Biology subjects

Hoffmeister, P.

Publications and source records attributed to Hoffmeister, P..

2 recordsLinked to original sources

bioRxiv2023

In vivo binding free energy landscape reveals kinetic control of transcription factor function

Transcription factors (TFs) such as the central DNA-binding hub in Notch signal transduction, RBPJ, bind to specific DNA sequences to regulate gene transcription. How the efficiency of gene regulation depends on the TF-DNA binding kinetics and cofactor interactions is mostly unknown. We determined the DNA binding kinetics and the transcriptional activity of RBPJ and several mutant variants by live-cell single-molecule tracking and reporter assays, and measured their genome-wide chromatin occupation by ChIP-Seq. We observed that cofactor binding, in addition to DNA binding, was required for target site specificity. Importantly, the target site search time of RBPJ was longer than its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired DNA binding, e.g. by mutation K195E related to Adams-Oliver-Syndrome, modulated not only dissociation, but also association to target sites. Impaired cofactor binding mainly altered the rates of unspecific binding and target site association. For other TFs, we also observed longer search than residence times, indicating that kinetic rather than thermodynamic stability of DNA binding might be a general feature of TFs in vivo. We propose that an effective in vivo binding energy landscape of TF-DNA interactions constitutes an instructive visualization of TF-DNA binding kinetics and the changes upon mutations.

biophysics↗

bioRxiv2021

A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

genetics↗