Biology subjects

Recchia, D. C.

Publications and source records attributed to Recchia, D. C..

3 recordsLinked to original sources

bioRxiv2025

A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

The dysregulation of bromodomain proteins, a family of "reader" proteins that recognize the critical post-translational modification of acylation, is implicated in diseases like cancer, making them important therapeutic targets. However, the development of specific small-molecule inhibitors is hindered by the lack of robust, high-throughput cellular assays to measure target engagement and off-target binding in living cells. To address this gap, we developed a modular platform of cell lines that stably express synthetic chromatin reader constructs, termed Acyl-eCRs, containing various bromodomains fused to eGFP. We demonstrate that these Acyl-eCRs recapitulate the same response to bromodomain inhibitors and PROTACs as endogenous proteins, allowing for the quantitative assessment of drug effects. We introduce two complementary flow cytometry-based assays to evaluate inhibitor-target engagement: a competitive binding assay leveraging PROTAC-induced degradation, and a nuclear retention assay that directly measures the displacement of bromodomains from chromatin. Our approach circumvents the need for laborious protein purification and in vitro characterization, providing a scalable and physiologically relevant method for assessing inhibitor potency and specificity. This platform represents a versatile tool for chemical biology, enabling the functional evaluation of chromatin-targeting drugs in a native cellular context.

molecular biology↗

bioRxiv2024

Probing DNA damage sites reveals context-dependent and novel DNA damage response factors

DNA damage is a constant threat to genome integrity and function. Diminished capacity for DNA repair is linked to many human diseases, therefore understanding the molecular pathways responding to DNA damage is key for developing novel therapies. Lack of unbiased probes to report DNA damage dynamics and the associated proteins in living cells and animals limit our current efforts to completely understand DNA repair processes. In this study we overcome these limitations by engineering protein probes containing the tandem-BRCT domain of MCPH1, which we show to have a specific affinity for the DNA-damage-associated histone mark {gamma}H2AX. We employ these probes to track DNA damage dynamics in living cells exposed to a panel of different genotoxic insults and to visualize programmed double strand breaks during gametogenesis in living animals. We further utilize the binding selectivity of our probe to tether TurboID biotin ligases to chromatin and identify the DNA damage-associated proteome via proximity ligation. By comparing five different DNA damaging agents, we reveal the proteome associated with specific lesions, and identify multiple novel proteins with potential implications in damage response and repair. Among these novel proteins, we characterize the ubiquitin ligase UBE3A, the methyl-binding and proteasome-recruiting protein L3MBTL3, and the spliceosomal factor U2SURP, as previously uncharacterized effectors of DNA damage response. These functional datasets reveal the DNA damage-dependent proteomes and reveal novel insights into DNA damage response.

molecular biology↗

bioRxiv2024

SETD2 promotes PAF1C interactions with the elongating RNA Pol II and is required for neuronal differentiation

Chromatin modifications are relevant for mammalian development and their aberrant deposition is associated with human disease. While the mechanisms that deposit and remove these modifications have been largely elucidated, their role in regulating gene activity during cellular differentiation have yet to be completely understood. By differentiating a panel of mouse embryonic stem cells lacking major chromatin regulators towards neuronal cells, we identified their requirement at different stages of cellular differentiation. We show that the H3K36me3 methyltransferase SETD2 is important for the establishment of neuronal gene expression during late stages of differentiation, but is dispensable once the cells have fully differentiated. This function is largely independent of the histone methyltransferase activity. By measuring the protein interaction network of elongating RNA Pol II, we identify a novel role for SETD2 in mediating interactions between the PAF1 complex and the elongating RNA Pol II, which is required to ensure optimal transcriptional processivity of neuronal genes.

molecular biology↗