Biology subjects

Jaynes, J. B.

Publications and source records attributed to Jaynes, J. B..

6 recordsLinked to original sources

bioRxiv2026

Sequence-to-function modeling uncovers the context-specific grammar of Drosophila chromatin insulation

Chromatin is organized into self-interacting topologically associating domains partitioned by boundary elements that insulate adjacent domains and restrict regulatory interactions. Yet, how sequence context and combinations of factors dictate boundary strength remains incompletely understood. Here we present Domino, a deep learning framework that maps genomic sequences to quantitative insulation scores defined directly from single-nucleosome resolution Drosophila melanogaster Micro-C data. Unlike traditional transcription factor motif scanning, Domino captures broad sequence context to resolve the functional contributions of individual sequence elements. We validate model predictions through experimental perturbations of insulator sequences. Model interpretation yields insulation-associated motifs genome-wide. Across 7,311 embryonic boundaries, Domino reveals a comprehensive insulation grammar defined by just 24 primary motifs that account for 59% of the boundaries, with an average of only two motifs per motif-containing boundary. Beyond known factors, we identify the zinc-finger proteins Trem, CG4854 and CG17385 as previously unreported insulation factors. We uncover distance- and orientation-dependent motif synergy, including a strict orientation preference of the prominent architectural factor M1BP. Finally, Domino traces tissue-specific shifts in the insulator landscape from the embryo to larval and adult brains, nominating new brain-specific insulation motifs. In sum, Domino provides a generalizable framework for decoding the regulatory logic of 3D genome architecture.

genomics↗

bioRxiv2025

homie and nhomie insulators compete with each other for interaction with distant copies, affecting enhancer-promoter interactions and eve function

Chromatin insulators, a.k.a. boundaries, separate regions of the chromosome with distinct chromatin characteristics, including distinct histone modifications. This activity affects gene expression by allowing chromatin domains to be stably regulated and maintained. They also block enhancer-promoter interactions and, somewhat paradoxically, can facilitate other enhancer-promoter interactions, particularly when they stitch together distant regions of the chromosome by pairing with specific insulator partners. Here we explore how long-range interactions facilitated by insulator pairing are affected by the presence of two specifically interacting partners. Our results show that when two partners are present, they compete, reducing each others effects, suggesting that interactions tend to be limited to two interacting partners at any given time. In particular, when a distant transgenic copy of an eve insulator (homie or nhomie) is present, it can interact with either endogenous insulator. But when one endogenous insulator is removed, the remaining one interacts more strongly with the transgenic copy, biasing the induced enhancer-promoter interactions toward those nearest the remaining endogenous insulator. We also show that removing one or both endogenous eve insulators significantly reduces endogenous eve function at a critical early stage of development, and that the eve Polycomb domain expands in both directions when its insulator boundaries are removed.

genetics↗

bioRxiv2024

The homie insulator has sub-elements with different insulating and long-range pairing properties

Chromatin insulators are major determinants of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers and promoters interact over long distances and between homologous chromosomes. Insulators can pair with copies of themselves in trans to facilitate homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops. Contrary to their canonical function of enhancer blocking, these loops can bring distant enhancers and promoters together to activate gene expression, while at the same time blocking other interactions in cis. The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3-dimensional architecture as a major functional determinant. Here we dissect the homie insulator from the Drosophila even skipped (eve) locus, to understand its substructure. We test pairing function based on homie-carrying transgenes interacting with endogenous eve. The assay is sensitive to both pairing strength and orientation. Using this assay, we found that a Su(Hw) binding site in homie is required for efficient long-range interaction, although some activity remains without it. This binding site also contributes to the canonical insulator activities of enhancer blocking and barrier function. Based on this and other results from our functional dissection, each of the canonical insulator activities, chromosomal loop formation, enhancer blocking, and barrier activity, are partially separable. Our results show the complexity inherent in insulator functions, which can be provided by an array of different proteins with both shared and distinct properties. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/578481v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@243157org.highwire.dtl.DTLVardef@234cfborg.highwire.dtl.DTLVardef@193012borg.highwire.dtl.DTLVardef@31d7e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Chromosome Structure II: Stem-loops and circle-loops

The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements:{lambda} DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: In MicroC experiments, the eve TAD is a "volcano" triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The{lambda} DNA replacement lacks boundary function: the endpoint of the "new" eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is instead flanked by a pair of "clouds" of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.

genetics↗

bioRxiv2023

Chromosome Structure I: Loop extrusion or boundary:boundary pairing?

Two different models have been proposed to explain how the endpoints of chromatin looped domains ("TADs") in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the "loop-extrusion" and the "boundary-pairing" models. Our findings are incompatible with the loop-extrusion model and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.

genetics↗

bioRxiv2017

Using competition assays to quantitatively model cooperative binding by transcription factors and other ligands

BACKGROUNDThe affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations.\n\nMETHODSWe use standard computational and mathematical methods, and develop novel methods as described in Results.\n\nRESULTSWe explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive expressions for the equilibria among the various complexes, and explore the limitations of binding experiments that model the system using a single parameter. We describe how to use single-ligand binding and ternary complex formation in tandem to determine parameters that have thermodynamic relevance. We develop an improved method for finding both single-ligand dissociation constants and concentrations simultaneously. We show how the cooperativity factor can be found when only one of the single-protein dissociation constants can be measured.\n\nCONCLUSIONSThe methods that we develop constitute an optimized approach to accurately model cooperative binding.\n\nGENERAL SIGNIFICANCEThe expressions and methods we develop for modeling and analyzing DNA binding and cooperativity are applicable to most cases where multiple ligands bind to distinct sites on a common substrate. The parameters determined using these methods can be fed into models of higher-order cooperativity to increase their predictive power.\n\nHIGHLIGHTSO_LIHill plots remain prominent in biology, but can mask cooperativity\nC_LIO_LIEffective modeling of binding by two ligands requires the use of 3 parameters\nC_LIO_LIWe develop novel ways to find these parameters for two cooperating ligands\nC_LIO_LIWe show how they can be used to enhance the power of established methods\nC_LIO_LIWe describe how this framework can be extended to multiple cooperating ligands\nC_LI

biochemistry↗