Soo-Kyung Lee - US grants

PROJECT SUMMARY Our long-term goal is to define gene networks that enable neural stem cells to produce remarkably divergent cell types in CNS development. The combinatorial action of transcription factors is a prevalent strategy to achieve cellular complexity in CNS. However, the mechanisms underlying combinatorial action of transcription factors in controlling the expression of unique set of terminal differentiation genes for a specific cellular identity remain unclear in vertebrate CNS. In this proposal, we wish to tackle this important issue by focusing on the gene networks for the specification of spinal motor neurons, in which the developmental transcription codes are relatively well understood. LIM homeodomain proteins Lhx3 and Isl1 regulate motor neuron specification in combination by forming a hexameric complex, named MN-hexamer. The key hypothesis of this proposal is that MN-hexamer directly controls a battery of genes that control wide aspects of MN identity, including cholinergic neurotransmission, by coordinating the actions of retinoid signal and chromatin modifying enzymes during spinal cord development. We will test this hypothesis using an ensemble of molecular and biochemical methods, genetically engineered embryonic stem cells, chick embryos and mutant mice. Three specific aims are proposed to dissect the hypothesis;1) To define the target genes of MN-hexamer that assign MN identity. 2) To investigate the regulation of cholinergic neuronal identity by similar hexameric complexes in spinal motor neurons and forebrain cholinergic neurons. 3) To define the role of RA in facilitating MN specification by MN-hexamer. Besides providing crucial insights into the generation of motor neurons and motor circuits, our studies will lay fundamental framework to study gene networks in creating the amazing cellular diversity during CNS development. These studies should also provide new tools for developing therapeutic strategies for the spinal cord injuries and diseases associated with impaired motor function, such as ALS (Lou Gehrig[unreadable]s disease), and the cognitive disorders resulting from the loss of forebrain cholinergic neurons, such as Alzheimer[unreadable]s diseases.

ABSTRACT Past studies on extrinsic signals and intrinsic transcription factors (TFs) have greatly enhanced our understanding of how the fate specification and differentiation of diverse neuronal cell types in CNS are genetically coded. The recent identification of diverse chromatin regulatory marks and the enzymes producing those marks allows us to integrate the potentially vital action of chromatin regulation with the activity of TFs in CNS development. However, this effort is riddled with a few challenges. 1) The developing CNS consists of profoundly heterogeneous cells at different developmental states, making it difficult to study how chromatin changes are orchestrated in each cell type. 2) While cell type-specific regulatory elements (herein referred to as cis-elements) are predicted to undergo the most functionally critical chromatin changes throughout development, such cis-elements are globally ill-defined. 3) Although multiple chromatin regulatory factors would be mobilized to cell type-specific cis-elements throughout development of each cell type, it is tough to identify the specific chromatin factors in action. Building on our prior effort to solve these limitations, the current study of the gene regulatory networks for spinal motor neuron (MN) development is a pioneering study in addressing the critical issue of chromatin regulation in CNS development. Our findings in the past funding cycles identified transcription codes and gene regulatory elements that underlie commitment and specification of MN fate, establishing an ideal cellular model to investigate chromatin regulation in CNS development. In the developing spinal cord, Olig2, a basic helix-loop-helix (bHLH) TF expressed in progenitors for MNs (pMNs), plays essential roles in establishing the pMN domain and keeping pMN cells from prematurely differentiating to MNs. As pMN cells begin to differentiate to MNs, the LIM homeodomain (HD) TFs Isl1 and Lhx3 are upregulated, along with the bHLH TF Ngn2. Isl1 and Lhx3 form a complex (Isl1-Lhx3), which directs MN fate via synergistic transactivation of MN genes with Ngn2. In this proposal to integrate these genetic programs with chromatin regulation, we hypothesize that during MN development, coordinated actions of the cell type-specific TFs (Olig2, Ngn2 and Isl1-Lhx3) and the chromatin modifiers (Ezh2, Jmjd3 and CBP/p300) orchestrate the chromatin changes in MN genes from transcriptionally poised/repressive to active state, enabling the timely acquisition of MN fate and cell differentiation. Based on our preliminary data, we specifically postulate that, in pMN cells, Olig2 recruits Ezh2 to MN genes, instituting the transcriptionally repressive chromatin mark, trimethylated histone H3-lysine 27 (H3K27me3). As Olig2 expression declines in differentiating pMN cells, Isl1-Lhx3 and Ngn2 seize MN-specific enhancers and recruit Jmjd3, which removes H3K27me3 and allows CBP/p300 to set up acetylated H3K27 (H3K27ac), a transcriptionally active chromatin mark. We will test our hypothesis using an ensemble of cellular, biochemical, genetic and genome-wide approaches.

Project Summary The cerebral cortex is responsible for higher cognitive and emotional functions, and has served as an ideal model to study CNS development due to enormous cellular complexity. Our long-term goal is to fully decode the genetic and epigenetic mechanisms by which transcription factors (TFs) and chromatin remodelers cooperate to regulate the cortex development and how disruption of such mechanisms leads to neurodevelopmental disorders with impaired cortical functions. To address these two critical issues, here we propose to study the role of the forkhead TF FoxG1 in corticogenesis and a human neurodevelopmental disorder FoxG1 syndrome (FS) (aka, a congenital variant of Rett syndrome, RTT), which results from inactivating mutations in one allele of the FoxG1 gene. Prominent clinical features of FS include microcephaly, agenesis of the corpus callosum, profound intellectual disability with autistic features and absent language, and seizures. Duplication of FoxG1 is also associated with developmental epilepsy, intellectual disability, and severe speech and social impairment. Overexpression of FoxG1 via unknown mechanism is also implicated in autism. These results indicate that brain development is highly sensitive to the dosage of FoxG1. The mechanisms underlying timely neurogenesis and production of diverse cortical neuronal types are beginning to be understood thanks to the discovery of TFs that are expressed with temporal and regional specificity within the neocortex. During CNS development, the neurogenic TFs are often expressed in multiple cell types, suggesting that neuronal TFs may acquire cell type-specific activity by regulating distinct sets of target genes in cell context-dependent manner. However, the molecular mechanisms by which neuronal TFs recognize and control cell type-specific transcription program in the developing cortex remain ill-defined. FoxG1 is strongly expressed in forebrain NPCs, in which it regulates self-renewal and a timing of neurogenesis. FoxG1 is downregulated during differentiation of NPCs, and then re-expressed in cortical neurons, in which FoxG1 promotes neuronal entry into the cortical plate (CP). While these results suggest cell context-dependent actions of FoxG1, the gene regulatory mechanisms by which FoxG1 controls the sequential steps of cortex development and how these mechanisms relate to FS pathology are unclear. Our unbiased comprehensive screening approaches (ChIPseq, RNAseq and proteomics) disclosed key clues for understanding the molecular actions of FoxG1 in the developing cortex. Based on these seminal findings, we hypothesize that FoxG1 regulates its target genes in a developmental timing sensitive manner by collaborating with cell type- specific partner TFs and chromatin regulatory factors in cortex development, and dysregulation of these processes leads to neurological deficits in FS. We will test this hypothesis using an ensemble of cellular, biochemical, genetic, and comprehensive genome-wide approaches.

The hypothalamus, consisting of multiple nuclei, regulates homeostasis crucial for survival and reproduction. In particular, the hypothalamic arcuate nucleus (ARC) has various neurons that control energy balance, reproduction, and growth and play key roles in sensing and processing peripheral cues due to its permeability and proximity to the peripheral bloodstream. Despite the vital roles of ARC neurons in the growth and energy homeostasis, the transcription factors (TFs) and epigenetic regulatory programs that orchestrate their development remain poorly understood. In this proposal, we wish to fill this gap by studying MLL4-complex (MLL4-C) and its partner TFs in ARC development. MLL4-C acts as an epigenetic coactivator of its partner TFs by utilizing its two histone H3-modifying enzyme subunits; the H3-lysine 4-methyltransferase (H3K4MT) MLL4 and the H3K27-demethylase (H3K27DM) UTX. In humans, mutations in MLL4 or UTX cause a developmental disorder Kabuki syndrome (KS) characterized by a unique facial feature, microcephaly, heart defects, intellectual disability, dwarfism and obesity. Our strong preliminary results suggest that the attenuated activity of MLL4-C in the ARC results in deficits in ARC neuronal development and contributes to the short stature and obesity observed in human KS, leading to the major hypothesis of this proposal: By interacting with partner TFs, MLL4-C is recruited to the genes critical for ARC neuronal fate determination and neurite/axonal growth and upregulates their expression using the chromatin-opening activities of MLL4/UTX. Using an ensemble of cell and molecular, biochemical, mouse genetics, and genome-wide approaches, we will test this hypothesis in three specific aims. This study will reveal fundamental principles of genetic and epigenetic regulatory programs that direct ARC neuronal development, establishing the concept that MLL4-C is a therapeutic target to treat various pathologies of KS.

Our overarching research goal is to comprehensively understand the gene regulatory network that directs the development of various motor columns (MCs) in the spinal cord and how each MC contributes to the neural circuitry for locomotion. This proposal is designed to study a MC named PreGanglionic MC (PGC, aka CT for Column of Terni in chick), which is found only in thoracic levels of the spinal cord and contains visceral motor neurons (vMNs) that control the activity of the sympathetic nervous system (SymNS). In sympathetic ganglia, the axons of PGC motor neurons (MNs) form synapse with sympathetic neurons, which then regulate the activity of smooth muscle fibers, cardiac muscles, and glands. Given that vMNs in the PGC control the SymNS, which targets various internal organs, it is tempting to speculate that PGC is not a homogenous cell population and instead consists of multiple subtypes. However, despite recent advances in our generation mechanisms of spinal MCs, little is known about either that specify PGC identity. This study the cellular composition of PGC understanding of the MNs or the molecular will interrogate these two major issues in the field. Our strong preliminary findings led to our hypothesis: PGC MNs have multiple subtypes and Jun is a vital transcription factor in the gene regulatory network that directs fate-specification, differentiation, diversification, cell body migration, and axonal projection of PGC MNs.? To dissect this hypothesis, we will employ an ensemble of cellular, biochemical, genetic and unbiased genome-wide approaches.

The ultimate research goal of this lab is to decipher the gene regulatory network that directs the development of various types of neurons in the mouse arcuate nucleus of the hypothalamus (ARC). Despite the physiological significance of many ARC neurons, the developmental gene regulatory programs for ARC neurons remain poorly understood. This lab has been pioneering this emerging area of studies by successfully combining mouse genetics and genome-wide studies. Notably, a close developmental link has been discovered among different types of ARC neurons. These intertwined developmental pathways are likely crucial to ensure the balanced production of different ARC neurons during embryogenesis, enabling a highly coordinated regulation of various homeostatic processes in later postnatal life, such as integration of feeding, reproduction, and growth. Key preliminary results in this grant include: i) Single cell RNA-seq (scRNA-seq) analyses reveal eight TFs enriched in developing growth hormone-releasing hormone (GHRH)-neurons in the ARC, which control linear growth; Prox1, Gsx1, Egr1, Foxp2, Pbx3, St18, Dlx1 and Dlx2. Notably, Dlx1/2, Foxp2 and Gsx1 have been shown to be important for the development of GHRH-neurons, indicating that the scRNA-seq approach is highly useful to identify TFs acting on neuronal lineage development. ii) GHRH-specific inactivation of Prox1 leads to dwarfism and reduced Ghrh expression in mice. iii) Further, ChIP-seq data for Dlx1 provides various new insights into the mechanism by which Dlx1 controls GHRH-neuronal development. Together, these results led to the central hypothesis Dlx1/2 and Prox1 play vital roles in acquiring GHRH-neuronal fate over other related ARC neuronal lineages in part by coordinating the expression of downstream TFs. This hypothesis will be tested in the two specific aims using an ensemble of biochemical and cellular methods, mouse genetics and genome-wide approaches. Completion of this innovative study will radically improve the understanding of how common progenitors are guided to gain a specific ARC lineage identity over other related cell fates, providing a critical mechanism contributing to the balanced production of diverse ARC neuronal types during development.