Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells

Abstract

Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K d ) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.fluorescence correlation spectroscopy | single-molecule sensitivity | homeodomain | Sex combs reduced | synthetic peptides T ranscription factor-DNA interactions, the assembly of functional transcriptional complexes, and the mechanisms involved in target site recognition are central to understanding gene regulation in vivo. In spite of such relevance, kinetic studies of transcription factor-DNA interactions are limited and have been performed only for a handful of proteins. Most prominent studies include the lac repressor in Escherichia coli (1-4), nuclear receptor-directed transcription (5), helicase translocation along DNA (6), and restriction enzymes-DNA interactions (7,8). The majority of these studies are carried out with naked DNA or reconstituted chromatin, reflecting the in vivo situation only to a limited extent. Chromatin structure in live cells is highly dynamic, involving multiple interactions and transformations (9)(10)(11). Moreover, a number of cofactors and coactivators/corepressors are involved in transcriptional regulation, suggesting that gene expression is accomplished through a dynamic, spatiotemporally entangled interplay between concomitant processesprotein searching for specific target sites, rearrangements of DNA conformation, and recruitment of cofactors and/or coregulators (5, 12, 13). Such complexity is not easily mimicked in solution, therefore necessitating live cell experimentation. However, these measurements are complex, and kinetic studies of protein-DNA interactions in live cells remain scarce (3,4,14,15), mainly due to the limited number of experimental approach...

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

“…Thus, D-values for HMG-17, SF2/ASF, and fibrillarin are about 20-40 times lower than values reported for free solutes in the nucleus or for GFP alone indicating that interactions of these proteins with nuclear components slow down the diffusion. Similar results are observed for Src peptides [50], where interactions with DNA reduced significantly the diffusion coefficients measured…”

“…Only recently, the kinetics of transcription factors have been studied in eukaryotes [45,46,50,107] revealing basic features of the facilitated diffusion of transcription factors in nuclei. The striking feature, as compared with prokaryotes, is a proportion of the time spent in 3D translocation and in interaction with non-specific DNA during the search process.…”

“…The studies of the crowding effects on particular steps of gene expression are concentrated on in vitro systems [102,103] with crowding agents and comparison with dilute solutions. There is a growing number of experimental studies concerning transcription factor kinetics in bacteria [104][105][106] and eukaryotes [45,46,50,107].…”

Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells

“…There are many applications of FCS for protein dynamics in single living cells [19,20]; however, the total amount of functional protein in a single cell is difficult to estimate because of photobleaching of fluorescent proteins and their heterogenous distribution. In contrast, the FCS-microwell system can be used to isolate the cell lysate from a single cell and total amount of functional protein can be determined without photobleaching.…”

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy

“…This phenomenon can lead to toxic chemical species and has largely prevented studies of protein diffusion in mammalian cells deep inside organisms, especially for proteins, such as TFs that are typically expressed at high levels 8,18 . Photobleaching has been minimized in culture conditions by selecting transfected cells that express minimal levels of labelled proteins 14,17 , using high-laser intensity to photobleach some of the initial fraction of fluorescent molecules 17 , or using heat-shock controllable promoters in Drosophila 19 . However, these approaches cannot be readily applied in developing mammalian embryos and ideally, FCS experiments should be independent of protein expression levels.…”