A quantitative metric of pioneer activity reveals that HNF4A has stronger in vivo pioneer activity than FOXA1.

BACKGROUND: We and others have suggested that pioneer activity - a transcription factor's (TF's) ability to bind and open inaccessible loci - is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci. RESULTS: Here, we present a quantitative in vivo measure of pioneer activity that captures the relative difference in a TF's ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure the binding of doxycycline-inducible TFs. For each location across the genome, we determine the concentration of doxycycline required for a TF to reach half-maximal occupancy; lower concentrations reflect higher affinity. We propose that the relative difference in a TF's affinity between ATAC-seq labeled accessible and inaccessible binding sites is a measure of its pioneer activity. We estimate binding affinities at tens of thousands of genomic loci for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has stronger pioneer activity than FOXA1. We show that both FOXA1 and HNF4A display higher binding affinity at inaccessible sites with more copies of their respective motifs. The quantitative analysis of binding suggests different modes of binding for FOXA1, including an anti-cooperative mode of binding at certain accessible loci. CONCLUSIONS: Our results suggest that relative binding affinities are reasonable measures of pioneer activity and support the model wherein most TFs have some degree of context-dependent pioneer activity.

A test of the pioneer factor hypothesis using ectopic liver gene activation.

The pioneer factor hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (non-PFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and non-PF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and 'pioneered' for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results, we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.

Cells only use a fraction of their genetic information to make the proteins they need. The rest is carefully packaged away and tightly bundled in structures called nucleosomes. This physically shields the DNA from being accessed by transcription factors ­ the molecular actors that can read genes and kickstart the protein production process. Effectively, the genetic sequences inside nucleosomes are being silenced. However, during development, transcription factors must overcome this nucleosome barrier and activate silent genes to program cells. The pioneer factor hypothesis describes how this may be possible: first, 'pioneer' transcription factors can bind to and 'open up' nucleosomes to make target genes accessible. Then, non-pioneer factors can access the genetic sequence and recruit cofactors that begin copying the now-exposed genetic information. The widely accepted theory is based on studies of two proteins ­ FOXA1, an archetypal pioneer factor, and HNF4A, a non-pioneer factor ­ but the predictions of the pioneer factor hypothesis have yet to be explicitly tested. To do so, Hansen et al. expressed FOXA1 and HNF4A, separately and together, in cells which do not usually make these proteins. They then assessed how the proteins could bind to DNA and impact gene accessibility and transcription. The experiments demonstrate that FOXA1 and HNF4A do not necessarily follow the two-step activation predicted by the pioneer factor hypothesis. When expressed independently, both transcription factors bound and opened inaccessible sites, activated target genes, and 'pioneered' for each other. Similar patterns were observed across the genome. The only notable distinction between the two factors was that FOXA1, the archetypal pioneering factor, required fewer copies of its target sequence to bind DNA than HNF4A. These findings led Hansen et al. to propose an alternative theory to the pioneer factor hypothesis which eliminates the categorical distinction between pioneer and non-pioneer factors. Overall, this work has implications for how biologists understand the way that transcription factors activate silent genes during development.

Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

Structural insights into peptide bond formation.

The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-A resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid-biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The alpha-NH(2) group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2' OH of the A-76 ribose in the P site or with the 2' OH of A2486 (2451). These interactions position the alpha amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.

The structures of four macrolide antibiotics bound to the large ribosomal subunit.

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.

A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.