. We will then map, by in situ hybridization to mouse chromosomes, the new genes isolated from the hybrid cell line and existing X-linked genes known to escape X inactivation in human. In addition to locating the genes in mouse, this analysis may reveal the presence of Y homologs. We will determine the inactivation status of the genes in adult mice in vivo, by exploiting a mouse X-autosome translocation where the normal X chromosome is inactive in all cells and the genetic variation between mouse species to evaluate allelic expression at a given locus by a reverse transcriptase polymerase chain reaction assay. We will extend these studies to the mouse embryo by taking advantage of the preferential paternal X- chromosome inactivation in extraembryonic membranes. We will follow the expression of Xist in embryos to see whether the onset of its expression correlates with that X inactivation. The expression of a transgene previously shown to escape X inactivation in adult mouse will be followed during embryogenesis to determine whether there is reactivation of the transgene or whether it escapes inactivation from the onset. Finally, we will look for regions of early replication, in the otherwise late-replicating inactive mouse X chromosome, that may delineate chromosomal regions that contain genes that escape X inactivation.

X chromosome inactivation is a form of chromosome-wide gene regulation which results in the silencing of one of the two X chromosomes in female mammals, thereby restoring equal dosage of gene expression between males and females. There are significant differences in this inactivation process between mammal groups, which probably reflects its evolution. One of the most striking difference is that in metatherian mammals (marsupials) only the paternal X chromosome is inactivated, whereas eutherian (placental) mammals show random inactivation in most tissues. X inactivation is initiated at the XIST locus in eutherians, but no homologous locus has yet been demonstrated in marsupials. X inactivation is less stable in marsupials than in eutherians, and is incomplete and tissue-specific. The molecular basis, too may be different. Although differences in histone acetylation have been demonstrated between active and inactive X chromosomes in marsupials as well as eutherians, DNA methylation of the promoter region of inactive genes have not, perhaps accounting for the less stable inactivation in this mammalian group. These differences between the X inactivation processes in marsupials and eutherian mammals suggest that marsupials may have retained a less complex and more primitive form of X inactivation. The differences between the groups offer a unique opportunity to dissect out the layers of the process of gene silencing at a chromosome level. Our goal is to investigate these differences by a series of molecular approaches. We plan first to isolate the XIST locus from marsupials, based on our preliminary finding of a female-specific RNA signal using a probe enriched in marsupial X chromosome DNA. We will compare eutherian and metatherian XIST sequence and methylation status to examine possible XIST imprinting in marsupials. Secondly, we will investigate the stability of X inactivation of marsupial genes in comparison to mouse by measuring expression from the active and inactive X chromosomes in individual cells. Thirdly, we will insert a marsupial gene into the mouse X chromosome to determine whether the gene is susceptible or resistant in vivo to epigenetic changes which characterize eutherian genes.

Epigenetic modifications modulate gene expression in normal development and in diseases, such as cancer. X chromosome regulation is an example of coordinate epigenetic regulation of gene expression at the level of an entire chromosome. Our goal is to investigate the epigenetic regulation of X-linked genes in mammals. Genes on the X chromosome are dosage compensated by two processes: upregulation of the active X chromosome in both sexes, and X inactivation in females. A subset of genes escapes X inactivation and thus has higher expression in females. The importance of these genes in normal development is illustrated by the phenotypic anomalies including embryonic lethality in Turner syndrome, which is associated with a single X chromosome. We will investigate the molecular mechanisms that allow genes that escape X inactivation to be expressed within the context of silenced chromatin. In Aim 1 we will establish a comprehensive list of all genes that escape X inactivation in mouse and determine their chromatin structure. We will use RNAsequencing to distinguish expression from each allele of X-linked genes in cells with two X chromosomes, each from a different mouse species, so that alleles can be distinguished based on polymorphisms between the species. We will map the distribution of chromatin modifications uniquely present on either the active or inactive X chromosomes using chromatin immunoprecipitation (ChIP) together with array and sequencing analyses. Based on these data we will investigate the role of specific enzymes that establish or remove specific histone modifications in relation to X inactivation or escape during female ES cell differentiation when X inactivation takes place. Our previous studies show that escape genes are flanked by binding sites for the chromatin insulator element CTCF that may protect them from adjacent heterochromatin. In Aim 2 we will determine the functional role of this element in regulating the chromatin structure of the X chromosome by constructing a mouse with mutations at CTCF binding sites that flank an escape gene (Jarid1c). The effects of these mutations will be examined in female ES cells and in mice to determine whether the expression and epigenetic features of Jarid1c are altered in the absence of CTCF binding. This study will advance understanding of the role of chromatin structure in the control of gene expression in normal biology and diseases.

DESCRIPTION (provided by applicant): Mammalian females have two X chromosomes and males have only one. This fundamental difference has lead to the evolution of dosage compensation mechanisms. An important problem that males face is a deficiency in X-linked gene expression. A well-known mechanism of dosage compensation is X inactivation, which equalizes gene expression dosage between the sexes. We recently obtained evidence of another form of dosage compensation, which doubles the global transcriptional output from the active X chromosome in males and females to achieve a similar expression level to that of autosomes. A crucial role of X up- regulation, the focus of the present proposal, is to avoid deleterious effects of haplo-insufficiency. This is similar to the situation in Drosophila where the male X is up-regulated. We used microarray analyses in several mammalian species to demonstrate that X up-regulation is established in early embryos, and is maintained in somatic tissues. We also found higher expression of X-linked genes in brain. The goal of the proposed research is to determine the molecular mechanisms of mammalian X up- regulation. We speculate that X up-regulation may result either from epigenetic modifications of the active X and/or from evolutionary modifications of the DMA sequence to increase gene expression. Our Aims are (1) to determine when and where X up-regulation is established during development, (2) to study global epigenetic modifications potentially associated with X up-regulation, including histone modifications and candidate proteins known to be involved in Drosophila dosage compensation, (3) to investigate the mechanisms of high expression of X-linked genes in specific regions of the brain, and (4) to perform functional studies of X up-regulation using a mouse model in which we have previously shown a doubling of expression from the chloride channel gene, Clcr>4, when it is located on the X compared to an autosome. Our research has implications for understanding the developmental and evolutionary biology of the X chromosome and the role of X-linked gene expression in sex chromosome disorders and mental retardation. The proposed research is relevant to the role of the X chromosome in human diseases. Particularly significant are our findings of overall increased X expression in specific regions of the brain as the prevalence of X-linked mental retardation is well documented. Maintenance of the balance of gene expression is critical for normal development, as can be seen from the presence of multiple congenital abnormalities in individuals with chromosomal imbalance due to autosomal monosomy.

? DESCRIPTION (provided by applicant): Chromosome aneuploidy represents a serious burden on human health. Congenital aneuploidy often results in birth defects while acquired aneuploidy is associated with diseases such as cancer. Additional or missing chromosomes can cause widespread gene expression dysregulation and epigenetic alterations throughout the genome. Our goal is to evaluate the molecular consequences of an abnormal number of X or Y chromosomes. We will focus on two common disorders, Klinefelter (47,XXY) and Turner (45,X) syndromes. In XXY men a single X chromosome remains active while the other is silenced by X inactivation. Despite this, these men have abnormal phenotypes including cognitive defects, in part due to over-expression of genes that escape X inactivation. Conversely, women with a single X chromosome have phenotypic anomalies including cognitive defects and heart abnormalities due to haploinsufficiency for these escape genes. Y-linked genes protect normal XY males from deleterious effects of X monosomy. Thus, both X-linked and Y-linked genes are critically implicated in sex chromosome aneuploidy. To rigorously determine the effects of sex chromosome aneuploidy on global gene expression and on the epigenetic environment we will derive isogenic induced pluripotent stem cell (iPSC) lines (1) by removal of the X or Y chromosome from parental iPSC lines with an XXY karyotype, and (2) by derivation of clonal iPSC lines from mosaic cultures with an aneuploid line. Comparisons between pairs of isogenic lines will circumvent the highly variable genetic background in the human population. To access cell types relevant to phenotypes observed in sex chromosome aneuploidy we will differentiate the iPSCs into neuronal cells and cardiomyocytes. We will then manipulate levels of two candidate X/Y gene pairs, KDM5C/KDM5D and KDM6A/UTY, in the new isogenic lines to delete or add a copy of the X or Y paralogs and determine whether we can recapitulate the effects of X or Y aneuploidy. These candidate genes are of special interest because they represent dosage-sensitive master regulators important for promoter and enhancer regulation and for neural cell and embryo development. We will extend our study in vivo by performing parallel gene expression and epigenetic analyses in brain and heart from mouse models of X aneuploidy including XXY and XO mice in which we will manipulate levels of Kdm5c and Kdm6a. By eliminating the noise of natural genomic variability between individuals we will more precisely assess perturbations of gene expression, epigenetic environment, and phenotypes in aneuploid cells and tissues. This study will contribute to a better understanding not only of aneuploidy but also of the role of the sex chromosomes in sex differences.

ABSTRACT ? PROJECT 3: UW-CNOF BIOLOGICAL VALIDATION DEVELOPMENT The complexity of hierarchical interactions within the nucleus demands that easy-to-use and accurate methods for mapping and modeling both close-range and long-range interactions be developed. In this project, the experimental and computational methods developed in Projects 1 and 2 will be put to the test to evaluate their ability to detect interactions at high resolution, as well as for the prediction of the sequence determinants of specific aspects of genome architecture. In Aim 1, we will apply newly developed methods to well-defined mouse and human biological systems in which the 4D structure of specific genomic regions and chromosomes can be anticipated. Our validation strategy will employ systems in which alleles can be identified to facilitate studies of diploid cells, i.e. tissues and cell lines from a mouse interspecific cross. Interactions between loci will be tested at enhancer/promoter regions, while interactions at topologically associated domains (TADs) will be tested by comparing the active and inactive X chromosomes in female cells. This functional validation approach will be complemented by high resolution DNA-FISH analyses to verify specific interactions. In Aim 2, to validate our approaches for generating a 4D view of dynamic changes in nuclear structure, we will measure interactions in single cells during the cell cycle and during mouse myoblast and embryonic stem (ES) cell differentiation. By focusing on relatively well understood aspects of these systems, we will achieve validation by linking dynamic changes in the nucleome to other layers of regulation. Analysis of mouse ESC differentiation will exploit the wealth of knowledge that surrounds X inactivation, a process central to nuclear remodeling in mammals, while skeletal myoblast differentiation is well characterized with respect to its transcriptional regulation. In Aim 3, we will validate our ability to predict the sequence determinants of genome architecture. Specifically, we will perform controlled manipulations of defined genomic regions, either by allele- specific heterozygous CRISPR/Cas9 targeting to generate cells with engineered deletions at promoter/enhancer interacting regions and at regions between TADs, or by Xist-mediated silencing of full autosomes. The biological validation work performed in all aims of this project will facilitate the progressive optimization of both bulk and single cell DNase Hi-C protocols (Project 1) and new approaches to modeling the 4D nucleome (Project 2), while also paving the way for biological model development and data generation (Project 4).  

Project Summary/Abstract Mammalian X chromosome inactivation is a complex epigenetic mechanism that ensures a near-balanced level of gene expression between males (XY) and females (XX). This chromosome-wide regulation makes the X an ideal model for studying heterochromatin regulation and structure. X inactivation affects a sizable portion of the mammalian genome and is crucial for normal development and normal lifespan. Indeed, X aberrations have been linked to birth defects and to age-related diseases. My research group has made several contributions to understanding the structure and regulation of the X by developing useful mouse models and studying human conditions with sex chromosome anomalies, while embracing novel technologies to advance the field. Here, I consider challenges related to the physical structure and location of the inactive X within the nucleus, with a focus on long non-coding RNAs (lncRNAs). By scaffolding proteins into flexible complexes lncRNAs have emerged as adaptable elements that organize chromatin and regulate gene expression, but many questions remain unanswered about their mechanisms of action. I will examine the cis- and trans- roles of conserved X-linked lncRNA loci and their transcripts in regulating the structure and location of the X chromosome within the nucleus. We found that Dxz4 shapes the unique bipartite structure of the inactive X in cis and Firre apparently controls the inactive X location and epigenetic features in trans. Here, I propose to introduce new approaches to manipulate these and other lncRNA loci and to relocate specific X chromosome regions within the nucleus. This will address the fundamental role of physical location in relation to heterochromatin formation and maintenance. Escape from X inactivation in females and the presence of a Y in males lead to sexual dimorphisms in cell physiology. Cell-type diversity within tissues is extensive but poorly understood; yet, novel exciting new technologies can measure gene expression and chromatin features in tens of thousands of single cells in vivo. To address the role of sex-linked genes at the cellular and organismal level I will establish an in vivo atlas of allelic features in single cells of male and female mouse tissues. Single-cell technology will also be applied to mouse and human tissues from fetuses and adults with sex chromosome aneuploidy to determine the impact on cell types. An interesting possibility is that an adaptive developmental epigenetic response to aneuploidy explains phenotypic variability. To explore this, I will compare epigenetic features in tissues to those obtained in isogenic cell lines with induced X chromosome loss. My goal is to understand the role of the sex chromosomes in sex differences and sex chromosome disorders in vivo.

Project Summary Women have a higher lifetime risk of developing Alzheimer's disease (AD) than men. This increased risk is not fully explained by differences in longevity, hormones or brain structure. Women who carry at least one copy of the APOE?4 allele, the strongest genetic risk factor for late onset AD (LOAD), have accelerated neuropathology. However, some studies suggest a faster decline in men, suggesting that sex bias may differ depending on the stage of the disease. Here, we will investigate how the sex chromosome complement and sex-linked genes influences sex differences in onset and progression of LOAD. Genome-wide association studies have identified genetic and epigenetic risk factors for LOAD, but the sex-chromosomes are often excluded in these studies meaning there is a lack of data on sex-linked genes. Males have unique Y-linked genes and females have higher expression of genes that escape X inactivation. Interestingly, many of the escape genes are related to immune function and neuroinflammation is a hallmark of AD, suggesting that these genes may directly contribute to disease progression. To address the impact of sex-linked genes combined with APOE?4 alleles on neuroinflammation in LOAD we will use unique cellular models and AD tissue for leveraging integrated omics and functional studies. We will evaluate the functional roles of sex chromosomes and sex-linked genes in brain cell types using human induced pluripotent stem cell (hiPSC) models. We have derived isogenic pairs of hiPSCs with a different number of sex chromosomes on the same genetic background (XXY/XY or XXX/X). These new hiPSC lines minimize variability between individuals, as well as environmental or hormonal confounders. We will generate isogenic pairs of these lines with ?3/3 or ?3/4 alleles by gene editing. After differentiation of hiPSC into neurons, microglia, and brain organoids we will employ a combination of `omic' analyses and functional assays focusing on neuroinflammation and neurodegeneration. This approach will identify sex-linked candidate genes, which will be tested for dosage effects by knockdown and overexpression. These in vitro studies will be validated in human tissue collected by the Precision Neuropathology Core from our Alzheimer's Disease Research Center brain bank. Using pathologically characterized AD brains we will employ myeloid-specific single-nucleus RNA sequencing to determine the effects of sex and APOE?4 genotypes on microglial subtypes and neuroimmune gene expression. Our new team combines expertise in hiPSC modeling, sex-linked genes, neuroinflammation, `omic analyses and neuropathology. This integrative study will help understand sex-specific genetic factors and how those factors interact with APOE?4 risk to modulate cellular dysfunction and pathology, thus providing novel insights into how to tailor a more effective treatment for AD.

Project Summary / Abstract A major shortcoming of most efforts to understand the 4D nucleome is that they have mainly focused on in vitro cell lines, rather than on dynamic, in vivo systems. Arguably, the most important in vivo system, which also happens to be the most dynamic, is development itself, wherein the nucleome both shapes and is shaped by the initial emergence of the myriad mammalian cell types. While these in vivo dynamics are presently poorly documented and understood, recently emerged technologies offer a path forward. Here we propose to establish the University of Washington 4-Dimensional Genomic Nuclear Organization of Mammalian Embryogenesis Center (UW 4D GENOME Center), which will address these massive gaps in our understanding by generating systematic datasets on nuclear morphology and associated molecular measurements in mammalian tissues and cell types. These datasets will be generated in the context of the leading model organism for mammalian development, the mouse. Our approach focuses on following nuclear structure, chromatin and gene expression changes at a ?whole organism? scale, using a combination of scalable single cell profiling and ?visual cell sorting? (VCS) methods, all well-established and mostly developed in our own labs. Our goal is to generate a high- resolution 4DN atlas of mouse embryogenesis for the community. The different types of data will be integrated, including cross-species imputation to integrate with human data, as well as models and navigable maps applied to pathways relevant to mammalian development.