ChromBPNet: bias factorized, base-resolution deep learning models of chromatin accessibility reveal cis-regulatory sequence syntax, transcription factor footprints and regulatory variants.

Despite extensive mapping of cis-regulatory elements (cREs) across cellular contexts with chromatin accessibility assays, the sequence syntax and genetic variants that regulate transcription factor (TF) binding and chromatin accessibility at context-specific cREs remain elusive. We introduce ChromBPNet, a deep learning DNA sequence model of base-resolution accessibility profiles that detects, learns and deconvolves assay-specific enzyme biases from regulatory sequence determinants of accessibility, enabling robust discovery of compact TF motif lexicons, cooperative motif syntax and precision footprints across assays and sequencing depths. Extensive benchmarks show that ChromBPNet, despite its lightweight design, is competitive with much larger contemporary models at predicting variant effects on chromatin accessibility, pioneer TF binding and reporter activity across assays, cell contexts and ancestry, while providing interpretation of disrupted regulatory syntax.

Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.

Functional analysis of non-coding variants associated with congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation.

Conserved -Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals.

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range.

Emx2 underlies the development and evolution of marsupial gliding membranes.

Phenotypic variation among species is a product of evolutionary changes to developmental programs. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation.

Rapid and Quantitative Functional Interrogation of Human Enhancer Variant Activity in Live Mice.

Functional analysis of non-coding variants associated with human congenital disorders remains challenging due to the lack of efficient models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice of any genetic background. We use this new technology to examine and measure the gain- and loss-of-function effects of enhancer variants linked to limb polydactyly, autism, and craniofacial malformation.

Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing.

Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expression, which is typically achieved through the concerted activity of multiple enhancers driving cell type-specific expression of a target gene. Mammalian genomes are organized in topologically associated domains, providing a preferred environment and framework for interactions between transcriptional enhancers and gene promoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancers, assessment of subregional enhancer activities via transgenic reporter assays in mice remains the gold standard for assigning enhancer activity in vivo.

Ultraconserved enhancer function does not require perfect sequence conservation.

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays.

Enhancer redundancy in development and disease.

Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control most developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering gene expression against mutations in non-coding regulatory regions of genes implicated in human disease.

Presynaptic Homeostasis Opposes Disease Progression in Mouse Models of ALS-Like Degeneration: Evidence for Homeostatic Neuroprotection.

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration.

Stable enhancers are active in development, and fragile enhancers are associated with evolutionary adaptation.

Background: Despite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers.

Results: We identify two classes of enhancers based on the density of nucleotides prone to deMs.

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes.

An Evolutionary Conserved Epigenetic Mark of Polycomb Response Elements Implemented by Trx/MLL/COMPASS.

Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear.

Using transgenic reporter assays to functionally characterize enhancers in animals.

Enhancers or cis-regulatory modules play an instructive role in regulating gene expression during animal development and in response to the environment. Despite their importance, we only have an incomplete map of enhancers in the genome and our understanding of the mechanisms governing their function is still limited. Recent advances in genomics provided powerful tools to generate genome-wide maps of potential enhancers.

Genome-scale functional characterization of Drosophila developmental enhancers in vivo.

Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis.

Deciphering the transcriptional cis-regulatory code.

Information about developmental gene expression resides in defined regulatory elements, called enhancers, in the non-coding part of the genome. Although cells reliably utilize enhancers to orchestrate gene expression, a cis-regulatory code that would allow their interpretation has remained one of the greatest challenges of modern biology. In this review, we summarize studies from the past three decades that describe progress towards revealing the properties of enhancers and discuss how recent approaches are providing unprecedented insights into regulatory elements in animal genomes.

Uncovering cis-regulatory sequence requirements for context-specific transcription factor binding.

The regulation of gene expression is mediated at the transcriptional level by enhancer regions that are bound by sequence-specific transcription factors (TFs). Recent studies have shown that the in vivo binding sites of single TFs differ between developmental or cellular contexts. How this context-specific binding is encoded in the cis-regulatory DNA sequence has, however, remained unclear.

HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature.

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. We tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, we found 102 (94%) to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes.

[Chromatin decompaction in the interbands of Drosophila polytene chromosomes does not correlate with high transcription level].

The search for correlation between structural organization of particular chromosome regions and their functions is among key directions of molecular cytogenetics. In this study, we used polytene chromosomes of Drosophila melanogaster as a convenient model for examining transcriptional activity of chromomeres (bands) and interchromomeres (interbands) in eukaryotic interphase chromosomes. Using cloning of the interband DNA sequences and determination of the molecular limits for some interbands, we analyzed the transcriptional activity of these regions and compared the band and interband transcriptional activity in polytene chromosomes.

[Functional organization of interbands in Drosophila polytene chromosomes].

The functional organization of particular chromosome regions is tightly associated with their function in eukaryotic cells. Details of this association are among the most topical problems of modem genetics. The paper characterizes the results of recent research of the specifics of the genetic organization and chromatin decondensation in interbands of Drosophila polytene chromosomes.