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Title: Enhancer activation via TCP and HD-ZIP and repression by Dof transcription factors mediate giant cell-specific expression
Abstract Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells.  more » « less
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The Plant Cell
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National Science Foundation
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  1. INTRODUCTION Neurons are by far the most diverse of all cell types in animals, to the extent that “cell types” in mammalian brains are still mostly heterogeneous groups, and there is no consensus definition of the term. The Drosophila optic lobes, with approximately 200 well-defined cell types, provides a tractable system with which to address the genetic basis of neuronal type diversity. We previously characterized the distinct developmental gene expression program of each of these types using single-cell RNA sequencing (scRNA-seq), with one-to-one correspondence to the known morphological types. RATIONALE The identity of fly neurons is determined by temporal and spatial patterning mechanisms in stem cell progenitors, but it remained unclear how these cell fate decisions are implemented and maintained in postmitotic neurons. It was proposed in Caenorhabditis elegans that unique combinations of terminal selector transcription factors (TFs) that are continuously expressed in each neuron control nearly all of its type-specific gene expression. This model implies that it should be possible to engineer predictable and complete switches of identity between different neurons just by modifying these sustained TFs. We aimed to test this prediction in the Drosophila visual system. RESULTS Here, we used our developmental scRNA-seq atlases to identify the potential terminal selector genes in all optic lobe neurons. We found unique combinations of, on average, 10 differentially expressed and stably maintained (across all stages of development) TFs in each neuron. Through genetic gain- and loss-of-function experiments in postmitotic neurons, we showed that modifications of these selector codes are sufficient to induce predictable switches of identity between various cell types. Combinations of terminal selectors jointly control both developmental (e.g., morphology) and functional (e.g., neurotransmitters and their receptors) features of neurons. The closely related Transmedullary 1 (Tm1), Tm2, Tm4, and Tm6 neurons (see the figure) share a similar code of terminal selectors, but can be distinguished from each other by three TFs that are continuously and specifically expressed in one of these cell types: Drgx in Tm1, Pdm3 in Tm2, and SoxN in Tm6. We showed that the removal of each of these selectors in these cell types reprograms them to the default Tm4 fate. We validated these conversions using both morphological features and molecular markers. In addition, we performed scRNA-seq to show that ectopic expression of pdm3 in Tm4 and Tm6 neurons converts them to neurons with transcriptomes that are nearly indistinguishable from that of wild-type Tm2 neurons. We also show that Drgx expression in Tm1 neurons is regulated by Klumpfuss, a TF expressed in stem cells that instructs this fate in progenitors, establishing a link between the regulatory programs that specify neuronal fates and those that implement them. We identified an intronic enhancer in the Drgx locus whose chromatin is specifically accessible in Tm1 neurons and in which Klu motifs are enriched. Genomic deletion of this region knocked down Drgx expression specifically in Tm1 neurons, leaving it intact in the other cell types that normally express it. We further validated this concept by demonstrating that ectopic expression of Vsx (visual system homeobox) genes in Mi15 neurons not only converts them morphologically to Dm2 neurons, but also leads to the loss of their aminergic identity. 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For instance, reduced levels of cut expression in Tm2 neurons, because of its negative regulation by pdm3 , controls the synaptic layer targeting of their axons. Knockdown of cut in Tm1 neurons is sufficient to redirect their axons to the Tm2 layer in the lobula neuropil without affecting other morphological features. CONCLUSION Our results support a model in which neuronal type identity is primarily determined by a relatively simple code of continuously expressed terminal selector TFs in each cell type throughout development. Our results provide a unified framework of how specific fates are initiated and maintained in postmitotic neurons and open new avenues to understanding synaptic specificity through gene regulatory networks. The conservation of this regulatory logic in both C. elegans and Drosophila makes it likely that the terminal selector concept will also be useful in understanding and manipulating the neuronal diversity of mammalian brains. Terminal selectors enable predictive cell fate reprogramming. Tm1, Tm2, Tm4, and Tm6 neurons of the Drosophila visual system share a core set of TFs continuously expressed by each cell type (simplified). The default Tm4 fate is overridden by the expression of a single additional terminal selector to generate Tm1 ( Drgx ), Tm2 ( pdm3 ), or Tm6 ( SoxN ) fates. 
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This is due to an overwhelming number of cases where nucleotides turn over at a high rate, but a similar combination of transcription factor binding sites and other sequence features can be maintained across millions of years of evolution, allowing the function of the enhancer to be conserved in a particular cell type or tissue. Experimentally measuring the function of orthologous enhancers across dozens of species is currently infeasible, but new machine learning methods make it possible to make reliable sequence-based predictions of enhancer function across species in specific tissues and cell types. RESULTS To overcome the limits of studying individual nucleotides, we developed the Tissue-Aware Conservation Inference Toolkit (TACIT). Rather than measuring the extent to which individual nucleotides are conserved across a region, TACIT uses machine learning to test whether the function of a given part of the genome is likely to be conserved. More specifically, convolutional neural networks learn the tissue- or cell type–specific regulatory code connecting genome sequence to enhancer activity using candidate enhancers identified from only a few species. This approach allows us to accurately associate differences between species in tissue or cell type–specific enhancer activity with genome sequence differences at enhancer orthologs. We then connect these predictions of enhancer function to phenotypes across hundreds of mammals in a way that accounts for species’ phylogenetic relatedness. We applied TACIT to identify candidate enhancers from motor cortex and parvalbumin neuron open chromatin data that are associated with brain size relative to body size, solitary living, and vocal learning across 222 mammals. 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