Specificity Determination by paralogous winged helix-turn-helix transcription factors

Transcription factors (TFs) localize to regulatory regions throughout the genome, where they exert physical or enzymatic control over the transcriptional machinery and regulate expression of target genes. Despite the substantial diversity of TFs found across all kingdoms of life, most belong to a relatively small number of structural families characterized by homologous DNA-binding domains (DBDs). In homologous DBDs, highly-conserved DNA-contacting residues define a characteristic ‘recognition potential’, or the limited sequence space containing high-affinity binding sites. Specificity-determining residues (SDRs) alter DNA binding preferences to further delineate this sequence space between homologous TFs, enabling functional divergence through the recognition of distinct genomic binding sites. This thesis explores the divergent DNA-binding preferences among dimeric, winged helix-turn-helix (wHTH) TFs belonging to the OmpR sub-family. As the terminal effectors of orthogonal two-component signaling pathways in Escherichia coli, OmpR paralogs bind distinct genomic sequences and regulate the expression of largely non-overlapping gene networks. Using high-throughput SELEX, I discover multiple sources of variation in DNA-binding, including the spacing and orientation of monomer sites as well as a novel binding ‘mode’ with unique half-site preferences (but retaining dimeric architecture). Surprisingly, given the diversity of residues observed occupying positions in contact with DNA, there are only minor quantitative differences in sequence-specificity between OmpR paralogs. Combining phylogenetic, structural, and biological information, I then define a comprehensive set of putative SDRs, which, although distributed broadly across the protein:DNA interface, preferentially localize to the major groove of the DNA helix. Direct specificity profiling of SDR variants reveals that individual SDRs impact local base preferences as well as global structural properties of the protein:DNA complex. This study demonstrates clearly that OmpR family TFs possess multiple ‘axes of divergence’, including base recognition, dimeric architecture, and structural attributes of the protein:DNA complex. It also provides evidence for a common structural ‘code’ for DNA-binding by OmpR homologues, and demonstrates that surprisingly modest residue changes can enable recognition of highly divergent sequence motifs. Importantly, well-characterized genomic binding sites for many of the TFs in this study diverge substantially from the presented de novo models, and it is unclear how mutations may affect binding in more complex environments. Further analysis using native sequences is required to build combined models of cis- and trans-evolution of two-component regulatory networks

Structural properties of the linkers connecting the n- and c- terminal domains in the mocr bacterial transcriptional regulators

Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties. © 2016 The Author(s

YCZR, a New Case of PLP-Dependent MOCR/GABR Type Transcription Regulator in Klebsiella Pneumonia

Increasing number of genes encoding PLP-dependent transcription regulators, MocR/GabR type regulators, have been identified in various bacterial genomes. However, only a handful of them, including MocR, PdxR and GabR have been studied experimentally. They control different aspects of the bacterial metabolism. Only GabR has reported crystallographic structures. MocR/GabR regulators possess a chimeric structure consisted of a WHTH DNA binding domain and an Aminotransferase-like regulation domain, which can bind PLP as an effector in transcription regulation. Such a chimeric construct presents an interesting case in molecular evolution. The regulation domains of All MocR/GabR type regulators loss their catalytic capacity during evolution and function as means of effector recognition and transcription regulation. A MocR/GabR homolog in Klebsiella pneumoniae has recent been studied; this homolog is currently named Duf161R (YczR), since it putatively controls a gene encoding a membrane protein annotated as domain of unknown function 161 . We have determined the three-dimensional crystal structure of the regulatory domain of YczR to a resolution of 1.79 Å. Our crystallographic studies has revealed the structure of a truncated regulation domain with PLP bound, and our spectroscopic studies has gained evidence to support at least partial transaminase-like catalytic activity of the regulation domain. Together with DNA binding studies, we start to shed light on a new case of MocR regulation and its currently unconfirmed biological function, which is likely augmenting pathogenesis via facilitating taurine trafficking in K. pneumoniae. During my Ph.D. program, I also worked on several other protein projects. I add two chapters to demonstrate the PTP1B project. Protein tyrosine phosphatase 1B (PTP1B) is an enzyme shown to play an important role in insulin regulation. PTP1B is a critical negative regulator of insulin and leptin signaling pathways by removing phosphate groups (PO43-) from insulin receptor and other post-receptor substrates. Previous studies have identified transition metal compounds that exhibit insulin mimetic effects. A plausible explanation is that vanadium-containing compounds and zinc-containing compounds inhibit PTP1B activity, which allows required phosphorylation reaction to proceed normally. Five specifically modified vanadium containing and zinc containing compounds have been synthesized. This research has determined the three-dimensional crystal structure of PTP1B and VO(acac)2 complex to a resolution of 2.2 Å. Furthermore, the kinetic data suggests a mixed inhibition because of the aqueous study of the vanadium complex

Characterization of histone acetyltransferase KAT6A and transcriptional repressor SAMD1 – two proteins possessing a novel class of unmethylated DNA-binding winged helix domains

This thesis addresses the functions of two transcriptional regulators; histone acetyltransferase and transcriptional activator KAT6A (MOZ), and transcriptional repressor SAMD1 (Atherin), in their respective molecular and cell type-specific roles. Firstly, we identified a novel winged helix domain at the N-terminal end of KAT6A as a distinct feature of the previously described NEMM (N-terminal part of Enok, MOZ or MORF) domain, a homologous region with other histone acetyltransferases. This KAT6A WH1 domain possesses a DNA-recognition motif, directly recruiting KAT6A to unmethylated CpG-rich DNA. Analyses of genome-wide ectopic KAT6A binding sites in HEK293 cells revealed a high correlation with unmethylated CpG islands (CGIs), particularly with CGIs associated with active promoters, decorated with the histone marks H3K4me3 and H3K9ac. We demonstrated that KAT6A WH1 is necessary for the observed recruitment to CGIs, but by itself not sufficient and dependent on KAT6A WH2 and DPF domains for effective KAT6A-CGI interactions. Indeed, mutations of the KAT6A WH1 domain caused a complete abrogation of KAT6A binding to CGIs, but increased the binding to gene body regions at a subset of target genes, indicating that recruitment mechanisms to these sites are independent of KAT6A WH1. This study, for the first time, demonstrates a direct chromatin recruitment mechanism of a histone acetyltransferase. Moreover, it provides new and more detailed insights into KAT6A chromatin recognition and target site association to facilitate histone acetylation and enforce transcription control. Secondly, we morphologically characterized homozygous and heterozygous SAMD1 knockout (KO) mouse embryos and their respective phenotypes in embryogenesis. The homozygous deletion of SAMD1 was embryonic lethal, while heterozygous SAMD1 KO mice were born alive. At embryonic day E14.5 severe degradation of internal organs and formation of subcutaneous oedema were visible in SAMD1 KO embryos, most likely due to disturbed blood vessel maturation and resulting hypoxia. The observed defects led to the conclusion that SAMD1 is required for functional angiogenesis and the development of the cardiovascular system. Furthermore, craniofacial defects upon SAMD1 KO indicated malfunctions in head and brain development. To gain further insight into the role of SAMD1 during neurogenesis, we established a directed neural differentiation approach of murine embryonic stem cells (mESCs). Transcriptional dysregulation and the global increase of H3K4me2 were observed in SAMD1 KO cells during this process, further supporting a functional role of SAMD1 in neural cell development. Overall, this study sheds light on the underlying molecular mechanism of KAT6A binding to chromatin and emphasizes the similarities of KAT6A WH1 to the previously described homologous SAMD1 WH domain, which directly interacts with unmethylated CGIs and facilitates transcription regulation, as well. Both KAT6A and SAMD1 are involved in a variety of developmental processes and dysregulations are associated with cellular abnormalities and the onset of cancer

An Omega-Based Bacterial One-Hybrid System for the Determination of Transcription Factor Specificity

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome

THE EVOLUTIONARY DYNAMICS OF TRANSCRIPTION FACTORS, OPERATORS, AND THEIR TARGET GENES ACROSS PROKARYOTES

In prokaryotes, transcriptional regulation commonly involves a transcription factor (TF) binding to a particular conserved sequence of nucleotides (operator). Binding elicits a transcriptional response, either activation or repression. The evolution of gene regulation has been identified as a primary driver of species diversity, making it an important area of research. This work examined the dynamics of the interactions between TFs and operators, and TFs and their primary target genes in attempt to assess the rapid evolution of transcriptional regulatory networks (TRNs) across a diverse set of prokaryotes. Using software packages, operator sequences from Escherichia coli K12 were compared to every bacterial and archaeal genome within the NCBI’s RefSeq database. This revealed that, based on genome composition, native TFs have a greater probability of interacting with sequences within their host’s genome than those of other species, indicating that appropriate operators may form spontaneously, and often, within a genome. TFs and target genes were assessed through co-occurrence patterns. Recently, research has shown that repeated co- occurrence of two genes is evidence for a functional interaction. Co-occurrence can be observed and quantified in phylogenetic profiles by measuring mutual information (MI); this is a metric of how often two genes co-occur adjusted for what is expected by chance. By measuring MI for all two-gene combinations from a subset of genomes from NCBI’s RefSeq database, results showed that, in \u3e 97% of the organisms observed, TFs form looser functional interactions than other genes, indicating that TFs do not form lasting associations on the evolutionary time scale. These results suggest regulatory interactions are not as specific or conserved as those between most other gene products. Together, these results suggest that TRNs evolve rapidly across most, if not all prokaryotes

STRUCTURAL AND FUNCTIONAL STUDIES OF THREE PROTEINS OF UNKNOWN FUNCTION ENCODED BY CHLAMYDIA TRACHOMATIS

Infections by chlamydial species are of significant impact to global public health, causing sexually transmitted infections, blinding trachoma and pneumonia. Despite its importance, there are many aspects of chlamydial biology that are not completely understood, including the mechanisms by which it infects, persists and replicates in its host cells. The reason for this ignorance of basic chlamydial biological processes is because there is an abundance of Open Reading Frames (ORFs) of unknown function present in chlamydial genomes, almost 30% of the entire genome in many species. This is likely due to the relatively large phylogenetic distance between Chlamydiae and better-understood bacteria such as E. coli and B. subtilis. Current strategies of genome annotation rely on the presence of homology to genes of known function and these approaches have not been effective in annotating chlamydial genomes. In an effort gain insight into the function of these chlamydial ORFs of unknown function, I utilized structural information (both computational and experimentally derived) about three proteins of interest. Based on these structural studies, hypotheses concerning the functions of these proteins were formed and then tested. Together, my findings not only provide valuable information about these proteins of unknown function, but they also serve to demonstrate both the strengths and shortcomings of the overall approach of utilizing structural information for functional prediction. One example of this approach is my work on the chlamydial ORF CT296. Although this protein was annotated as having an unknown function (due to insignificant homology to proteins of known function), it had been experimentally characterized as an iron-dependent transcription factor. Having an experimentally characterized function allowed me to test my approach of utilizing structural information to predict function on a protein with a relatively well-understood function. Surprisingly, structural information of this protein suggested that it functions as a Fe(II) 2-oxoglutarate-dependent enzyme and not as a transcription factor. Subsequent functional analyses of the protein were unable to reproduce previous reports of its DNA binding. Together, my findings suggest that this protein may not function as a transcription factor. A second example of my structure-function approach was applied to the chlamydial protein CT584. This protein was first experimentally described as interacting with the chlamydial Type Three Secretion System (T3SS) needle protein in an interactome study. This observation, combined with a subsequent biophysical characterization of the protein lead to an initial hypothesis that the protein may be a chlamydial T3SS needle tip protein. However, results of structural studies on the protein reveal a structure that is not similar to any of the known T3SS needle tip proteins. Additionally, functional studies on the protein focusing on identifying its localization on chlamydial organisms revealed localization patterns not consistent with its proposed role as a T3SS needle protein. Together, my studies suggest that this protein may not function as a needle tip protein. A final example of the utility of structural information for informing function concerns chlamydial ORF CT009. This protein was annotated in many chlamydial species as a protein of unknown function; however, bioinformatics analyses had identified it as a helix-turn-helix containing transcription factor. Results of computational and experimental structures of this protein show structural similarity to the protein RodZ, a key component of the bacterial morphogenic complex. Subsequent functional analyses of CT009 demonstrate that this protein has the characteristics of a chlamydial homolog to RodZ

Structural insights into phosphoprotein chaperoning of nucleoprotein in measles virus

Instruct Biennial Structural Biology Conference Abstract BookletMeasles virus is an important, highly contagious, human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein, P, to prevent newly synthesized N from binding to cellular RNA. Here, we pulled down an N01-408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal, P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0 binding peptide, P1-48, to the C-terminus of an N021-408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at 2.7 Å resolution. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0 preventing both self-assembly of N0 and its binding to RNA. We compare the structure of an N0-P complex to atomic model of helical ribonucleocapsid. We thus propose a model how P may help to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of the virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development not only to treat measles but also potentially other paramyxovirus diseases.Non peer reviewe