The Self-Organizing Genome: Principles of Genome Architecture and Function.

Pioneer transcription factors have the intrinsic biochemical ability to scan partial DNA sequence motifs that are exposed on the surface of a nucleosome and thus access silent genes that are inaccessible to other transcription factors. Pioneer factors subsequently enable other transcription factors, nucleosome remodeling complexes, and histone modifiers to engage chromatin, thereby initiating the formation of an activating or repressive regulatory sequence. Thus, pioneer factors endow the competence for fate changes in embryonic development, are essential for cellular reprogramming, and rewire gene networks in cancer cells. Recent studies with reconstituted nucleosomes in vitro and chromatin binding in vivo reveal that pioneer factors can directly perturb nucleosome structure and chromatin accessibility in different ways. This review focuses on our current understanding of the mechanisms by which pioneer factors initiate gene network changes and will ultimately contribute to our ability to control cell fates at will.

Pioneer Transcription Factors Initiating Gene Network Changes

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

Structure and mechanism of the RNA polymerase II transcription machinery.

What Is a Transcriptional Burst

Differential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal cis-regulatory sequences that govern spatiotemporal and quantitative expression dynamics of target genes. Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.

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Mechanisms of enhancer action: the known and the unknown

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

/pdf/live-cell-imaging-reveals-the-interplay-between-1snbfjh38t.pdf

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

/pdf/single-molecule-imaging-reveals-the-interplay-between-3zowr8trsh.pdf

Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

We present a microscopy technique, orbital particle tracking, in which the scanner scans orbits around species, unlike a raster imaging technique in which the scanner scans an area one line at a time. By analyzing the fluorescence emission intensity variation along an orbit, the location of a species in the orbit can be determined with precision of a tenth of a nanometer in a millisecond time scale, and the orbit can be moved to the new location of the species through a feedback loop if any movement is detected. This technique can be extended to two scanning orbits, one above and one below the sample plane to track the sample in 3D space. It can be used in vitro or in vivo to track a motion of a sample or to understand the dynamics of the sample. Additional detectors can help reveal the correlation between events with different emission spectrums. We have performed two different experiments with the system to show the capability of the technique. In the first example, we track a transcription site to understand the relationship between transcription factor - DNA binding and RNA transcription [1, 2]. By labeling a transcription factor with Halo-JF646 and nascent RNA with PP7-GFP, we were able to cross correlate fluorescence intensity to discover temporal coordination between transcription factor DNA binding and resulting gene activation. In the second experiment, we tracked lysosomes in live cells to understand the nature of the transport whether it is an active transport or a free diffusion [3]. Trajectories of a total of 24 lysosomes are recorded during the experiment. The mean squared displacement (MSD) curves of the trajectories showed some clear differences between the behaviors of the lysosomes which were attributed to the active transport along microtubules as opposed to freely diffusing lysosomes.

Anh Huynh

Papers

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility

Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

Nano-Resolution in Vivo 3D Orbital Tracking System to Study Cellular Dynamics and Bio-Molecular Processes