Mechanisms governing target search and binding dynamics of hypoxia-inducible factors

Abstract

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, another main determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches we also confirmed IDR-dependent binding and activation of a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.

Transcription factor clusters enable target search but do not contribute to target gene activation

“…Both these regions have been predicted and shown to contain disordered regions 15,58,59 (Figure S5A). Since IDRs have previously been linked to cluster formation and target search 13,24,25 , we wondered how the CR and AD of Gal4 contribute to cluster formation and localization.…”

TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1α axis in melanoma

“…S3 ). Interestingly, TIPE can dramatically activate the hypoxia response element(HRE) reporter activity, with which HIF-1α is more prone to recognize the specific DNA motif to regulate metabolic gene transcription(Chen et al, 2022b)( Fig. 1L ).…”

Single-molecule tracking (SMT): a window into live-cell transcription biochemistry

“…An approach recently developed in our lab relies on Bayesian inference of posterior probabilities over a large two-dimensional array of states with different values of diffusion coefficient and localization error. Discrete populations are distinguishable as peaks in the resulting probability distribution [68][69][70].…”