3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors.

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.

3D enhancer-promoter interactions and multi-connected hubs: Organizational principles and functional roles.

The spatiotemporal control of gene expression is dependent on the activity of cis-acting regulatory sequences, called enhancers, which regulate target genes over variable genomic distances and, often, by skipping intermediate promoters, suggesting mechanisms that control enhancer-promoter communication. Recent genomics and imaging technologies have revealed highly complex enhancer-promoter interaction networks, whereas advanced functional studies have started interrogating the forces behind the physical and functional communication among multiple enhancers and promoters. In this review, we first summarize our current understanding of the factors involved in enhancer-promoter communication, with a particular focus on recent papers that have revealed new layers of complexities to old questions. In the second part of the review, we focus on a subset of highly connected enhancer-promoter "hubs" and discuss their potential functions in signal integration and gene regulation, as well as the putative factors that might determine their dynamics and assembly.

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.

MFP1/MSD-1 and MFP2/NSPH-2 co-localize with MSP during C. elegans spermatogenesis.

Until recently, the only verified component of Fibrous Bodies (FBs) within Caenorhabditis elegans spermatocytes was the Major Sperm Protein (MSP), a nematode-specific cytoskeletal element. Earlier studies in the pig parasite Ascaris suum had identified accessory proteins that facilitate MSP polymerization and depolymerization within the pseudopod of crawling spermatozoa. In this study, we show that C. elegans homologs of the two Ascaris accessory proteins MFP1 and MFP2 co-localize with MSP in both the pseudopods of C. elegans sperm and the FBs of C. elegans spermatocytes.

The intrinsically disordered protein SPE-18 promotes localized assembly of MSP in Caenorhabditis elegans spermatocytes.

Many specialized cells use unconventional strategies of cytoskeletal control. Nematode spermatocytes discard their actin and tubulin following meiosis, and instead employ the regulated assembly/disassembly of the Major Sperm Protein (MSP) to drive sperm motility. However, prior to the meiotic divisions, MSP is sequestered through its assembly into paracrystalline structures called fibrous bodies (FBs). The accessory proteins that direct this sequestration process have remained mysterious. This study reveals SPE-18 as an intrinsically disordered protein that is essential for MSP assembly within FBs. In spe-18 mutant spermatocytes, MSP forms disorganized cortical fibers, and the cells arrest in meiosis without forming haploid sperm. In wild-type spermatocytes, SPE-18 localizes to pre-FB complexes and functions with the kinase SPE-6 to localize MSP assembly. Changing patterns of SPE-18 localization uncover previously unappreciated complexities in FB maturation. Later, within newly individualized spermatids, SPE-18 is rapidly lost, yet SPE-18 loss alone is insufficient for MSP disassembly. Our findings reveal an alternative strategy for sequestering cytoskeletal elements, not as monomers but in localized, bundled polymers. Additionally, these studies provide an important example of disordered proteins promoting ordered cellular structures.

Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila.

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.

Lysine 27 of replication-independent histone H3.3 is required for Polycomb target gene silencing but not for gene activation.

Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.

Hormone-dependent control of developmental timing through regulation of chromatin accessibility.

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.