Single molecule biophysics in living cells
Binding of a transcription factor to a regulatory sequence on DNA is the first step in gene expression, accompanied by binding of different cofactors and enzymes, assembly of the transcription complex and final read-out of the genetic information stored in genomic DNA. Interestingly, the interactions between initiating factors and DNA are short-lived and stochastic. This raises questions about the temporal coordination between binding events as well as the coupling between transcription factor binding and the onset of transcription:
How does the cell carry forward the information of a first binding event to eventual expression of a gene?
How do cofactors influence the temporal sequence?
Does binding of a transcription factor initiate an independently running process, or is continuous transcription accompanied by repeated transcription factor binding?
We approach these intriguing questions by following individual biomolecules involved in gene expression in their natural environment of a living cell and study their temporal interplay with other components of transcription using multi color single molecule fluorescence microscopy.
→ Chromatin Architecture
Cell-cycle-dependent interactions between CTCF and Chromatin
The three-dimensional arrangement of chromatin encodes regulatory traits important for nuclear processes such as transcription and replication. Chromatin topology is in part mediated by the architectural protein CCCTC-binding factor (CTCF) that binds to the boundaries of topologically associating domains. Whereas sites of CTCF interactions are well characterized, little is known on how long CTCF binds to chromatin and how binding evolves during the cell cycle. We monitored CTCF-chromatin interactions by live cell single molecule tracking in different phases of the cell cycle. In G1-, S-, and G2-phases, a majority of CTCF molecules was bound transiently (∼0.2 s) to chromatin, whereas minor fractions were bound dynamically (∼4 s) or stably (>15 min). During mitosis, CTCF was mostly excluded from chromatin. Our data suggest that CTCF scans DNA in search for two different subsets of specific target sites and provide information on the timescales over which topologically associating domains might be restructured. During S-phase, dynamic and stable interactions decreased considerably compared to G1-phase, but were resumed in G2-phase, indicating that specific interactions need to be dissolved for replication to proceed.
→ Transcription Regulation
Facilitated diffusion in a network of binding sites
The response time of a gene and its transcription level are vital parameters of its regulatory function. We present a model of transcription factor (TF) target site search which accounts for both of these parameters. We find that the criterion of a high transcription level constraints the optimization of the response time in a search process of facilitated diffusion. This constraint strongly depends on the specific binding time. Next, we consider the optimal TF search process in the presence of further species. By discussing a gene activated by two dimerizing species we find that specific binding sites on DNA may speed up protein dimerization by more than an order of magnitude. If a competitor is present, unspecific and specific binding times of the competitor influence the optimal response time and the transcription level of a gene. Finally, we find that facilitated dissociation of the TF occurring in a crowded environment stabilizes the transcription level of the gene with respect to variations in the unspecific binding time of the TF.
DNA residence time is a regulatory factor of transcription repression
Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation.
Live cell single-molecule imaging of transcription factor binding to DNA
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.
Super-resolved spatial organization of RNA polymerase II
Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.