Jane E. Johnson - US grants

DESCRIPTION (provided by applicant): An increasing number of diseases of the brain are being linked to deviations from the normally carefully calibrated balance between excitatory and inhibitory neuronal activities. A critical choice point for establishing this inhibitory/excitatory neuronal balance is governed by the transcription factor Ptf1a. Ptf1a is a bHLH transcription factor that is required for GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. In the absence of Ptf1a, neural progenitor cells fail to generate inhibitory neurons and aberrantly assume an excitatory neuronal phenotype. Uncovering the transcriptional control of Ptf1a expression and the function of its downstream targets will provide molecular insight into developmental processes regulating the neuronal circuitry in multiple regions of the central nervous system. Because of the timing and the mechanism of PTf1a function, it provides a unique opportunity to uncover the molecular mechanisms that couple neuronal differentiation and neuronal subtype specification. Identification of cis-regulatory sequences in the Ptf1a gene locus revealed separable elements controlling transcription initiation, autoregulation, restriction to dI4/dIL progenitors, and downregulation as the cells differentiate to inhibitory neurons. In addition, direct targets of Ptf1a have been identified that serve as candidates for mediating inhibitory neuronal identity while suppressing excitatory neuron identity. The goal of the current project is to build on these findings to 1) identify trans-acting upstream factors and signaling pathways that function through the cis-regulatory sequences to regulate Ptf1a, and 2) determine the function of a downstream target of Ptf1a in specification of interneurons in the dorsal horn, cerebellum, and retina. Success in this program will impact understanding of how stem/progenitor cells transition to mature cell types and generate the neuronal diversity required for circuit formation. Identifying the pathways that direct cells down a specific lineage may have therapeutic value in stem cell manipulation and treatment of neurological disorders. PUBLIC HEALTH RELEVANCE: Alterations in the balance of inhibitory and excitatory neurons are thought to underlie diverse neurological disorders from epilepsy to autism to hyperalgesia. Ptf1a is an essential regulator of this balance in multiple regions of the nervous system, and thus, factors and signaling pathways regulating Ptf1a and regulated by Ptf1a will control the formation of these balanced neuronal networks. Pathways identified here may serve as targets for manipulating the fate of stem cells for regenerative purposes, and are likely to provide fundamental principles of gene regulation common to developing systems.

DESCRIPTION (provided by applicant): Mash1 is an essential transcription factor in neural development throughout multiple regions of the central and peripheral nervous systems. Mash1 expression is tightly regulated; it is expressed in proliferating neural stem cells and is down-regulated as the cells become post-mitotic and mature into neurons. Synthesizing results from multiple investigators on Mash1 expression and function, we hypothesize that (1) Mash1 expression is controlled by signals instructing stem cells to begin the differentiation program, (2) the specificity of function for a particular bHLH involves interacting factors, and (3) in a neural progenitor cell, Mash1 functions in transcriptional control of some but not all pathways required for neuronal differentiation. Experiments here will address these hypotheses, and identify specific molecular components upstream and downstream of this essential neural differentiation factor. We will identify transcription factors that bind the Mash1 enhancer to control Mash1 expression in the spinal neural tube. We will identify structural domains in Mash1 required for specific functions in neurogenesis and screen for interacting factors that modulate these functions. And finally, we will identify the specific regulatory pathways during neuronal differentiation controlled by Mash1 by analysis of gene expression profiles in multiple loss- and gain-of-function paradigms. Success in this research program will increase our understanding of molecular mechanisms involved in neural precursor proliferation, differentiation, and specification. This understanding is important for future therapeutic strategies in treating neurological disorders involving neuronal cell death such as Parkinson's disease, and in regenerative strategies for treatment of brain and spinal cord damage.

[unreadable] DESCRIPTION (provided by applicant): Math1 is a bHLH transcription factor that functions during neurogenesis and neuronal specification, and is essential for the development of multiple neuronal lineages including granule cells in the cerebellum, sensory hair cells in the inner ear, and dl1 dorsal commissural interneurons in the spinal cord. In order to fulfill these essential functions, Math1 expression is precisely controlled both spatially and temporally, since mis-expression of Math1 leads to mis-specification of neurons and lethality. Thus, both the regulation and function of Math1 are critical for normal neural development. The goal of this proposal is to define molecular mechanisms controlling neurogenesis and neuronal specification using Math1 as a model regulatory gene. To attain this goal, multiple strategies will be pursued. First, upstream regulators of Math1 expression will be identified that function through interactions with cis-regulatory sequences in the Math1 enhancer. These regulatory genes may be important factors for initiating differentiation in neural stem/progenitor cells, and for patterning the neural tube in the dorsal/ventral axis. To understand the molecular rationale underlying neuronal differentiation and neuronal specification, genomic approaches will be used to identify targets of Math1 transcriptional activity. And finally, to understand the in vivo specificity of function elicited by the neural bHLH factors, proteins that interact with Math1 will be identified. Success in this research program will increase our understanding of molecular mechanisms involved in neural precursor proliferation, differentiation, and specification. This understanding is important for future use of neural stem cells in treating neurological disorders involving loss of neurons from disease or trauma. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abnormal development of the nervous system is thought to underlie many complex neurological disorders. In the past decade, research in several vertebrate organisms has shown the bHLH transcription factor Ascl1 (previously Mash1) is essential for neuronal differentiation and sub-type specification for the generation of multiple neuronal cell-types throughout the brain, spinal cord, and autonomic nervous system, as well as neuroendocrine and sensory cells. Recent studies show that Ascl1 is also present in cells fated to become oligodendrocytes. The focus of this research project is to understand the regulation and function of Ascl1 in the generation of these diverse neural cell-types. First, elucidation of mechanisms regulating Ascl1 levels is critical for understanding cell number control since Ascl1 function is placed at a critical point in the transition between cycling progenitor cells and post-mitotic neural cells. Experiments testing the importance of sequences conserved across multiple species will be used to identify the regulatory mechanisms controlling Ascl1 expression. Second, Ascl1 is one of the few transcription factors known to regulate diversity in the CNS, however, because Ascl1 expression is transient, and because cells can undergo extensive migrations as they differentiate, it has been difficult to identify the full complement of cell-types in the adult brain that are in the Ascl1 lineage. An in vivo inducible genetic fate-mapping strategy will be used to identify the full complement of cell-types in the brain that have their origin in an Ascl1-expressing cell. And finally, the function of Ascl1 in oligodendrogenesis will be examined for comparison to its function in neurogenesis. We will identify and compare transcriptional targets of Ascl1 activity in spatially and temporally distinct Ascl1- expressing tissues to uncover the mechanism of Ascl1 function. These experiments will address intrinsic molecular mechanisms used to generate the correct number and type of neurons and oligodendrocytes in the CNS. Relevance: Since Ascl1 is critical in transitioning cells from a progenitor state to a differentiated state, studies of the regulation and function of Ascl1 hold significance for their fundamental contributions to multiple areas of concern to public health such as 1) manipulation of neural stem cells, 2) the underlying biology in multiple developmental disorders of the nervous system such as autism and schizophrenia, and 3) in cancers originating in neural tissue such as glioblastoma and neuroblastoma.

DESCRIPTION (provided by applicant): Ptf1a is required for GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. In the absence of Ptf1a, neural progenitor cells fail to generate inhibitory neurons and aberrantly assume an excitatory neuronal phenotype. Ptf1a is a class II bHLH factor that can heterodimerize with class I bHLH factors (E-proteins) to bind E-box DNA consensus sequences in vitro. However, the in vivo function of Ptf1a is in a unique transcription factor complex, called PTF1-J, that contains Ptf1a and an E-protein, plus a third factor Rbpj. This complex binds a bipartite DNA sequence containing the E-box plus a TC- box. Rbpj has a distinct function as the transcriptional effector of Notch signaling. Notch-Rbpj signaling inhibits neuronal differentiation, while Ptf1a-Rbpj functions in neuronal subtype specification. The proposal is to use in vivo chromatin immunoprecipitation combined with massive parallel sequencing platforms (ChIP-seq) to delineate the function of Ptf1a by genome wide identification of its transcription targets. ChIP-seq identified target genes of Ptf1a in vivo will be validated and characterized by combining results from expression profiling of Ptf1a cells in the dorsal neural tube, evaluating changes in expression in Ptf1a mutants, and by testing target regulatory regions in reporter assays in chick neural tube with and without exogenous Ptf1a. Identifying targets of Ptf1a will provide molecular insight into developmental processes controlling the balance of inhibitory and excitatory neurons. PUBLIC HEALTH RELEVANCE: Normal nervous system function requires the correct balance of inhibitory and excitatory neurons. Alterations in this balance are thought to underlie diverse neurological disorders from epilepsy to autism to hyperalgesia. Successful completion of these aims will substantially advance efforts to define transcriptional networks regulating the balance of inhibitory and excitatory neuronal specification. This is a unique opportunity to combine multiple technical advances to begin to tackle the challenging goal of defining the molecular rationale controlling the generation of neuronal diversity.

DESCRIPTION (provided by applicant): Voltage-gated potassium (Kv) channels form a large and diverse family of ion channels that are involved in regulating the resting membrane potential, the action potential waveform, neurotransmitter release and rhythmic firing patterns of neurons. Their pivotal role is highlighted by several inherited human diseases caused by mutations in Kv channel genes. Among the many different types of Kv channels, Kv3-type channels display unique biophysical properties: very rapid activation and deactivation kinetics, high thresholds of activation and large unit conductances, properties that enable neurons to fire narrow actions potentials at extremely high frequencies. Among Kv3-type channels, subunits for Kv3.1 and Kv3.3 are highly expressed in the cerebellum, and some of the behaviorally observed phenotypes in Kv3-null mutant mice are characteristic of cerebellar dysfunction such as impaired motor performance and, of relevance to this proposal, very high alcohol sensitivity. We have previously shown that altered firing patterns of cerebellar Purkinje cells are responsible for impaired motor function yet not for heightened alcohol sensitivity. Here, we propose experiments to test the hypothesis that the extreme alcohol sensitivity of Kv3.1/Kv3.3-double mutants originates from changes in granule cell physiology, neurons that normally express high levels of Kv3.1 and Kv3.3 channel subunits. We will use a molecular biological approach to localize the neuronal origin of high alcohol sensitivity in the cerebellum of Kv3-mutant mice. In future work, this approach will enable us to study the altered neuronal physiology in brain-slice preparations and to correlate changes in neuronal firing patterns with the corresponding behavioral alterations, in particular with the intoxicating effects of alcohol. PUBLIC HEALTH RELEVANCE: We have recently developed potassium channel-mutant mice that are very sensitive to low concentrations of alcohol. Hence, these mice will serve as well-defined rodent models to study the electrophysiological changes, i.e., altered neuronal firing patterns, that cause extreme alcohol sensitivity.

DESCRIPTION (provided by applicant): The bHLH transcription factor Ascl1 (previously Mash1) is essential for neuronal differentiation and sub- type specification of multiple neuronal cell-types throughout the brain, spinal cord, and autonomic nervous system, as well as neuroendocrine cels and cels in sensory systems such as the retina and olfactory epithelia. Ascl1 function is balanced with that of Notch signaling to control progenitor proliferation and differentiation. In this respect, it is not surprising that Ascl1 expression is aberrantly present n neural and neuroendocrine tumors, and has also been identified as a key factor in directly reprogramming fibroblasts to neurons. With these important functions attributed to Ascl1, it is of fundamental importance to understand how Ascl1 functions as a transcription factor in these processes. There has been a tremendous advance in defining the importance of genomic landscape and epigenetics in controlling lineage specific gene expression programs. However, in most cases, connecting the chromatin modifications with site-specific DNA binding factors like Ascl1 is lacking. At the end of the previous funding period, we succeeded in generating a map of Ascl1 bound sites across the genome in vivo during neural tube development using chromatin immunoprecipitation followed by sequencing (ChIP-seq). These data revealed the identity of multiple factors that may play important roles as collaborating factors modulating Ascl1 activity. In the next funding period, we propose to test models for how Ascl1 interfaces with the genome and with collaborating factors to regulate lineage specific gene expression required to understand and manipulate neural lineage specific gene programs. Project goals are to 1) identify transcription factors collaborating with Ascl1 in neural development, 2) distinguish between models of chromatin accessibility and sequence specific mechanisms for how Ascl1 functions to activate a neural gene expression program, and 3) test the hypothesis that a novel Ascl1-interacting factor connects site-specific DNA binding factors like Ascl1 with higher order chromatin modifications to regulate downstream gene expression programs. Understanding how Ascl1 functions as a transcription factor and identifying co-factors that modulate its activity and the gene programs it regulates will provide key entrance points for improving the efficiency of generating specific types of neurons in vitro and in vivo, and for targeting tumor generating cells

Summary The balance between inhibitory and excitatory neurons is established early in development in a process dominated by the interplay between the transcriptional activator PTF1A and the repressor PRDM13 in multiple regions of the nervous system. Initial cell fate decisions that ultimately give rise to inhibitory neurons in the dorsal spinal cord, cerebellum, and retina depend on the early activity of these fate-specifying transcription factors (TFs). PTF1A, like other early- acting basic helix-loop-helix (bHLH) factors, acts as a `master regulator' by triggering downstream genetic cascades. Such TFs have profound effects by restricting progenitor developmental potential long before the appearance of mature neurons. In the absence of PTF1A, neural progenitors fail to generate inhibitory neurons and aberrantly assume an excitatory neuronal fate. Thus, the spatial and temporal control of PTF1A expression controls the formation of the inhibitory/excitatory balance in multiple neuronal circuits. In Aim 1 we will examine the in vivo requirement for a dorsal neural tube specific enhancer for Ptf1a at the molecular, cellular, and behavioral levels. PRDM13, a transcriptional repressor and a direct target of PTF1A, ensures correct specification of dorsal spinal cord inhibitory neurons by repressing genes essential for specifying the alternative excitatory neuronal fates. Because PRDM factors can have methyltransferase activity and/or can recruit other chromatin modifying enzymes, and PRDM13 may bind to bHLH TFs, PRDM13 may provide a molecular link between these factors and accompanying changes in the epigenetic landscape during neuronal subtype- specification. Indeed, PRDM13 binds many similar genomic sites as PTF1A and another bHLH factor ASCL1. In Aims 2 and 3, we will probe PRDM13 functions in the developing nervous system, and test the hypothesis that PRDM13 is recruited to bHLH bound sites to facilitate repressive chromatin modifications to repress transcription through these sites.

The bHLH transcription factor ASCL1 (HASH1/MASH1) is essential for neuronal differentiation and sub-type specification of multiple neuronal cell-types throughout the brain, spinal cord, and autonomic nervous system, as well as cells in sensory systems such as the retina and olfactory epithelia. ASCL1 function is balanced with NOTCH signaling activity to control progenitor proliferation and differentiation. ASCL1 has also been identified as a pioneering factor and a key component of cocktails directly reprogramming fibroblasts to neurons. With these important functions attributed to ASCL1, and its requirement for controlled spatial and temporal expression in vivo for viability postnatally, it is surprising how little is known about regulation of ASCL1 gene transcription. This gap in knowledge reflects past technical challenges in identifying and manipulating cis-regulatory elements (REs) found at large distances from the gene of interest. REs functioning at long-distances to control key developmental genes are being discovered using advances in technologies that can interrogate and manipulate the spatial genome. Here we will exploit these technologies to gain much needed insights into transcriptional control of ASCL1 using cell culture and in vivo models of neural development. Each model has a particular strength that allows unique aspects of ASCL1 regulation to be uncovered. Aims include identifying and testing functions of long-range REs controlling ASCL1 during neuronal differentiation in mouse (in vivo) and human (in vitro) models. Success in these aims will provide functional non-coding regulatory sequences controlling ASCL1 expression. This is important for future projects to identify molecular components of the signaling complexes working through these REs to reach the goal of providing an understanding of how a key lineage defining transcriptional regulator is controlled during development and disease.