Biology subjects

Rabinowitz, A.

Publications and source records attributed to Rabinowitz, A..

5 recordsLinked to original sources

bioRxiv2024

Contextualising transcription factor binding during embryogenesis using natural sequence variation

Understanding how genetic variation impacts transcription factor (TF) binding remains a major challenge, limiting our ability to model disease-associated variants. Here, we used a highly controlled system of F1 crosses with extensive genetic diversity to profile allele-specific binding of four TFs at several embryonic time-points, using Drosophila as a model. Using a combined haplotype test, we identified 9-18% of TF bound regions impacted by genetic variation. By expanding WASP (a tool for allele-specific read mapping) to examine INDELs, we increased detection of allele imbalanced (AI) peaks by 30-50%. This fine-grained mutagenesis could reconstruct functionalized binding motifs of all factors. To prioritise potential causal variants, we trained a convolutional neural network (Basenji) to predict TF binding from DNA sequence. The model could accurately predict experimental AI for strong effect variants, providing a mechanistic interpretation for how genetic variation impacted TF binding. This revealed unexpected relationships between TFs, including potential cooperative pairs, and mechanisms of tissue specific recruitment of the ubiquitous factor CTCF.

genetics↗

bioRxiv2022

New enhancer-promoter interactions are gained during tissue differentiation and reflect changes in E/P activity

To regulate gene expression, enhancers must come into proximity with their target gene. At some loci the timing of enhancer-promoter proximity is uncoupled from gene activation, while at others it is tightly linked. Here, we assessed this more globally for 600 characterized enhancers or promoters (E/P) with tissue-specific activity in Drosophila embryos, by performing Capture-C and insulator ChIP in FACS-purified myogenic or neurogenic cells at different stages of embryogenesis. This high-resolution view enabled direct comparison between E/P interactions and activity across 5 developmental conditions. This revealed largely invariant E/P contacts between the blastoderm and cell fate specification stages, despite changes in activity. However, E/P interactions diverge during terminal tissue differentiation when many tissue-specific interactions are gained on top of a pre-existing topology. Changes in E/P proximity reflect changes in enhancer activity and gene activation, and are generally not accompanied by changes in insulator binding. Using transgenes and deletions, we show that many tissue-specific interactions represent functional E-P pairs. Our results reveal a shift in E-P landscapes as embryogenesis proceeds, from largely pre-formed topologies at early stages to more distal tissue-specific loops during differentiation, when E/P proximity appears coupled to activation.

developmental biology↗

bioRxiv2022

CTCF, BEAF-32 and CP190 are not required for the initial establishment of TADs in early Drosophila embryos, but have locus specific roles

The boundaries of Topologically-Associating Domains (TADs) are delimited by insulators and active promoters, however how they are initially established during embryogenesis remains unclear. Here, we examined this during the first hours of Drosophila embryogenesis. DNA-FISH on individual embryos indicates that domains form during zygotic genome activation (ZGA), but have extensive cell-to-cell heterogeneity compared to later stages. Most newly formed boundaries are occupied by combinations of CTCF, BEAF- 32 and/or CP190. Depleting each insulator from chromatin revealed that TADs can still establish during ZGA, although with lower insulation, with particular boundaries being more sensitive. Some weakened boundaries have aberrant gene expression, however the majority of mis-expressed genes have no obvious relationship to changes in domain-boundary insulation. Deletion of an active promoter (thereby blocking transcription) at one boundary had a greater impact compared to deleting the insulator-bound region itself. These results suggest cross-talk between insulators and transcription might reinforce domain formation during embryogenesis.

developmental biology↗

bioRxiv2018

AAK1 inhibits WNT signaling by promoting clathrin-mediated endocytosis of LRP6

{beta}-catenin-dependent WNT signal transduction governs normal development and adult tissue homeostasis. Inappropriate pathway activity mediates a vast array of human diseases, including bone density disorders, neurodegeneration and cancer. Although several WNT-directed therapeutics are in clinical trials, new targets, compounds and strategies are needed. We performed a gain-of-function screen of the human kinome to identify new druggable regulators of {beta}-catenin-dependent transcription. We found that over-expression of the AP2 Associated Kinase 1 (AAK1) strongly inhibited WNT signaling. Reciprocally, silencing of AAK1 expression or pharmacological inhibition of AAK1 kinase activity using a new, selective and potent small molecule inhibitor activated WNT signaling. This small molecule is a cell active dual AAK1/BMP2K inhibitor that represents the best available tool to study AAK1-dependent signaling pathways. We report that AAK1 and the WNT co-receptor LRP6 physically co-complex and that AAK1 promotes clathrin-mediated endocytosis of LRP6. Collectively, our data support a WNT-induced negative feedback loop mediated by AAK1-driven, clathrin-mediated endocytosis of LRP6.\n\nSummary StatementA gain-of-function screen of the human kinome revealed AAK1 as a negative regulator of WNT signaling. We show that AAK1 promotes clathrin-mediated endocytosis of LRP6, resulting in downregulation of WNT signaling. We use a new selective and potent AAK1/BMP2K small molecule probe to validate our findings.

cell biology↗

bioRxiv2017

Activity-Dependent Control Of Inhibitory Interneuron Number In The Mammalian Cortex

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic, cell type specific and can be reduced in a cell autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

neuroscience↗