CONTROL OF POSTMITOTIC NEURONAL CHROMATIN ACCESSIBILITY

Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. We have developed efficient protocols for bulk and single cell ATAC-seq, RNA-seq, histone mark ChIPmentation, and TF CUT&RUN using either flow sorted 5-HT neurons or affinity purified 5-HT neuron nuclei. Using these methods, we have found that unique distal enhancers define the Pet1 neuron lineage that generates serotonin (5-HT) neurons in mice. Our single cell analyses showed that heterogeneous single cell chromatin landscapes are established early in postmitotic Pet1 neurons. These analyses have also revealed the putative regulatory programs driving Pet1 neuron subtype identities. We found that distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. This led to our discovery that Pet1 and Lmx1b control chromatin accessibility to select Pet1-lineage specific enhancers for 5-HT neurotransmission. Additionally, we found these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. A current project is focused on defining the requirement for continual TF input in the maintenance of postmitotic neuron chromatin accessibility. This will be accomplished by targeting Pet1 and Lmx1b at different stages of life followed by ATAC-seq, ChIPmentation, and CUT&RUN to see if loss of these TFs destabilizes the 5-HT chromatin landscape.

DEVELOPMENT OF SEROTONIN NEURON DIVERSITY

Although significant progress has been made in understanding the genetic origins of neurodevelopmental disorders, it remains unclear what specific molecular steps are disrupted and in which specific neurons types they are dysfunctional or inoperable. One potential reason for this lack of understanding is that neurons, originally classified according the type of transmitter produced, are now well known to possess substantial molecular, cellular, and functional heterogeneity. It seems plausible that neurodevelopmental disorders may arise not only from developmentally altered identities of an entire population of one particular neuron type but also from altered development of one of its specific molecular or functional subtype(s). There has been a decades-long intense interest in the regulatory mechanisms controlling 5-HT neurons as 5-HT has wide-ranging modulatory effects on central neural circuitry and dysfunction of the serotonergic system has been implicated in several neuropsychiatric diseases including depression, stress-related anxiety disorders, autism, intellectual disability, OCD, and schizophrenia. 5-HT neurons possess tremendous molecular, morphological, and electrophysiological heterogeneity. However, developmental trajectories of 5-HT neuron subtypes are currently unknown as are the regulatory mechanisms that govern their development.  We are using single cell RNA-seq and single cell ATAC-seq to comprehensively define the spatiotemporal developmental trajectories of 5-HT neuron subtype transcriptomes and chromatin accessibility. We are combining recent advances in single-cell genomics methods together with our well-established serotonergic transgenic tools, our extensive experience in flow sorting mouse 5-HT neurons, and our bioinformatics expertise to investigate the development of single-cell 5-HT neuron transcriptomes and chromatin accessibility throughout fetal to early postnatal maturation. We are also investigating at the single cell level, the hypothesis that two disease-associated terminal selector transcription factors in 5-HT neurons, Pet1 and Lmx1b, function to determine postmitotic 5-HT neuron subtypes through differential regulation of subtype-specific gene expression, subtype-specific chromatin accessibility and control of downstream subtype-specific transcription factor codes. Pet1 is of special interest as homozygous knockout mutations in FEV, the human ortholog of Pet1, were recently linked to intellectual Disability and Autism Spectrum Disorder. This project will lead to a greater understanding of the subtype-specific gene regulatory networks that generate 5-HT neuron subtypes, which may help illuminate specific neurogenetic pathways that are disrupted in neurodevelopmental disorders such as ASD

ADULT TRANSCRIPTION FACTORS FUNCTIONS

The mechanisms by which transcription factors (TFs) regulate early neuronal development and differentiation is fairly well established, however very little is understood about the exact role of these TFs in controlling postmitotic neuron function.  Many TFs that are critical to the fate specification and neurotransmitter identity of neurons continue to be expressed throughout life, but what their “late functions” are remain largely unidentified. Recently, cell-type specific genetic tools have been developed that allowed us to study the importance of continued expression of these traditionally “developmental TFs”. Recent studies have suggested that the continued expression of some of these developmental TFs is required in postnatal life to maintain neuron identity.  Importantly, findings have also suggested that deficiencies in these TFs in later life can cause neuronal dysfunction and abnormal behavioral phenotypes such as anxiety and neurodegenerative locomotor defects.   The maintenance of the serotonin (5-hydroxytryptamine, 5-HT) neurotransmitter identity is of particular interest because of its role in modulating an enormous array of behaviors including sleeping feeding, learning, mood and aggression.  Additionally, 5-HT system  dysfunction has been implicated in several neurodevelopmental psychiatric disorders such as major depression, schizophrenia, and anxiety. Recent studies on 5-HT neuron differentiation factors, Pet-1 and Lmx1b show that these developmentally crucial TFs continue to function throughout the life of 5-HT neurons.  However, the exact role these factors play in postnatal 5-HT neurons remains unclear.  

ADULT-STAGE MAINTENANCE MECHANISMS SAFEGUARDING SYNAPSES AND AXONS

Degeneration of synapses and axons in the brain occurs naturally as we age and is accelerated in many neurodegenerative diseases. Why neuronal connectivity is vulnerable to breakdown is not understood. It seems likely that this vulnerability arises from the life-long cell biologic challenge to maintain extraordinarily long axonal membranes and their constituent components against constant macromolecular turnover in postmitotic neurons. We are using serotonin (5-HT) neurons as an in vivo model to discover how the integrity of neuronal synapses and axons are maintained in adulthood. In considering this question, we imagine that analogous to gene regulatory networks that act during development to build neuronal connectivity there are distinct gene regulatory networks that function strictly after development to preserve synaptic and axonal integrity. In pursuit of this notion, we discovered an intrinsic transcriptional program that functions strictly in adulthood to homeostatically maintain 5-HT synapses and axons and safeguard them from progressive degeneration. The positive autoregulating TFs, Lmx1b and Pet1, govern this adult stage connectivity survival program. They do so by maintaining expression of hundreds of genes encoding synaptic, axonal, and mitochondrial (SAM) components. These new findings led us to propose that breakdown of adult stage connectivity survival programs, brought about by either genetic or environmental factors, may subvert the integrity of synapses and axons and consequently impose vulnerability to age-related neurodegenerative disorders.

A large literature supports a progressive breakdown of the 5-HT system in Parkinson’s disease (PD). Degeneration of 5-HT connectivity has been reported in idiopathic PD as well as in rare monogenic forms of PD such as one form caused by the human a-synuclein mutation, hA53T. Despite clear 5-HT system pathology, little is known about the molecular mechanisms underlying the breakdown of 5-HT connectivity in PD. In a new project, we are investigating whether genetic weakening of the Lmx1b/Pet1 connectivity survival program worsens degeneration of 5-HT synapses and axons caused by A53T overexpression. Does hA53T induces 5-HT axon pathology by inhibiting maintenance of Lmx1b, 5-HT neurotransmission genes and SAM connectivity genes? We are investigating this question with FACS purified Td-Tomato-labeled 5-HT neurons to perform bulk RNA-seq analyses at different time points after AAV-hA53T injection. We are also testing the hypothesis that hA53T disrupts Lmx1b-occupied open chromatin landscapes required for expression of Lmx1b controlled SAM genes. For this experiment, we are using the Sun1-GFP based INTACT affinity purification protocol to isolate adult stage 5-HT neuron nuclei from mouse brains injected with AAV-hA53T. These isolated nuclei will then be used for i) ATAC-seq to determine the impact of hA53T on 5-HT neuron accessible chromatin, ii) ChIPmentation of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 marks to determine how hA53T overexpression impacts maintenance of histone posttranslational modifications associated with transcriptional activation and repression, and iii) CUT&RUN to determine whether hA53T disrupts Lmx1b genomic occupancy at Lmx1b controlled 5-HT connectivity genes..