Manganese-dependent transcription regulation by MntR and PerR in Thermus thermophilus HB8.

Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA-binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA-binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies in Thermus thermophilus HB8. By homology search, we classify the DtxR homolog as a manganese-specific, MntR (TtMntR), and the FUR homolog as a peroxide-sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes in vivo, and TtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We show TtPerR and TtMntR bind DNA in the presence of manganese in vitro and in vivo; however, TtPerR is unable to bind DNA in the presence of iron, likely due to iron-mediated histidine oxidation. Unlike canonical PerR homologs, TtPerR does not appear to contribute to peroxide detoxification. Instead, the TtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation.

A CsoR family transcriptional regulator, TTHA1953, controls the sulfur oxidation pathway in Thermus thermophilus HB8.

Transcription regulation is a critical means by which microorganisms sense and adapt to their environments. Bacteria contain a wide range of highly conserved families of transcription factors that have evolved to regulate diverse sets of genes. It is increasingly apparent that structural similarities between transcription factors do not always equate to analogous transcription regulatory networks. For example, transcription factors within the copper-sensing operon repressor (CsoR)-resistance to cobalt and nickel repressor family have been found to repress a wide range of gene targets, including various metal efflux genes, as well as genes involved in sulfide and formaldehyde detoxification machinery. In this study, we identify the preferred DNA-binding sequence for the CsoR-like protein, TTHA1953, from the model extremophile Thermus thermophilus HB8 using the iterative selection approach, restriction endonuclease, protection, selection, and amplification. By mapping significant DNA motifs to the T. thermophilus HB8 genome, we identify potentially regulated genes that we validate with in vitro and in vivo methodologies. We establish TTHA1953 as a master regulator of the sulfur oxidation pathway, providing the first link between CsoR-like proteins and Sox regulation.

SWAN pathway-network identification of common aneuploidy-based oncogenic drivers.

Haploinsufficiency drives Darwinian evolution. Siblings, while alike in many aspects, differ due to monoallelic differences inherited from each parent. In cancer, solid tumors exhibit aneuploid genetics resulting in hundreds to thousands of monoallelic gene-level copy-number alterations (CNAs) in each tumor. Aneuploidy patterns are heterogeneous, posing a challenge to identify drivers in this high-noise genetic environment. Here, we developed Shifted Weighted Annotation Network (SWAN) analysis to assess biology impacted by cumulative monoallelic changes. SWAN enables an integrated pathway-network analysis of CNAs, RNA expression, and mutations via a simple web platform. SWAN is optimized to best prioritize known and novel tumor suppressors and oncogenes, thereby identifying drivers and potential druggable vulnerabilities within cancer CNAs. Protein homeostasis, phospholipid dephosphorylation, and ion transport pathways are commonly suppressed. An atlas of CNA pathways altered in each cancer type is released. These CNA network shifts highlight new, attractive targets to exploit in solid tumors.

BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract.

The tumor suppressor BRCA1 is considered a master regulator of genome integrity. Although widely recognized for its DNA repair functions, BRCA1 has also been implicated in various mechanisms of chromatin remodeling and transcription regulation. However, the precise role that BRCA1 plays in these processes has been difficult to establish due to the widespread consequences of its cellular dysfunction. Here, we use nucleoplasmic extract derived from the eggs of Xenopus laevis to investigate the role of BRCA1 in a cell-free transcription system. We report that BRCA1-BARD1 suppresses transcription initiation independent of DNA damage signaling and its established role in histone H2A ubiquitination. BRCA1-BARD1 acts through a histone intermediate, altering acetylation of histone H4K8 and recruitment of the chromatin reader and oncogene regulator BRD4. Together, these results establish a functional relationship between an established (BRCA1) and emerging (BRD4) regulator of genome integrity.

Identification of the Preferred DNA-Binding Sequence and Transcription Regulatory Network for the Thermophilic Zinc Uptake Regulator TTHA1292.

D-block metal cations are essential for most biological processes; however, excessive metal exposure can be deleterious to the survival of microorganisms. To tightly control heavy metal regulation, prokaryotic organisms have developed several mechanisms to sense and adapt to changes in intracellular and extracellular metal concentrations. The ferric uptake regulator superfamily of transcription factors associates with DNA when complexed with a regulatory metal cofactor and often represses the transcription of genes involved in metal transport, thus providing a genomic response to an environmental stressor. Although extensively studied in mesothermic organisms, there is little information describing ferric uptake regulator homologs in thermophiles. In this study, we biochemically characterize the ferric uptake regulator homolog TTHA1292 in the extreme thermophile Thermus thermophilus HB8. We identify the preferred DNA-binding sequence of TTHA1292 using the combinatorial approach, restriction endonuclease, protection, selection, and amplification (REPSA). We map this sequence to the Thermus thermophilus HB8 genome and identify the TTHA1292 transcription regulatory network, which includes the zinc ABC transporter subunit genes TTHA0596 and TTHA0453/4. We formally implicate TTHA1292 as a zinc uptake regulator and show that zinc coordination is critical for the multimerization of TTHA1292 dimers on DNA in vitro and transcription repression in vivo. IMPORTANCE Discovering how organisms sense and adapt to their environments is paramount to understanding biology. Thermophilic organisms have adapted to survive at elevated temperatures (>50°C); however, our understanding of how these organisms adapt to changes in their environment is limited. In this study, we identify a zinc uptake regulator in the extreme thermophile Thermus thermophilus HB8 that provides a genomic response to fluctuations in zinc availability. These results provide insights into thermophile biology, as well as the zinc uptake regulator family of proteins.

Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359.

Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5'-AWTGTRA(N)6TYACAWT-3', which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

Cell-free transcription in Xenopus egg extract.

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that Xenopus nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts.

Adsorption of amino acids on graphene: assessment of current force fields.

We compare the free energies of adsorption (ΔAads) and the structural preferences of amino acids on graphene obtained using the non-polarizable force fields-Amberff99SB-ILDN/TIP3P, CHARMM36/modified-TIP3P, OPLS-AA/M/TIP3P, and Amber03w/TIP4P/2005. The amino acid-graphene interactions are favorable irrespective of the force field. While the magnitudes of ΔAads differ between the force fields, the relative free energy of adsorption across amino acids is similar for the studied force fields. ΔAads positively correlates with amino acid-graphene and negatively correlates with graphene-water interaction energies. Using a combination of principal component analysis and density-based clustering technique, we grouped the structures observed in the graphene adsorbed state. The resulting population of clusters, and the conformation in each cluster indicate that the structures of the amino acid in the graphene adsorbed state vary across force fields. The differences in the conformations of amino acids are more severe in the graphene adsorbed state compared to the bulk state for all the force fields. Our findings suggest that the force fields studied will give qualitatively consistent relative strength of adsorption across proteins but different structural preferences in the graphene adsorbed state.

Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors.

Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism's genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro, iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms' transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods.

Biolayer interferometry for DNA-protein interactions.

Biolayer interferometry (BLI) is a widely utilized technique for determining macromolecular interaction dynamics in real time. Using changes in the interference pattern of white light reflected off a biosensor tip, BLI can determine binding parameters for protein-protein (e.g., antibody-substrate kinetics) or protein-small molecule (e.g., drug discovery) interactions. However, a less-appreciated application for BLI analysis is DNA-protein interactions. DNA-binding proteins play an immense role in cellular biology, controlling critical processes including transcription, DNA replication, and DNA repair. Understanding how proteins interact with DNA often provides important insight into their biological function, and novel technologies to assay DNA-protein interactions are of broad interest. Currently, a detailed protocol utilizing BLI for DNA-protein interactions is lacking. In the following protocol, we describe the use of BLI and biotinylated-DNA probes to determine the binding kinetics of a transcription factor to a specific DNA sequence. The experimental steps include the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis by scientific graphing and statistical software (e.g., GraphPad Prism). Although the example experiment used throughout this protocol involves a prokaryotic transcription factor, this technique can be easily translated to any DNA-binding protein. Pitfalls and potential solutions for investigating DNA-binding proteins by BLI are also presented.

BRD4 promotes resection and homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are one of the most toxic forms of DNA damage and represent a major source of genomic instability. Members of the bromodomain and extra-terminal (BET) protein family are characterized as epigenetic readers that regulate gene expression. However, evidence suggests that BET proteins also play a more direct role in DNA repair. Here, we establish a cell-free system using Xenopus egg extracts to elucidate the gene expression-independent functions of BET proteins in DSB repair. We identify the BET protein BRD4 as a critical regulator of homologous recombination and describe its role in stimulating DNA processing through interactions with the SWI/SNF chromatin remodeling complex and resection machinery. These results establish BRD4 as a multifunctional regulator of chromatin binding that links transcriptional activity and homology-directed repair.

Influence of carbon nanomaterial defects on the formation of protein corona.

In any physiological media, carbon nanomaterials (CNM) strongly interact with biomolecules leading to the formation of biocorona, which subsequently dictate the physiological response and the fate of CNMs. Defects in CNMs play an important role not only in material properties but also in the determination of how materials interact at the nano-bio interface. In this article, we probed the influence of defect-induced hydrophilicity on the biocorona formation using micro-Raman, photoluminescence, infrared spectroscopy, electrochemistry, and molecular dynamics simulations. Our results show that the interaction of proteins (albumin and fibrinogen) with CNMs is strongly influenced by charge-transfer between them, inducing protein unfolding which enhances conformational entropy and higher protein adsorption.