Navigating the crowd: visualizing coordination between genome dynamics, structure, and transcription

Abstract

The eukaryotic genome is hierarchically structured yet highly dynamic. Regulating transcription in this environment demands a high level of coordination to permit many proteins to interact with chromatin fiber at appropriate sites in a timely manner. We describe how recent advances in quantitative imaging techniques overcome caveats of sequencing-based methods (Hi-C and related) by enabling direct visualization of transcription factors and chromatin at high resolution, from single genes to the whole nucleus. We discuss the contribution of fluorescence imaging to deciphering the principles underlying this coordination within the crowded nuclear space in living cells and discuss challenges ahead.

Fig. 1

Advances in imaging techniques allow rich information about chromatin beyond Hi-C. Chromatin is constantly remodeled during time (top to bottom), which is illustrated on the basis of a short polymer which changes its configuration over time (left). A single-cell Hi-C-like type of data over time of this polymer would reveal relatively few contacts at each time point for single cells as the technique relies on proximity ligation (middle column). In contrast, imaging offers the determination of actual positions and distances between any two loci in three dimensions and thus reveals a more complete picture than “C” methods (right column). The illustrative maps were created by tracing the contour of the polymer shown on the left and computing the pairwise distance between any two loci which is shown in the imaging-like matrix. The Hi-C map is a thresholded version of the distance map and shows contacts only at small spatial distances. Yet, note that there may be a broad distance distribution underlying measured Hi-C contacts [36], and as such, the illustration is highly simplified. The Hi-C map is a thresholded version of the distance map and shows contacts only at small spatial distances. While imaging chromatin at many loci simultaneously is currently, with a few exceptions, done in fixed cells, it has the potential to advance toward analysis in living cells in the future. However, a single-cell time evolution of chromatin structure by Hi-C cannot be obtained since Hi-C is a destructive method

Fig. 2

Labeling and imaging strategies to image chromatin. Conventional labeling using stably expressed fluorescent proteins or organic dyes usually covers the whole genome unspecifically (modality 1). Due to the high density of chromatin, only a spatial resolution well above the diffraction limit can be achieved, while fast imaging is generally possible. Super-resolution imaging of chromatin overcomes the diffraction limit but is challenging since chromatin in vivo is not well structured (modality 2). Usually, resolution is gained for the expense of acquisition time and thus cells must be fixed. While conventional and super-resolution whole-genome labeling is sequence-unspecific, single loci can be targeted using fluorescence in situ hybridization (FISH; in case of fixed cell imaging), ANCHOR, a CRISPR/Cas9 system, or others (modality 3). Single locus labeling can be done in fixed as well as in living cells. To cover an extended region of chromatin for which sequence information is available, a sequential FISH or oligopaint strategy can be followed in fixed cells (modality 4). Using oligonucleotides which are designed for the specific genomic region, the chromatin configuration is sampled by sequential hybridization rounds and computationally reconstructed

Fig. 3

Mechanisms for chromatin organization and dynamics during transcription. (A) The transcriptional hubs are formed by liquid-liquid phase separation of transcription factors, which in turn is mediated by the local crowding conditions. TFs binding to both enhancer and promoter mediate enhancer-promoter contacts by an effective attraction exerted by the LLPS property of transcription factories. Once transcription is initiated by these enhancer-promoter contacts, transcription elongation proceeds by reeling the transcribed gene body along the transcription factory. Loop extrusion by cohesin can additionally establish transient enhancer-promoter contacts, and the turnover time of cohesin and CTCF regulates the frequency of these contacts. Loop extrusion dynamics and the placement of (semi-) permeable border elements may thus regulate transcription. (B) The nucleus is sprinkled with transcription factories to which chromatin is tethered. The resulting network of transcriptional hubs restricts chromatin motion and induces a stiffness to chromatin, which is expressed in long-range correlations of chromatin dynamics (colored arrows). (C) The local molecular crowding reduction of chromatin mobility upon transcription. This transition is associated to high molecular crowding, eventually facilitating the formation of transcription factories to which chromatin is tethered