Guoping Fan, Ph.D. - US grants

DESCRIPTION (provided by applicant): DNA methylation is a major epigenetic factor involved in gene regulation, genomic imprinting, and X-chromosome inactivation. Aberrant DNA methylation has been associated with several human mental retardation disorders including Rett, ICF, Fragile-X, and ATRX syndromes. However, little is known about how DNA methylation changes perturb neural function and lead to neurological disorders. The long-term objective of our research is to elucidate the role of DNA methylation in neural development and function. Using the conditional gene knockout approach, we have recently constructed a strain of mutant mice in which the maintenance methyltransferase gene Dnmtl is deleted exclusively in precursor cells of the central nervous system (CNS). Dnmtl deficiency in CNS precursor cells causes significant demethylation in differentiating neurons and glial cells. Mutant embryos carrying 95 percent of hypomethylated CNS cells die immediately after birth, indicating that hypomethylation disrupts vital CNS function for animal survival. In mosaic animals carrying 30 percent of Dnmtl-/- cells in the embryonic CNS, mutant cells are selectively eliminated during postnatal maturation, showing that methylation is also important for the survival of postnatal CNS cells. The goal of this proposal is to characterize the neural defects in the hypomethylated brain and define the molecular mechanism by which DNA hypomethylation influences the survival and differentiation of neurons and glia. Our working hypothesis is that DNA hypomethylation results in inappropriate expression of many neural genes, which subsequently leads to multiple defects during CNS development. We therefore propose the following Specific Aims: 1. To determine the effect of DNA hypomethylation on the cell fate determination of CNS precursor cells. 2. To determine whether DNA hypomethylation affects neuronal maturation and synaptic function. 3. To define the mechanism of cell death triggered by DNA hypomethylation in the postnatal CNS environment. The proposed study may provide fundamental insights into the role of DNA methylation in neural development as well as the disease mechanism underlying certain mental retardation disorders.

DESCRIPTION (provided by applicant): In mammals DNA cytosine methylation is one of the major epigenetic factors that regulate many cellular events including developmental gene expression and genomic imprinting. Alternations in DNA methylation machinery have been linked to several mental retardation disorders, including Rett, ICF, Fragile-X, and ATRX syndromes, suggesting that methylation is important for neuronal development and function. To investigate the function of DNA methylation in the central nervous system (CNS), we have recently applied the cre/loxP system to delete the maintenance DNA methyltransferase Dnmtl gene exclusively in the CNS. By crossing the Emx1-cre transgene with the Dnmtl conditional allele (Dnmt2lox), we have obtained conditional knockout mice with Dnmtl deficiency restricted to the cortex and hippocampus. Emx1-cre mediated Dnmtl gene deletion is initiated in pallial cortical precursor cells at embryonic day (E) 9-10, resulting in DNA hypomethylation in embryonic and postnatal cortical projection neurons. Mutant mice are viable in adulthood but exhibit obvious behavioral defects such as hyperactivity and hind limb clasping upon tail suspension. Morphological studies indicate that Emx1-cre;Dnmtl mutant mice exhibit massive loss of cortical volume, thus become a valuable animal model for studying the effect of DNA hypomethylation on cortical degeneration. In this proposal, we plan to first examine the time course of cortical neuronal cell death and determine the gross histological and behavioral defects in the mutant mice. Further experiments are designed to determine the effect of DNA hypomethylation on the proliferation and differentiation of precursor cells, as well as dendritic arborization of Dnmtl-/- neurons. Finally, we plan to determine the mechanism by which DNA hypomethylation induces neuronal cell death in the mutant cortex. It is known that levels of DNA methylation decrease with aging and in age-related neurodegenerative disorders such as Alzheimer's disease. Understanding the mechanism of cell death in hypomethylated cortical neurons may help us develop therapeutic strategies to prevent hypomethylation-induced neuronal degeneration.

DESCRIPTION (provided by applicant): Epigenetic gene regulation through DNA methylation and histone modifications has been shown to be a crucial mechanism for the development and function of the nervous system, ranging from cell differentiation to neuronal plasticity and from learning &memory to behavior. The deregulation of the epigenome could lead to various neuropsychiatric disorders. To address the role of DNA methylation in the development and function of cortex and hippocampus, we have examined expression of DNA methyltransferases (Dnmts including Dnmt1, Dnmt3a, and Dnmt3b) in the developing and adult central nervous system (CNS). Intriguingly, we found that Dnmts such as Dnmt1 and Dnmt3a are still highly expressed in postmitotic neurons. We hypothesize that expression of Dnmts in postmitotic CNS neurons is to maintain and modulate DNA methylation patterns, which can subsequently regulate long-term changes of neuronal gene expression. To test this hypothesis, in Aim 1, we plan to generate conditional mutants with the Cre/LoxP system in which both Dnmt1 and Dnmt3a are absent in postmitotic cortical and hippocampal neurons. This unique mouse model system allows us to examine methylation changes under seizure condition in the presence or absence of both Dnmt1 and Dnmt3a. In Aim 2, we will map genome-wide methylation patterns in hippocampal dentate gyrus neurons in control and seizure conditions through shotgun bisulfate sequencing (BS-Seq) and determine the potential alteration of DNA methylation patterns in Dnmt-deficient hippocampal neurons. This BS-Seq approach has been successfully applied to decipher methylomes at single nucleotide resolution in plants and human cells. With the advent of next-generation and third generation sequencers, we would obtain methylomes in mammalian hippocampal neurons at a very reasonable cost. By mapping DNA methylation in CNS neurons under normal and seizure conditions in the presence and absence of Dnmts, we will gain insight into the role of Dnmts and DNA methylation in postmitotic neurons. Our findings will lay a solid foundation for future study to understand the involvement of abnormal methylation in neurological disorders. PUBLIC HEALTH RELEVANCE: This grant proposes to understand regulatory mechanisms of gene expression in brain cells - specifically, to examine the pattern of DNA modification (namely DNA methylation) in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression and it is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use high throughput sequencing technique to identify DNA methylation patterns in the developing brain cells and examine the consequence of the perturbation of DNA methylation patterns on brain development and function, thus impacting public health by paving the way for understanding pathological mechanisms of mental retardation disorders due to the perturbation of DNA methylation.

DESCRIPTION (provided by applicant): Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is an autosomal recessive disorder affecting multiple organ systems. Recent human genetic studies have identified that most ICF syndrome patients carry genetic mutations in DNMT3B encoding a de novo DNA methyltransferase. Consequently, cells derived from affected patients exhibited significantly reduced methyltransferase activity and abnormal hypomethylation of CpG sites within pericentromeric satellite, subtelomeric, and X chromosome regions. Transgenic mouse models that mirror genetic mutations in human ICF syndrome do not fully recapitulate the human pathogenesis of the syndrome. For this purpose, we propose to develop a human stem cell model that is better suited to understand the molecular basis of ICF syndrome. In Specific Aim 1, we will generate and characterize human iPSCs carrying ICF mutations and determine whether DNMT3B deficiency affects cell proliferation and genome stability. ICF-iPSCs will be characterized for expression of pluripotency markers and multi-potentials of cell differentiation through embryoid formation and teratoma formation. Specific Aim 2 is designed to compare directed differentiation of control and ICF mutant iPSCs into hematopoietic progenitor cells (HPCs) and neural crest lineage cells (NCs) to determine the impact of DNMT3B deficiency on lineage-specific cell differentiation. In Specific Aim 3, we will attempt to rescue ICF phenotypes by introducing wild-type DNMT3B expression in ICF mutant iPSCs, HPCs, and NCs. We will focus on understanding whether DNMT3B expression can rescue stage-specific DNA hypomethylation on the genome stability and gene expression during cell differentiation. Our proposed research will provide a novel approach to understanding the pathogenesis of ICF syndrome, thus potentially develop a new approach to cure ICF syndrome through stem cell therapy.

Abstract Human overgrowth syndrome is a class of diseases characterized by systemic or regional excess growth compared to peers of the same age. Recent human genetic studies have revealed a series of missense mutations in epigenetic regulators including a de novo DNA methyltransferase enzyme DNMT3A. It is suggested that cells derived from affected patients would exhibit defects in epigenetic modifications such as DNA methylation and histone modifications, leading to the alteration of specific gene expression. However, the molecular mechanism underlying pathogenesis of overgrowth syndrome is largely unknown. We therefore propose to develop stem cell models that are better suited to understand the molecular basis of DNMT3A mutations in overgrowth syndrome with intellectual disability (coined Tatton-Brown-Rahman Syndrome in OMIM). In Specific Aim 1, we will generate isogenic human and mouse ESCs (hESCs and mESCs) carrying either wild-type DNMT3 allele or a spectrum of specific DNMT3A point mutations via CRISPR/Cas9 mediated gene editing technology. DNMT3A mutant hESCs and mESCs will be extensively characterized for potential defects in cell cycle regulation, DNA methylation, H3K27me3 and RNA transcriptome in order to determine convergent molecular pathways associated different types of DNMT3A mutations. To directly examine the defects in cell lineages involved in craniofacial development, in Specific Aim 2, we will examine the impact of DNMT3A mutations on sequential differentiation of mutant ESCs into neural precursor cells (NPCs), cortical- like neurons, glial cells, as well as neural crest derivatives. By performing analysis of DNA methylation, H3K27me3, and RNA transcriptome at different stage of cell differentiation, we will define shared molecular changes and regulatory pathways in RNA transcriptome and DNA methylation that underlie pathogenesis of craniofacial and brain development in vitro. In Aim 3, we will generate transgenic mice carrying DNMT3A mutations, and examine mouse mutant phenotypes relevant to overgrowth. Molecular characterization of brain neurogenesis and craniofacial development in vivo in mutant mice will lead to the identification of either novel or known signaling pathways (such as PTEN/mTOR/IGF signaling) associated with overgrowth phenotype. Moreover, we will determine whether the defects of cell growth, proliferation, and differentiation in vivo will be consistent with what we observed in vitro in both human and mouse stem cell differentiation model in Aim 2. Finally, we will also perform learning and memory behavioral tests to determine the association of DNMT3A mutations with potential learning and memory deficits. Our proposed research will provide a novel approach to understanding the molecular pathogenesis of human DNMT3A overgrowth syndrome, potentially leading to the development of a therapeutic approach to prevent or cure human overgrowth disorders.