Using synthetic genome readers/regulators to interrogate chromatin processes: A brief review.

Aberrant gene expression underlies numerous human ailments. Hence, developing small molecules to target and remedy dysfunctional gene regulation has been a long-standing goal at the interface of chemistry and medicine. A major challenge for designing small molecule therapeutics aimed at targeting desired genomic loci is the minimization of widescale disruption of genomic functions. To address this challenge, we rationally design polyamide-based multi-functional molecules, i.e., Synthetic Genome Readers/Regulators (SynGRs), which, by design, target distinct sequences in the genome. Herein, we briefly review how SynGRs access chromatin-bound and chromatin-free genomic sites, then highlight the methods for the study of chromatin processes using SynGRs on positioned nucleosomes in vitro or disease-causing repressive genomic loci in vivo.

Domain-Selective BET Ligands Yield Next-Generation Synthetic Genome Readers/Regulators with Nonidentical Cellular Functions.

SynTEF1, a prototype synthetic genome reader/regulator (SynGR), was designed to target GAA triplet repeats and restore the expression of frataxin (FXN) in Friedreich's ataxia patients. It achieves this complex task by recruiting BRD4, via a pan-BET ligand (JQ1), to the GAA repeats by using a sequence-selective DNA-binding polyamide. When bound to specific genomic loci in this way, JQ1 functions as a chemical prosthetic for acetyl-lysine residues that are natural targets of the two tandem bromodomains (BD1 and BD2) in bromo- and extra-terminal domain (BET) proteins. As next-generation BET ligands were disclosed, we tested a select set with improved physicochemical, pharmacological, and bromodomain-selective properties as substitutes for JQ1 in the SynGR design. Here, we report two unexpected findings: (1) SynGRs bearing pan-BET or BD2-selective ligands license transcription at the FXN locus, whereas those bearing BD1-selective ligands do not, and (2) rather than being neutral or inhibitory, an untethered BD1-selective ligand (GSK778) substantively enhances the activity of all active SynGRs. The failure of BD1-selective SynGRs to recruit BRD4/BET proteins suggests that rather than functioning as "epigenetic/chromatin mimics," active SynGRs mimic the functions of natural transcription factors in engaging BET proteins through BD2 binding. Moreover, the enhanced activity of SynGRs upon cotreatment with the BD1-selective ligand suggests that natural transcription factors compete for a limited pool of nonchromatin-bound BET proteins, and blocking BD1 directs pan-BET ligands to more effectively engage BD2. Taken together, SynGRs as chemical probes provide unique insights into the molecular recognition principles utilized by natural factors to precisely regulate gene expression, and they guide the design of more sophisticated synthetic gene regulators with greater therapeutic potential.

CueR activates transcription through a DNA distortion mechanism.

The MerR-family transcription factors (TFs) are a large group of bacterial proteins responding to cellular metal ions and multiple antibiotics by binding within central RNA polymerase-binding regions of a promoter. While most TFs alter transcription through protein-protein interactions, MerR TFs are capable of reshaping promoter DNA. To address the question of which mechanism prevails, we determined two cryo-EM structures of transcription activation complexes (TAC) comprising Escherichia coli CueR (a prototype MerR TF), RNAP holoenzyme and promoter DNA. The structures reveal that this TF promotes productive promoter-polymerase association without canonical protein-protein contacts seen between other activator proteins and RNAP. Instead, CueR realigns the key promoter elements in the transcription activation complex by clamp-like protein-DNA interactions: these induce four distinct kinks that ultimately position the -10 element for formation of the transcription bubble. These structural and biochemical results provide strong support for the DNA distortion paradigm of allosteric transcriptional control by MerR TFs.

The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription.

The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αßß'ωε) RNA Polymerase (RNAP) core enzyme, σA, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the -35 element to the -10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal -35/-10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy.

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as 'zinc sparks.' The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as 'hardening', which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using X-ray fluorescence microscopy, transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. This adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.

TRANSCRIPTION. Allosteric transcriptional regulation via changes in the overall topology of the core promoter.

Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-form DNA-like conformation. These factors switch on and off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.

Zinc, insulin, and the liver: a ménage à trois.

Insulin and Zn2+ enjoy a multivalent relationship. Zn2+ binds insulin in pancreatic ß cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological signals such as increased blood glucose. This transition metal is an essential cofactor in insulin-degrading enzyme and several key Zn2+ finger transcription factors that are required for ß cell development and insulin gene expression. Studies are increasingly revealing that fluctuations in Zn2+ concentration can mediate signaling events, including dynamic roles that extend beyond that of a static structural or catalytic cofactor. In this issue of the JCI, Tamaki et al. propose an additional function for Zn2+ in relation to insulin: regulation of insulin clearance from the bloodstream.