Biology subjects

Eisen, M. B.

Publications and source records attributed to Eisen, M. B..

At least 19 recordsLinked to original sources

bioRxiv2026

A million orthopteran singers from a year of continuously operating field recorders detected, tracked and organized by song

Continuous acoustic monitoring has become an invaluable tool in ecology and conservation biology, with hundreds of thousands of automated recording units deployed worldwide. While semi-manual annotation is adequate for some uses, fully automated methods to segregate and identify the source of animal sounds are essential to utilize these data to their full extent. Significant progress has been made for birds, whose calls are generally discrete and structured, and where large annotated reference libraries are available. However these methods do not extend neatly to insects, whose choruses dominate nighttime, and often daytime, soundscapes across most of the world. While many approaches have been proposed to fill this gap, an efficient end-to-end system for annotating insect calls that can operate at the scale of available data has not emerged. Here we describe an unsupervised method that uses the intrinsic, highly repetitive structure of the songs of many insects, especially orthopterans, to identify and separate distinct songs in narrow (minute) time windows, connect them across windows for as long as entire nights as their carrier frequencies and syllable repeat rates vary with time, temperature and other factors, and assemble them across days and locations into a catalog of singers and song types across time, space and conditions. We apply this method to thirteen months (November 2024 to November 2025) of recordings from 27 autonomous recording units in different microhabitats at a field site in southwestern Arkansas, 175,896 recorder-hours in all. We detected 10.9 million singers in five-minute windows and linked them, within each night or day, into 911,005 tracks, of which 103,802 lasted two hours or more (71,770 at night, 32,032 by day), a total of 335,882 hours of song. In the plane of carrier frequency against syllable repeat rate these singers fall on discrete lines along which each slides with temperature, and we use this organization to define 45 song types, 37 trilling and 8 chirping, each composed of dozens to thousands of singer-nights. Within singers, syllable rate rises by about 3 percent per degree and carrier frequency by about 1.5 percent in nearly every type, recapitulating and extending classical observations on the temperature dependence of orthopteran song. Published and reference recordings placed on the same plane fall on these lines, and species of Anaxipha recorded by Walker and Funk land on single types at the right temperatures. More generally, this method and the data it produces provide a robust means of studying changes in insect song over time and space, and of understanding the behavioral and physical forces that shape its production.

ecology↗

bioRxiv2026

Automated localization of calling birds with small passive acoustic arrays in complex soundscapes

Accurately localizing vocalizing animals from passive acoustic recordings remains challenging in complex outdoor soundscapes. Although automated detection and classification of bird calls have advanced rapidly, reliable spatial localization has lagged, particularly for small arrays of autonomous recorders operating without manual intervention. Here we describe a fully automated pipeline for three-dimensional localization of bird vocalizations using distributed networks of 4 to 6 GPS-synchronized recorders deployed in heterogeneous forest environments. Our framework integrates automated multi-recorder event matching, frequency-selective FFT-based cross-correlation for time-difference-of-arrival (TDOA) estimation, geometric cycle-consistency filtering to resolve ambiguous cross-correlation peaks, and nonlinear optimization of source location and effective sound speed. Applied to multi-year datasets from three field sites, the localizations exhibit strong concordance of localizations with independently known landscape features and species-specific habitat associations. These analyses indicate that small, practical arrays can recover ecologically meaningful spatial structure in complex soundscapes without manual curation. This preprint documents the current state of the system and its performance under realistic field conditions.

ecology↗

bioRxiv2024

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

genetics↗

bioRxiv2023

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

evolutionary biology↗

bioRxiv2023

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high concentration clusters within nuclei. Here we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high throughput imaging screen in Drosophila S2 cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

molecular biology↗

bioRxiv2023

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

developmental biology↗

bioRxiv2019

Whole genome sequences of 23 species from the Drosophila montium species group (Diptera: Drosophilidae): A resource for testing evolutionary hypotheses

Large groups of species with well-defined phylogenies are excellent systems for testing evolutionary hypotheses. In this paper, we describe the creation of a comparative genomic resource consisting of 23 genomes from the species-rich Drosophila montium species group, 22 of which are presented here for the first time. The montium group is uniquely positioned for comparative studies. Within the montium clade, evolutionary distances are such that large numbers of sequences can be accurately aligned while also recovering strong signals of divergence; and the distance between the montium group and D. melanogaster is short enough so that orthologous sequence can be readily identified. All genomes were assembled from a single, small-insert library using MaSuRCA, before going through an extensive post-assembly pipeline. Estimated genome sizes within the montium group range from 155 Mb to 223 Mb (mean=196 Mb). The absence of long-distance information during the assembly process resulted in fragmented assemblies, with the scaffold NG50s varying widely based on repeat content and sample heterozygosity (min=18 kb, max=390 kb, mean=74 kb). The total scaffold length for most assemblies is also shorter than the estimated genome size, typically by 5 - 15 %. However, subsequent analysis showed that our assemblies are highly complete. Despite large differences in contiguity, all assemblies contain at least 96 % of known single-copy Dipteran genes (BUSCOs, n=2,799). Similarly, by aligning our assemblies to the D. melanogaster genome and remapping coordinates for a large set of transcriptional enhancers (n=3,457), we showed that each montium assembly contains orthologs for at least 91 % of D. melanogaster enhancers. Importantly, the genic and enhancer contents of our assemblies are comparable to that of far more contiguous Drosophila assemblies. The alignment of our own D. serrata assembly to a previously published PacBio D. serrata assembly also showed that our longest scaffolds (up to 1 Mb) are free of large-scale misassemblies. Our genome assemblies are a valuable resource that can be used to further resolve the montium group phylogeny; study the evolution of protein-coding genes and cis-regulatory sequences; and determine the genetic basis of ecological and behavioral adaptations.

genomics↗

bioRxiv2018

Effects of the maternal factor Zelda on zygotic enhancer activity in the Drosophila embryo

The maternal factor Zelda is broadly bound to zygotic enhancers during early fly embryogenesis, and has been shown to be important for the expression of a large number of genes. However, its function remains poorly understood. Here, we carried out detailed analysis of the functional role of Zelda on the activities of a group of enhancers that drive patterned gene expression along the anterior -posterior axis. We found that among these enhancers, only one lost its activity entirely when all its Zelda bind sites were mutated. For all others, mutations of all of their Zelda binding sites only had limited effect, which varied temporally and spatially. These results suggest that Zld may exert a quantitative effect on a broad range of enhancers, which presumably is critical to generate highly diverse spatial and temporal expression patterns for different genes in the developmental gene network in fly embryo. Lastly, we found that the observed effect of Zelda site mutations was much stronger when a mutant enhancer was tested using a BAC based reporter construct than a simple reporter construct, suggesting that the effect of Zld is dependent on chromatin environment.

developmental biology↗

bioRxiv2018

Zelda potentiates transcription factor binding to zygotic enhancers by increasing local chromatin accessibility during early Drosophila melanogaster embryogenesis.

The maternally deposited transcription factor Zelda binds to and is required for the activation of a large number of genes in early Drosophila development, and has been suggested to act as a pioneer factor. In this study, we investigated the temporal dynamics of Zelda binding along with the maternal patterning factors Dorsal and Caudal during early embryogenesis. We found in regions bound by Zelda and either Dorsal or Caudal, Zelda binding was detected, and reached maximum levels, earlier than Caudal and Dorsal, providing support of its role as a pioneer factor. We found that Dorsal and Caudal binding correlated strongly with Zelda binding at mitotic cycle 12, suggesting that Zelda is important for early binding by these factors and early onset of their target gene expression. At the same time, we show that among Dorsal target enhancers, the dorsal and ventral ectoderm enhancers are much more strongly associated with Zelda than mesoderm enhancers, revealing an additional function of Zelda in coordinating spatial activity of enhancers. We have also investigated the role of Zelda on chromatin structure. We found that in early embryos, before Dorsal and Caudal are bound at significant levels, Zelda binding is associated with histone acetylation and local histone depletion. These chromatin associated changes accompanied with increased local chromatin accessibility were also detected around Zelda peaks in coding sequences that do not appear to play a role in subsequent transcription factor binding. These findings suggest that Zelda binding itself can lead to chromatin structural changes. Finally, we found that Zelda motifs, both bound and unbound, tend to be associated with positioned nucleosomes, which we suggest may be important for the regulatory specificity of enhancers.

developmental biology↗

bioRxiv2018

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet it remains poorly understood how transcription factors facilitate these multiple functions. Here we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key patterning factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid dependent transcription. Based on our observations we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.

developmental biology↗

bioRxiv2018

Entomophthovirus: An insect-derived iflavirus that infects a behavior manipulating fungal pathogen of dipterans

We discovered a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Entomophthovirus, is a capsid-forming, positive-strand RNA virus in the viral family iflaviridae, whose known members almost exclusively infect insects. We show that the virus RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and that virus particles are present in E. muscae. Two close relatives of the virus had been previously described as insect viruses based on the presence of viral genomes in transcriptomes assembled from RNA extracted from wild dipterans. By analyzing sequencing data from these earlier reports, we show that both dipteran samples were co-infected with E. muscae. We also find the virus in RNA sequencing data from samples of two other species of dipterans, Musca domestica and Delia radicum, known to be infected with E. muscae. These data establish that Entomophthovirus is widely, and seemingly obligately, associated with E. muscae. As other members of the iflaviridae cause behavioral changes in insects, we speculate on the possibility that Entomophthovirus plays a role in E. muscae involved host manipulation.

microbiology↗

bioRxiv2018

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

developmental biology↗

bioRxiv2018

The ecology of the Drosophila-yeast mutualism in wineries

The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila.

ecology↗

bioRxiv2018

Convergent evolution of gene expression in two high-toothed stickleback populations

Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in cis regulatory elements that act in an allele-specific manner, or changes to the overall trans regulatory environment that interacts with cis regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene expression associated with convergently evolved morphological evolution? Do cis or trans regulatory changes contribute more to the evolutionary gain of a morphological trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing cis and trans regulatory changes using phased gene expression data from F1 hybrids, we found that trans regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while cis regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar trans regulatory environments.\n\nAuthor SummaryConvergent evolution, where a similar trait evolves in different lineages, provides an opportunity to study the repeatability of evolution. Convergent morphological evolution has been well studied at multiple evolutionary time scales ranging from ancient, to recent, such as the gain in tooth number in freshwater stickleback fish. However, much less is known about the accompanying evolved changes in gene regulation during convergent evolution. Here we compared evolved changes in gene expression in dental tissue of ancestral low-toothed marine fish to fish from two independently derived high-toothed freshwater populations. We also partitioned gene expression changes into those affecting a genes regulatory elements (cis), and those affecting the overall regulatory environment (trans). Both freshwater populations have evolved similar gene expression changes, including a gain of expression of putative dental genes. These similar gene expression changes are due mainly to shared changes to the trans regulatory environment, while the cis changes are largely population specific. Thus, during convergent evolution, overall similar and perhaps predictable transcriptome changes can evolve despite largely different underlying genetic bases.

genomics↗

bioRxiv2017

A fungal pathogen that robustly manipulates the behavior of Drosophila melanogaster in the laboratory

Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of a model system with advanced tools for molecular manipulation. We recently discovered a strain of the behavior-manipulating fungal pathogen Entomophthora muscae infecting wild Drosophila, and established methods to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides and affix in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. We found that E. muscae invades the fly nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, we believe this new system will enable rapid progress in understanding the mechanistic basis of E. muscaes behavioral manipulation in the fly.

animal behavior and cognition↗

bioRxiv2017

Microbiome-by-ethanol interactions impact Drosophila melanogaster fitness and physiology

Ethanol is one of the worlds most abused drugs yet the impacts of chronic ethanol consumption are debated. Ethanol is a prevalent component in the diets of diverse animals and can act as a nutritional source, behavior modulator, and a toxin. The source of ethanol is microbes, which can both produce and degrade ethanol, and the gut microbiome has been associated with differential health outcomes in chronic alcoholism. To disentangle the various and potentially interacting roles of bacteria and ethanol on host health, we developed a model for chronic ethanol ingestion in the adult fruit fly, Drosophila melanogaster, which naturally consumes a diet between 0 and 5% ethanol. We took advantage of the tractability of the fly microbiome, which can be experimentally removed to separate the direct and indirect effects of commensal microbes. We found that moderate to heavy ethanol ingestion decreased lifespan and reproduction, without causing inebriation. These effects were more pronounced in flies lacking a microbiome, but could not be explained by simple bacterial degradation of ethanol. However, moderate ethanol ingestion increased reproduction in bacterially-colonized flies, relative to bacteria-free flies. Ethanol decreased intestinal stem cell turnover in bacterially-colonized flies and decreased intestinal barrier failure and increased fat content in all flies, regardless of microbiome status. Analysis of host gene expression finds that ethanol triggers the innate immune response, but only in flies colonized with bacteria. Taken together we show that, chronic ethanol ingestion negatively impacts fly health in a microbiome-dependent manner.

microbiology↗

bioRxiv2017

Spatially uniform establishment of chromatin accessibility in the early Drosophila embryo

As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However, it remains unclear whether open chromatin is a product of activity - transcription at promoters and patterning transcription factor binding at enhancers - or whether it is established by independent mechanisms. Recent work has implicated the ubiquitously expressed, maternal factor Zelda in this process. To assess the relative contribution of activity in the establishment of chromatin accessibility, we have probed chromatin accessibility across the anterior-posterior axis of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is remarkably similar between the two halves. Promoters and enhancers that are active in exclusively one half of the embryo have open chromatin in the other half, demonstrating that chromatin accessibility is not a direct result of activity. However, there is a small skew at enhancers that drive transcription exclusively in either the anterior or posterior half of the embryo, with greater accessibility in the region of activity. Taken together these data support a model in which regions of chromatin accessibility are defined and established by ubiquitous factors, and fine-tuned subsequently by activity.

developmental biology↗

bioRxiv2017

Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo

Evidence has emerged in recent years linking insulators and the proteins that bind them to the higher order structure of animal chromatin, but the precise nature of this relationship and the manner by which insulators influence chromatin structure have remained elusive. Here we present high-resolution genome-wide chromatin conformation capture (Hi-C) data from early Drosophila melanogaster embryos that allow us to map three-dimensional interactions to 500 base pairs. We observe a complex, nested pattern of regions of chromatin self-association, and use a combination of computational and manual annotation to identify boundaries between these topological associated domains (TADs). We demonstrate that, when mapped at high resolution, boundaries resemble classical insulators: short (500 - 1000 bp) genomic regions that are sensitive to DNase digestion and strongly bound by known insulator proteins. Strikingly, we show that for regions where the banding pattern of polytene chromosomes has been mapped to genomic position at comparably high resolution, there is a perfect correspondence between polytene banding and our chromatin conformation maps, with boundary insulators forming the interband regions that separate compacted bands that correspond to TADs. We propose that this precise, high-resolution relationship between insulators and TADs on the one hand and polytene bands and interbands on the other extends across the genome, and suggest a model in which the decompaction of insulator regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.

genomics↗