To loop or not to loop: what is the role of TADs in enhancer function and gene regulation?

The past decade has seen a huge jump in the resolution and scale at which we can interrogate the three-dimensional properties of the genome. This revealed different types of chromatin structures including topologically associating domains, partitioning genes and their enhancers into interacting domains. While the visualisation of these topologies and their dynamics has dramatically improved, our understanding of their underlying mechanisms and functional roles in gene expression has lagged behind.

Predictive features of gene expression variation reveal mechanistic link with differential expression.

For most biological processes, organisms must respond to extrinsic cues, while maintaining essential gene expression programmes. Although studied extensively in single cells, it is still unclear how variation is controlled in multicellular organisms. Here, we used a machine-learning approach to identify genomic features that are predictive of genes with high versus low variation in their expression across individuals, using bulk data to remove stochastic cell-to-cell variation.

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation.

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X.

Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy.

Dual functionality of -regulatory elements as developmental enhancers and Polycomb response elements.

Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio-temporal expression, and Polycomb response elements (PREs), which maintain stable gene silencing. These two -regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the pleiohomeotic repressive complex (PhoRC) during embryogenesis, we revealed extensive co-occupancy at developmental enhancers.

Promoter shape varies across populations and affects promoter evolution and expression noise.

Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. Here we used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape).

Genetic variants regulating expression levels and isoform diversity during embryogenesis.

Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown.

Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network.

Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.

Chromatin Immunoprecipitation for Analyzing Transcription Factor Binding and Histone Modifications in Drosophila.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is an invaluable technique to assess transcription factor binding and histone modifications in a genome-wide manner, an essential step towards understanding the mechanisms that govern embryonic development. Here, we provide a detailed protocol for all steps involved in generating a ChIP-seq library, starting from embryo collection, fixation, chromatin preparation, immunoprecipitation, and finally library preparation. The protocol is optimized for Drosophila embryos, but can be easily adapted for any model organism.

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

Background: The yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI.

Qualitative Dynamical Modelling Can Formally Explain Mesoderm Specification and Predict Novel Developmental Phenotypes.

Given the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. Here, we delineate a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature.

Next-generation sequencing-based detection of germline L1-mediated transductions.

Background: While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.

Results: Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome.

FourCSeq: analysis of 4C sequencing data.

Motivation: Circularized Chromosome Conformation Capture (4C) is a powerful technique for studying the spatial interactions of a specific genomic region called the 'viewpoint' with the rest of the genome, both in a single condition or comparing different experimental conditions or cell types. Observed ligation frequencies typically show a strong, regular dependence on genomic distance from the viewpoint, on top of which specific interaction peaks are superimposed. Here, we address the computational task to find these specific peaks and to detect changes between different biological conditions.

Conservation of transcription factor binding specificities across 600 million years of bilateria evolution.

Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data.

Ultrasensitive proteome analysis using paramagnetic bead technology.

In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3).

Enhancer loops appear stable during development and are associated with paused polymerase.

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed.

Coordinated repression and activation of two transcriptional programs stabilizes cell fate during myogenesis.

Molecular models of cell fate specification typically focus on the activation of specific lineage programs. However, the concurrent repression of unwanted transcriptional networks is also essential to stabilize certain cellular identities, as shown in a number of diverse systems and phyla. Here, we demonstrate that this dual requirement also holds true in the context of Drosophila myogenesis.

A conserved role for Snail as a potentiator of active transcription.

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation.

Subtle changes in motif positioning cause tissue-specific effects on robustness of an enhancer's activity.

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos.

Logical modelling of Drosophila signalling pathways.

A limited number of signalling pathways are involved in the specification of cell fate during the development of all animals. Several of these pathways were originally identified in Drosophila. To clarify their roles, and possible cross-talk, we have built a logical model for the nine key signalling pathways recurrently used in metazoan development.

Predicting spatial and temporal gene expression using an integrative model of transcription factor occupancy and chromatin state.

Precise patterns of spatial and temporal gene expression are central to metazoan complexity and act as a driving force for embryonic development. While there has been substantial progress in dissecting and predicting cis-regulatory activity, our understanding of how information from multiple enhancer elements converge to regulate a gene's expression remains elusive. This is in large part due to the number of different biological processes involved in mediating regulation as well as limited availability of experimental measurements for many of them.

Impact of genomic structural variation in Drosophila melanogaster based on population-scale sequencing.

Genomic structural variation (SV) is a major determinant for phenotypic variation. Although it has been extensively studied in humans, the nucleotide resolution structure of SVs within the widely used model organism Drosophila remains unknown. We report a highly accurate, densely validated map of unbalanced SVs comprising 8962 deletions and 916 tandem duplications in 39 lines derived from short-read DNA sequencing in a natural population (the "Drosophila melanogaster Genetic Reference Panel," DGRP).

Fragmentation of DNA in a sub-microliter microfluidic sonication device.

Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step. Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip.