Biology subjects

Duffy, T.

Publications and source records attributed to Duffy, T..

3 recordsLinked to original sources

bioRxiv2025

Discovery of a VHL molecular glue degrader of GEMIN3 by Picowell RNA-seq

Targeted protein degradation (TPD) is an emerging therapeutic modality in which small molecules are used to recruit targets to the natural protein degradation machinery of the cell. Molecular glue degraders (MGD) are monovalent small molecules that accomplish this by redirecting E3 ubiquitin ligases to target proteins, offering the potential to degrade previously unliganded and "undruggable" proteins in cancer, neurodegenerative, and other diseases. While attractive due to their drug-like properties, MGDs are exceptionally hard to discover and have largely been identified serendipitously. The Von Hippel-Lindau (VHL) E3 ligase is the second most widely used effector for TPD, though current VHL-based degraders are primarily large heterobifunctional PROTACs (proteolysis-targeting chimeras) designed using target-based ligands. Here, we have instead pursued target-agnostic discovery of VHL MGDs leveraging proprietary ultra-miniaturized microfluidics devices (Picowells) to facilitate unbiased RNA-seq screening of a biased E3-focused library. This resulted in dGEM3, a novel VHL molecular glue that targets the survival of motor neuron (SMN) complex member GEMIN3 for degradation. Through a combination of cellular, biochemical, and biophysical assays, we have characterized the GEMIN3 degron within its helicase ATP-binding domain, and how the kinetics of ternary complex formation impact degradation. These findings provide insights on the re-programmability of VHL for novel targets using drug-like molecular glues.

biochemistry↗

bioRxiv2019

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation

O_LIPlants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24-hour period, enabling the anticipation of these events. C_LIO_LIWe used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. C_LIO_LIApproximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore we identified sequence motifs enriched in the promoters of similarly-phased genes, some potentially associated with transcription factors. C_LIO_LIWhen considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species. C_LI

plant biology↗

bioRxiv2018

Nuclear receptor HNF4A trans-represses CLOCK:BMAL1 and acts as a core component of tissue-specific circadian networks

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly trans-represses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed co-occupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose and amino acid homeostasis. Taken together, we establish that HNF4A defines a novel feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.\n\nSignificanceInterlocked feedback loops promote robustness and stability in a system and are a feature of circadian clocks in both animal and plants. The mammalian circadian clock is known to consist of two transcriptional feedback loops, relying on the transcriptional activity of the master complex CLOCK:BMAL1 and the feedback regulation by its target genes. Our research extends this knowledge by establishing a novel feedback loop in peripheral circadian oscillators and highlights the underlying mechanisms mediated by the unappreciated CLOCK:BMAL1 trans-repression activity of the circadian nuclear receptor HNF4A.

molecular biology↗