Biology subjects

Hart, C. M.

Publications and source records attributed to Hart, C. M..

4 recordsLinked to original sources

bioRxiv2026

Drosophila co-insulator proteins Pzg and Chro but not CP190 interact with promoter-proximal insulator-binding protein BEAF from a distance

Chromosomes in Drosophila melanogaster are organized into distinct topologically associated domains delimited by boundaries bound by insulator proteins. The insulator protein BEAF (Boundary Element-Associated Factor of 32kDa) plays roles in both chromatin organization and transcriptional regulation, yet its precise molecular mechanisms remain elusive. To examine the role of BEAF in insulator function we used yeast 2-hybrid assays, pull-down assays with bacterially expressed proteins, and bimolecular fluorescence complementation assays in S2 cells to characterize interactions between BEAF and three co-insulator proteins: CP190, Pzg, and Chro. Our studies pinpoint minimal regions of CP190, Pzg, and Chro that directly interact with BEAF, as well as parts of BEAF that are crucial for its interactions with these co-insulator proteins. Functional analyses in transfected S2 cells revealed distinct regulatory roles for BEAF in association with CP190, Pzg, and Chro. CP190 showed a weak interaction with BEAF, and CP190 bound 2.3 kb upstream of promoter-proximal BEAF could not loop out the intervening DNA to effectively communicate with BEAF for luciferase reporter gene activation. In contrast, more robust interactions were observed between BEAF and both Pzg and Chro. Both could also effectively interact with BEAF from a distance to activate the reporter gene. It is likely that the role of CP190 in long-range insulator interactions is mediated by insulator proteins other than BEAF, although BEAF could help stabilize interactions. On the other hand, we propose that BEAF can directly collaborate with Pzg and Chro to mediate long-range chromatin interactions.

molecular biology↗

bioRxiv2025

Characterization of core promoter activation by the Drosophila insulator-binding protein BEAF

There are distinctions between housekeeping and regulated genes in terms of core promoter motif usage and architecture. The Boundary Element-Associated Factor of 32 kDa, BEAF, was identified as a chromatin domain insulator protein that affects chromatin structure and plays a role in insulator function. Genome-wide mapping subsequently found that it usually binds near transcription start sites of housekeeping genes found at topologically associating domain (TAD) boundaries, suggesting roles in both insulator function and gene activation. This was substantiated when it was found to activate the RpS12 and aurA promoters, and that BEAF-dependent promoter activation could be separated from BEAF-dependent insulator activity. Here, we use luciferase assays after transfection of Drosophila S2 cells to show that BEAF activates housekeeping promoters without showing a preference for particular housekeeping core promoter motifs. BEAF also activates core promoters lacking motifs or with only an Inr. Regulated core promoters with a TATA box or with an Inr plus MTE or DPE or both are not activated. Activation by BEAF does not correlate with promoter basal activity. We additionally show that BEAF activates promoters synergistically with DRE or Motif 1 housekeeping promoter motifs. This establishes BEAF as an activator for a large set of promoters.

molecular biology↗

bioRxiv2024

The Tofu mutation restores female fertility to Drosophila with a null BEAF mutation

Compensatory mutations offer clues to deciphering the role of a particular protein in cellular processes. Here we investigate an unknown compensatory mutation, present in the BEAFNP6377 fly line, that provides sufficient rescue of the defective ovary phenotype caused by null BEAF alleles to allow maintenance of fly stocks lacking the chromatin domain insulator proteins Boundary Element-Associated Factors BEAF-32A and BEAF-32B. We call this mutation Tofu. We employ both classical genetics and genomic sequencing to attempt to identify the mutation. We find evidence that points to a mutation in a predicted Polycomb response element upstream of the ribbon gene, which may lead to aberrant rib expression.

genetics↗

bioRxiv2022

PPARγ/ETV2 Axis Regulates Endothelial-to-Mesenchymal Transition in Pulmonary Hypertension

Endothelial-to-mesenchymal transition (EndoMT) plays an important role in pulmonary hypertension (PH). Also, the molecular mechanisms regulating EndoMT in PH remain to be defined. In this study, we first showed that reduced expression of the transcription factors ETV2 (ETS variant 2) and PPAR{gamma} (Peroxisome Proliferator-Activated Receptor gamma) along with reduced endothelial markers and increased EndoMT markers were consistently observed in lungs and pulmonary artery endothelial cells (PAECs) of idiopathic pulmonary arterial hypertension (IPAH) patients, in hypoxia-exposed mouse lungs, human PAECs, and in induced EndoMT cells. Base on this observation, we aimed to investigate the function of ETV2 and PPAR{gamma} in EndoMT. We have explored the function of ETV2 and PPAR{gamma} and its mechanism in PH using in Etv2+/- mice or PPAR{gamma} KO mice. Etv2+/- mice spontaneously developed PH and right ventricular hypertrophy, associated with increased EndoMT markers and decreased EC markers. PPAR{gamma} transcriptionally activated the ETV2 promoter. Endothelial PPAR{gamma} expression in mice is positively correlated with ETV2 expression, but inversely with EndoMT markers. Overexpression of ETV2 in hypoxia-exposed rat pulmonary artery led to vascular relaxation. We conclude that PPAR{gamma}-ETV2 signaling can function as a novel pathway in PH pathogenesis by attenuating EndoMT.

physiology↗