Cell-type-directed design of synthetic enhancers.

Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes1. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. Here we show that deep learning models2-6, can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. We evaluate the function of fully synthetic enhancers to specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. We further exploit enhancer design to create 'dual-code' enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, we characterize enhancer codes through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, we apply the same strategies to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states.

Phenotypic and transcriptomic impact of expressing mammalian TET2 in the Drosophila melanogaster model.

Ten-Eleven Translocation (TET) proteins have recently come to light as important epigenetic regulators conserved in multicellular organisms. TET knockdown studies in rodents have highlighted the critical role of these proteins for proper brain development and function. Mutations in mammalian mTET proteins and mTET2 specifically are frequent and deregulated in leukaemia and glioma respectively. Accordingly, we examined the role of mTET2 in tumorigenesis in larval haemocytes and adult heads in Drosophila melanogaster. Our findings showed that expression of mutant and wild type mTET2 resulted in general phenotypic defects in adult flies and accumulation of abdominal melanotic masses. Notably, flies with mTET2-R43G mutation at the N-terminus of mTET2 exhibited locomotor and circadian behavioural deficits, as well as reduced lifespan. Flies with mTET2-R1261C mutation in the catalytic domain, a common mutation in acute myeloid leukaemia (AML), displayed alterations affecting haemocyte haemostasis. Using transcriptomic approach, we identified upregulated immune genes in fly heads that were not exclusive to TET2 mutants but also found in wild type mTET2 flies. Furthermore, inhibiting expression of genes that were found to be deregulated in mTET2 mutants, such as those involved in immune pathways, autophagy, and transcriptional regulation, led to a rescue in fly survival, behaviour, and hemocyte number. This study identifies the transcriptomic profile of wild type mTET2 versus mTET2 mutants (catalytic versus non-catalytic) with indications of TET2 role in normal central nervous system (CNS) function and innate immunity.

Drosophila Tet Is Required for Maintaining Glial Homeostasis in Developing and Adult Fly Brains.

Ten-eleven translocation (TET) proteins are crucial epigenetic regulators highly conserved in multicellular organisms. TETs' enzymatic function in demethylating 5-methyl cytosine in DNA is required for proper development and TETs are frequently mutated in cancer. Recently, Drosophila melanogaster Tet (dTet) was shown to be highly expressed in developing fly brains and discovered to play an important role in brain and muscle development as well as fly behavior. Furthermore, dTet was shown to have different substrate specificity compared with mammals. However, the exact role dTet plays in glial cells and how ectopic TET expression in glial cells contributes to tumorigenesis and glioma is still not clear. Here, we report a novel role for dTet specifically in glial cell organization and number. We show that loss of dTet affects the organization of a specific glia population in the optic lobe, the "optic chiasm" glia. Additionally, we find irregularities in axon patterns in the ventral nerve cord (VNC) both, in the midline and longitudinal axons. These morphologic glia and axonal defects were accompanied by locomotor defects in developing larvae escalating to immobility in adult flies. Furthermore, glia homeostasis was disturbed in dTet-deficient brains manifesting in gain of glial cell numbers and increased proliferation. Finally, we establish a Drosophila model to understand the impact of human TET3 in glia and find that ectopic expression of hTET3 in dTet-expressing cells causes glia expansion in larval brains and affects sleep/rest behavior and the circadian clock in adult flies.

Hydrop enables droplet-based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.

Single-cell RNA-seq and single-cell assay for transposase-accessible chromatin (ATAC-seq) technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost and pave the way for more specialized multiome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyze a small population of fluorescence-activated cell sorted neurons from the Drosophila brain, confirming the protocol's applicability to low input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.

Scientists are now able to determine the order of chemical blocks, or nucleic acids, that make up the genetic code. These sequencing tools can be used to identify which genes are active within a biological sample. They do this by extracting and analysing open chromatin (regions of DNA that are accessible to the cell's machinery), or sequences of RNA (the molecular templates cells use to translate genes into working proteins). Initially, most sequencing tools could only provide an 'averaged-out' profile of the genes activated in bulk pieces of tissue which contain multiple types of cell. However, advances in technology have led to new methods that can extract and analyse open chromatin or RNA from individual cells. First, the cells are separated, via a technique called microfluidics, into tiny droplets of water along with a single bead that carries a unique barcode. The cell is then broken apart inside the droplet and the barcode within the bead gets released and attaches itself to the genetic material extracted from the cell. All the genetic material inside the droplets is then pooled together and sequenced. Researchers then use the barcode tags to identify which bits of RNA or DNA belong to each cell. Single-cell sequencing has many advantages, including being able to pinpoint precise genetic differences between healthy and abnormal cells, and to create cell atlases of whole organisms, tissues and microbial communities. But existing methods for extracting chromatin are very expensive, and there were no openly available tools for processing thousands of cells at speed. Furthermore, while several single-cell RNA sequencing tools are already freely available, they are not very sensitive or practical to use. Here, De Rop et al. have developed a new open-source platform called HyDrop that overcomes these barriers. The method entails a new type of barcoded bead and optimised elements of existing microfluidics protocols using open-source reagents. These changes created a more user-friendly workflow and increased sensitivity of sequencing at no additional cost. De Rop et al. used their new platform to screen the RNA and open chromatin of thousands of individuals cells from the brains of mice and flies. HyDrop outperformed other open-source methods when working in RNA-sequencing mode. It also provides the first open-source tool for sequencing open chromatin in single cells. Further improvements are expected as researchers tweak the platform, which for now provides an affordable alternative to existing methods.

Decoding gene regulation in the fly brain.

The Drosophila brain is a frequently used model in neuroscience. Single-cell transcriptome analysis1-6, three-dimensional morphological classification7 and electron microscopy mapping of the connectome8,9 have revealed an immense diversity of neuronal and glial cell types that underlie an array of functional and behavioural traits in the fly. The identities of these cell types are controlled by gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. Here, to characterize GRNs at the cell-type level in the fly brain, we profiled the chromatin accessibility of 240,919 single cells spanning 9 developmental timepoints and integrated these data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which 70,000 are linked to developmental trajectories involving neurogenesis, reprogramming and maturation. For 40 cell types, uniquely accessible regions were associated with their expressed transcription factors and downstream target genes through a combination of motif discovery, network inference and deep learning, creating enhancer GRNs. The enhancer architectures revealed by DeepFlyBrain lead to a better understanding of neuronal regulatory diversity and can be used to design genetic driver lines for cell types at specific timepoints, facilitating their characterization and manipulation.

Ten-eleven translocation proteins and their role beyond DNA demethylation - what we can learn from the fly.

Ten-eleven Translocation (TET) proteins have emerged as a family of epigenetic regulators that are important during development and have been implicated in various types of cancers. TET is a highly conserved protein that has orthologues in almost all multicellular organisms. Here, we review recent literature on the novel substrate specificity of this family of DNA 5-methylcytosine demethylases on DNA 6-methyladenine and RNA 5-methylcytosine that were first identified in the invertebrate model Drosophila. We focus on the biological role of these novel epigenetic marks in the fruit fly and mammals and highlight TET proteins' critical function during development specifically in brain development.

Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development.

Ten-Eleven Translocation (TET) proteins are important epigenetic regulators that play a key role in development and are frequently deregulated in cancer. Drosophila melanogaster has a single homologous Tet gene (dTet) that is highly expressed in the central nervous system during development. Here, we examined the expression pattern of dTet in the third instar larval CNS and discovered its presence in a specific set of glia cells: midline glia (MG). Moreover, dTet knockdown resulted in significant lethality, locomotor dysfunction, and alterations in axon patterning in the larval ventral nerve cord. Molecular analyses on dTet knockdown larvae showed a downregulation in genes involved in axon guidance and reduced expression of the axon guidance cue Slit. Our findings point toward a potential role for dTet in midline glial function, specifically the regulation of axon patterning during neurodevelopment.