Dual roles of histone H3 lysine-4 in antagonizing Polycomb group function and promoting target gene expression.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo . Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms.

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.

Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function.

Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoan models is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such RD histone gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms have been developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array ( HisC ), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.

The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development.

Chromatin remodeling is central to the dynamic changes in gene expression that drive cell fate determination. During development, the sets of enhancers that are accessible for use change globally as cells transition between stages. While transcription factors and nucleosome remodelers are known to work together to control enhancer accessibility, it is unclear how the short stretches of DNA that they individually unmask yield the kilobase-sized accessible regions characteristic of active enhancers. Here, we performed a genetic screen to investigate the role of nucleosome remodelers in control of dynamic enhancer activity. We find that the Drosophila Switch/Sucrose Non-Fermenting complex, BAP, is required for repression of a temporally dynamic enhancer, brdisc. Contrary to expectations, we find that the BAP-specific subunit Osa is dispensable for mediating changes in chromatin accessibility between the early and late stages of wing development. Instead, we find that Osa is required to constrain the levels of brdisc activity when the enhancer is normally active. Genome-wide profiling reveals that Osa directly binds brdisc as well as thousands of other developmentally dynamic regulatory sites, including multiple genes encoding components and targets of the Notch signaling pathway. Transgenic reporter analyses demonstrate that Osa is required for activation and for constraint of different sets of target enhancers in the same cells. Moreover, Osa loss results in hyperactivation of the Notch ligand Delta and development of ectopic sensory structures patterned by Notch signaling early in development. Together, these findings indicate that proper constraint of enhancer activity is necessary for regulation of dose-dependent developmental events.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

A tissue dissociation method for ATAC-seq and CUT&RUN in Drosophila pupal tissues.

Chromatin accessibility, histone modifications, and transcription factor binding are highly dynamic during Drosophila metamorphosis and drive global changes in gene expression as larval tissues differentiate into adult structures. Unfortunately, the presence of pupa cuticle on many Drosophila tissues during metamorphosis prevents enzyme access to cells and has limited the use of enzymatic in situ methods for assessing chromatin accessibility and histone modifications. Here, we present a dissociation method for cuticle-bound pupal tissues that is compatible for use with ATAC-Seq and CUT&RUN to interrogate chromatin accessibility and histone modifications. We show this method provides comparable chromatin accessibility data to the non-enzymatic approach FAIRE-seq, with only a fraction of the amount of input tissue required. This approach is also compatible with CUT&RUN, which allows genome-wide mapping of histone modifications with less than 1/10th of the tissue input required for more conventional approaches such as Chromatin Immunoprecipitation Sequencing (ChIP-seq). Our protocol makes it possible to use newer, more sensitive enzymatic in situ approaches to interrogate gene regulatory networks during Drosophila metamorphosis.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing.

Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or replication-independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

Disentangling the developmental origins of a novel phenotype: enhancement versus reversal of environmentally induced gene expression.

Increasing evidence suggests that many novel traits might have originated via plasticity-led evolution (PLE). Yet, little is known of the developmental processes that underpin PLE, especially in its early stages. One such process is 'phenotypic accommodation', which occurs when, in response to a change in the environment, an organism experiences adjustments across variable parts of its phenotype that improve its fitness. Here, we asked if environmentally induced changes in gene expression are enhanced or reversed during phenotypic accommodation of a novel, complex phenotype in spadefoot toad tadpoles (Spea multiplicata). More genes than expected were affected by both the environment and phenotypic accommodation in the liver and brain. However, although phenotypic accommodation primarily reversed environmentally induced changes in gene expression in liver tissue, it enhanced these changes in brain tissue. Thus, depending on the tissue, phenotypic accommodation may either minimize functional disruption via reversal of gene expression patterns or promote novelty via enhancement of existing expression patterns. Our study thereby provides insights into the developmental origins of a novel phenotype and the incipient stages of PLE.

Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses.

Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.

Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

Memes: A motif analysis environment in R using tools from the MEME Suite.

Identification of biopolymer motifs represents a key step in the analysis of biological sequences. The MEME Suite is a widely used toolkit for comprehensive analysis of biopolymer motifs; however, these tools are poorly integrated within popular analysis frameworks like the R/Bioconductor project, creating barriers to their use. Here we present memes, an R package that provides a seamless R interface to a selection of popular MEME Suite tools. memes provides a novel "data aware" interface to these tools, enabling rapid and complex discriminative motif analysis workflows. In addition to interfacing with popular MEME Suite tools, memes leverages existing R/Bioconductor data structures to store the multidimensional data returned by MEME Suite tools for rapid data access and manipulation. Finally, memes provides data visualization capabilities to facilitate communication of results. memes is available as a Bioconductor package at https://bioconductor.org/packages/memes, and the source code can be found at github.com/snystrom/memes.

Transcriptomic bases of a polyphenism.

Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.

An online repository of solvation thermodynamic and structural maps of SARS-CoV-2 targets.

SARS-CoV-2 recently jumped species and rapidly spread via human-to-human transmission to cause a global outbreak of COVID-19. The lack of effective vaccine combined with the severity of the disease necessitates attempts to develop small molecule drugs to combat the virus. COVID19_GIST_HSA is a freely available online repository to provide solvation thermodynamic maps of COVID-19-related protein small molecule drug targets. Grid inhomogeneous solvation theory maps were generated using AmberTools cpptraj-GIST, 3D reference interaction site model maps were created with AmberTools rism3d.snglpnt and hydration site analysis maps were created using SSTMap code. The resultant data can be applied to drug design efforts: scoring solvent displacement for docking, rational lead modification, prioritization of ligand- and protein- based pharmacophore elements, and creation of water-based pharmacophores. Herein, we demonstrate the use of the solvation thermodynamic mapping data. It is hoped that this freely provided data will aid in small molecule drug discovery efforts to defeat SARS-CoV-2.

Damage-responsive, maturity-silenced enhancers regulate multiple genes that direct regeneration in Drosophila.

Like tissues of many organisms, Drosophila imaginal discs lose the ability to regenerate as they mature. This loss of regenerative capacity coincides with reduced damage-responsive expression of multiple genes needed for regeneration. We previously showed that two such genes, wg and Wnt6, are regulated by a single damage-responsive enhancer that becomes progressively inactivated via Polycomb-mediated silencing as discs mature (Harris et al., 2016). Here we explore the generality of this mechanism and identify additional damage-responsive, maturity-silenced (DRMS) enhancers, some near genes known to be required for regeneration such as Mmp1, and others near genes that we now show function in regeneration. Using a novel GAL4-independent ablation system we characterize two DRMS-associated genes, apontic (apt), which curtails regeneration and CG9752/asperous (aspr), which promotes it. This mechanism of suppressing regeneration by silencing damage-responsive enhancers at multiple loci can be partially overcome by reducing activity of the chromatin regulator extra sex combs (esc).

An online repository of solvation thermodynamic and structural maps of SARS-CoV-2 targets.

SARS-CoV-2 recently jumped species and rapidly spread via human-to-human transmission to cause a global outbreak of COVID-19. The lack of effective vaccine combined with the severity of the disease necessitates attempts to develop small molecule drugs to combat the virus. COVID19_GIST_HSA is a freely available online repository to provide solvation thermodynamic maps of COVID-19-related protein small molecule drug targets. Grid Inhomogeneous Solvation Theory maps were generated using AmberTools cpptraj-GIST and Hydration Site Analysis maps were created using SSTmap code. The resultant data can be applied to drug design efforts: scoring solvent displacement for docking, rational lead modification, prioritization of ligand- and protein- based pharmacophore elements, and creation of water-based pharmacophores. Herein, we demonstrate the use of the solvation thermodynamic mapping data. It is hoped that this freely provided data will aid in small molecule drug discovery efforts to defeat SARS-CoV-2.

Expression of E93 provides an instructive cue to control dynamic enhancer activity and chromatin accessibility during development.

How temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that expression of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility.

Structure-Based and Property-Driven Optimization of N-Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.