Local Gene Regulation Details a Recognition Code within the LacI Transcriptional Factor Family

The specific binding of regulatory proteins to DNA sequences exhibits no clear patterns of association between amino acids (AAs) and nucleotides (NTs). This complexity of protein-DNA interactions raises the question of whether a simple set of wide-coverage recognition rules can ever be identified. Here, we analyzed this issue using the extensive LacI family of transcriptional factors (TFs). We searched for recognition patterns by introducing a new approach to phylogenetic footprinting, based on the pervasive presence of local regulation in prokaryotic transcriptional networks. We identified a set of specificity correlations –determined by two AAs of the TFs and two NTs in the binding sites– that is conserved throughout a dominant subgroup within the family regardless of the evolutionary distance, and that act as a relatively consistent recognition code. The proposed rules are confirmed with data of previous experimental studies and by events of convergent evolution in the phylogenetic tree. The presence of a code emphasizes the stable structural context of the LacI family, while defining a precise blueprint to reprogram TF specificity with many practical applications.Ministerio de Ciencia e Innovación, Spain (Formación de Profesorado Universitario fellowship)Ministerio de Ciencia e Innovación, Spain (grant BFU2008-03632/BMC)Madrid (Spain : Region) (grant CCG08-CSIC/SAL-3651

Classification and structure-based inference of transcriptional regulatory proteins

Dissertação de mestrado em BioinformáticaTranscription factors (TFs) are proteins that mediate the cellular response to the changes of the surrounding environment. Studying their functional domains and protein structure is fundamental in order to gain insight of the way they are triggered and how they shape genetic transcription. The current work aimed for classifying both TFs and functional domains, understanding which features can be related to the different functions of the TFs. By using UniProtJAPI, a JAVA library that allows remote access to UniProt, the information of 200 Escherichia coli’s (E. coli) TFs has been retrieved. This data was manually curated, in order to remove domain duplicates and other excess information, and to add missing domains. The obtained functional domains were classified according to their molecular function, while the TFs were classified according to their regulatory function. TFs that exclusively induce gene expression were classified as activators, while TFs that only perform gene repression were classified as repressors. On the other hand, TFs that perform both the activation and repression of transcription were classified as duals. The information was then analysed altogether in order to understand what relationships between the TFs’ function and functional domains could exist. Several analysis were performed, which include statistical tests and clustering methods. Along with the analysis of the full list of TFs, TFs that are part of two-component signal transduction systems and global TFs were given special focus, due to their important role in cellular function. The results showed that there is a relationship between the functional domains and the regulatory function of the different TFs. This may be related to the evolutionary relationships between repressors and activators. It is also understandable that dual regulators are closely related to activators and repressors than what activators and repressors are to each other. Moreover, TFs of two-component signal transduction systems are similar to each other, given that they perform similar functions. Their domain architectures are also predictable and do not vary from what was expected of these TFs. However, in global TFs the results are opposite of the ones obtained for two-component system TFs: their structures are very different from each other and each TF is specific. The amount of different domains is high when comparing to the full sample of TFs, since the number of domains exceeds the number of TFs. Domains of all classification types are present in their structure and the domain architectures are varied, which reflects their different activities within the cell.Os factores de transcrição (TFs) são proteínas que mediam resposta celular perante alterações do meio em que se inserem. Estudar os seus domínios funcionais e estrutura proteica é fundamental para compreender a forma como as suas funções são desencadeadas e como moldam a regulação da transcrição. Este trabalho teve como objectivos a classificação dos TFs de acordo com a sua função, assim como a classificação dos domínios funcionais. Através do uso da UniProtJAPI, uma biblioteca de JAVA que permite o acesso remoto à UniProt, foi recolhida informação de 200 TFs da Escherichia coli (E. coli). Estes dados foram curados manualmente, com o objectivo de remover domínios duplicados e outra informação em excesso, assim como de adicionar domínios em falta. Os domínios funcionais obtidos foram classificados de acordo com a sua função molecular, enquanto que os TFs foram classificados de acordo com a sua função regulatória. TFs que exclusivamente induzem a expressão genética foram classificados como activadores, enquanto que TFs que apenas reprimem a expressão genética foram classificados como repressores. Por usa vez, TFs que tanto induzem como reprimem a expressão genética foram classificados como duais. A informação dos domínios e dos TFs foi considerada como um todo de forma a compreender quais as possíveis relações entre a função regulatória dos TFs e os domínios funcionais. Várias análises foram efectuadas, das quais testes estatísticos e métodos de clustering. Para além da análise de todos os TFs, foi também feita uma análise de TFs que fazem parte de two-component transduction systems e TFs globais, devido à sua importância na actividade celular. Os resultados demonstram que existe uma relação entre os domínios funcionais e a função regulatória dos TFs. Esta pode ter a ver com as relações evolucionárias dos activadores e repressores. É, também, perceptível que os reguladores duais relacionam-se com mais proximidade dos activadores e dos repressores do que os activadores e os repressores se relacionam entre si. Para além disso, TFs de two-component transduction systems têm estruturas semelhantes , uma vez que desempenham funções idênticas. As duas arquitecturas de domínios também são previsíveis e não variam do que era esperado. Contudo, para os TFs globais, os resultados são antagónicos: as suas estruturas são diferentes umas das outras e cada TF é específico. A quantidade de domínios diferentes é elevada em comparação com a amostra completa de TFs, uma vez que o número de domínios excede o número de TFs. Domínios de todas as classificações estão presentes na estrutura dos TFs globais e as arquitecturas de domínios são variadas, o que reflecte as suas actividades específicas na célula

Consequences of local and global chromatin mechanics to adaption and genome stability in the budding yeast Saccharomyces cerevisiae

Le génome de la levure de boulanger Saccharomyces cerevisiae a évolué à partir d'un ancêtre chez lequel une profonde décompaction du génome s'est produite à la suite de la perte de la méthylation de la lysine 9 de l'histone H3, il y a environ 300 millions d'années. Il a été proposé que cette décompaction du génome a entraîné une capacité accrue des levures à évoluer par des mécanismes impliquant des taux de recombinaison méiotique et de mutation exceptionnellement élevés. La capacité à évoluer accrue qui en résulte pourrait avoir permis des adaptations uniques, qui en ont fait un eucaryote modèle idéal et un outil biotechnologique. Dans cette thèse, je présenterai deux exemples de la façon dont les adaptations locales et globales du génome se reflètent dans les changements des propriétés mécaniques de la chromatine qui, à leur tour, indiquent un phénomène de séparation de phase causée par les modifications post-traductionnelles des histones et des changements dans les taux d'échange des histones. Dans un premier manuscrit, je présente des preuves d'un mécanisme par lequel la relocalisation du locus INO1, gène actif répondant à la déplétion en inositol, du nucléoplasme vers l'enveloppe nucléaire, augmente la vitesse d'adaptation et la robustesse métabolique aux ressources fluctuantes, en augmentant le transport des ARNm vers le cytosol et leur traduction. La répartition d'INO1 vers l'enveloppe nucléaire est déterminée par une augmentation locale des taux d'échange d'histones, ce qui entraîne sa séparation de phase du nucléoplasme en une phase de faible densité plus proche de la périphérie nucléaire. J'ai quantifié les propriétés mécaniques de la chromatine du locus du gène dans les états réprimé et actif en analysant le déplacement de 128 sites LacO fusionnés au gène liant LacI-GFP en calculant diffèrent paramètres tel que la constante de ressort effective et le rayons de confinement du locus. De plus, j'ai mesuré l'amplitude et le taux d'expansion en fonction du temps du réseau LacO et j'ai observé une diminution significative du locus à l'état actif, ce qui est cohérent avec le comportement de ressort entropique de la chromatine décompactée. J'ai montré que les séquences d'éléments en cis dans le promoteur du locus, essentielles à la séparation de phase, sont des sites de liaison pour les complexes de remodelage de la chromatine effectuant l'acétylation des histones. Ces modifications de la chromatine entraînent une augmentation des taux d'échanges des sous-unités des complexes d'histones, et une séparation de phase locale de la chromatine. Enfin, je présente l’analyse de simulations in silico qui montrent que la séparation de phase locale de la chromatine peut être prédite à partir d'un modèle de formation/disruption des interactions multivalentes protéine-protéine et protéine-ADN qui entraîne une diminution de la dynamique de l'ADN. Ces résultats suggèrent un mécanisme général permettant de contrôler la formation rapide des domaines de la chromatine, bien que les processus spécifiques contribuant à la diminution de la dynamique de l'ADN restent à étudier. Dans un second manuscrit, je décris comment nous avons induit la « retro-évolution » de la levure en réintroduisant la méthylation de la lysine 9 de l'histone H3 par l'expression de deux gènes de la levure Schizosaccaromyces pombe Spswi6 et Spclr4. Le mutant résultant présente une augmentation de la compaction de la chromatine, ce qui entraîne une réduction remarquable des taux de mutation et de recombinaison. Ces résultats suggèrent que la perte de la méthylation de la lysine 9 de l'histone H3 pourrait avoir augmenté la capacité à l'évoluer. La stabilité inhabituelle du génome conférée par ces mutations pourrait être utile pour l'ingénierie métabolique de S. cerevisiae, dans laquelle il est difficile de maintenir des gènes exogènes intégrés pour les applications de nombreux processus biotechnologiques courants tels que la production de vin, de bière, de pain et de biocarburants. Ces résultats soulignent l'influence des propriétés physiques d'un génome sur son architecture et sa fonction globales.The genome of the budding yeast Saccharomyces cerevisiae evolved from an ancestor in which a profound genome decompaction occurred as the result of the loss of histone H3 lysine 9 methylation, approximately 300 million years ago. This decompaction may have resulted in an increased capacity of yeasts to evolve by mechanisms that include unusually high meiotic recombination and mutation rates. Resultant increased evolvability may have enabled unique adaptations, which have made it an ideal model eukaryote and biotechnological tool. In this thesis I will present two examples of how local and global genome adaptations are reflected in changes in the mechanical properties of chromatin. In a first manuscript, I present evidence for a mechanism by which partitioning of the active inositol depletion-responsive gene locus INO1 from nucleoplasm to the nuclear envelope increases the speed of adaptation and metabolic robustness to fluctuating resources, by increasing mRNA transport to the cytosol and their translation. Partitioning of INO1 to the nuclear envelope is driven by a local increase in histone exchange rates, resulting in its phase separation from the nucleoplasm into a low-density phase closer to the nuclear periphery. I quantified the mechanical properties of the gene locus chromatin in repressed and active states by monitoring mean-squared displacement of an array of 128 LacO sites fused to the gene binding LacI-GFP and calculating effective spring constants and radii of confinement of the array. Furthermore, I measured amplitude and rate of time-dependent expansion of the LacO array, and observed a significant decrease for the active-state locus which is consistent with entropic spring behavior of decompacted chromatin. I showed that cis element sequences in the promoter and upstream of the locus that are essential to phase separation are binding sites for chromatin remodeling complexes that perform histone acetylation among other modifications that result in increased histone complex exchange rates, and consequent local chromatin phase separation. Finally, I present analytical simulations that show that local phase separation of chromatin can be predicted from a model of formation/disruption of multivalent protein-protein and protein-DNA interactions that results in decreased DNA dynamics. These results suggest a general mechanism to control rapid formation of chromatin domains, although the specific processes contributing to the decreased DNA dynamics remain to be investigated. In a second manuscript, I describe how we retro-evolutionarily engineered yeast by reintroducing histone H3 lysine 9 methylation through the expression of two genes from the yeast Schizosaccaromyces pombe Spswi6 and Spclr4. This mutant shows an increase in compaction, resulting in remarkable reduced mutation and recombination rates. These results suggest that loss of histone H3 lysine 9 methylation may have increased evolvability. The unusual genome stability imparted by these mutations could be of value to metabolically engineering S. cerevisiae, in which it is difficult to maintain integrated exogenous genes for applications for many common biotechnological processes such as wine, beer, bread, and biofuels production. These results highlight the influence of the physical properties of a genome on its overall architecture and function

Engineering Transcriptional Control and Synthetic Gene Circuits in Cell Free systems

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. In contrast to cell-based systems, cell free extracts offer a flexible and well-characterized context in which to implement predictable gene circuits. Critical to these efforts is the availability of a library of ligand sensitive gene regulatory systems. Here, I describe efforts to develop molecular tools to control gene expression and implement a negative feedback circuit in E.coli cell extracts. First, a strategy to regulate T7 RNA polymerase using DNA aptamers is detailed. I test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. A DNA aptamer that binds thrombin is used as a model system for demonstrating feasibility of the approach. I show that for the hybrid T7-aptamer promoter, thrombin addition results in up to a 5-fold reduction in gene expression. I further demonstrate that gene expression be tuned by altering the position of the aptamer relative to the transcription start site. I then devised a mechanism to engineer dual regulation of T7 promoters using LacI and TetR repressor proteins. To achieve this, a LacI binding site (lacO) was positioned 92bp upstream from a T7lacO promoter, which resulted in an increased repression from T7lacO promoters presumably by a looping based mechanism. TetR binding sites were introduced into this framework to disrupt the DNA looping to create T7 promoters that respond to both LacI and TetR. I show that positioning a tetO operator between the upstream lacO and the T7lacO promoter results in relieving lacO mediated repression by TetR. Finally, a negative feedback circuit was realized using T7lacO promoters. To this end, mono-cistronic and bi-cistronic system assembly approaches for system assembly are examined leading to the realization of an inducible negative feedback circuit in cell free systems. Collectively, the tools developed in this work pave the way for expanding the library of ligands that can be used for regulating gene expression, enabling signal integration at T7 promoters and facilitating engineering of gene networks in cell free systems

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria

Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate-active enzymes including two multimodular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1) domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate-binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi-modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi-modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments

Robust Algorithms for Detecting Hidden Structure in Biological Data

Biological data, such as molecular abundance measurements and protein sequences, harbor complex hidden structure that reflects its underlying biological mechanisms. For example, high-throughput abundance measurements provide a snapshot the global state of a living cell, while homologous protein sequences encode the residue-level logic of the proteins\u27 function and provide a snapshot of the evolutionary trajectory of the protein family. In this work I describe algorithmic approaches and analysis software I developed for uncovering hidden structure in both kinds of data. Clustering is an unsurpervised machine learning technique commonly used to map the structure of data collected in high-throughput experiments, such as quantification of gene expression by DNA microarrays or short-read sequencing. Clustering algorithms always yield a partitioning of the data, but relying on a single partitioning solution can lead to spurious conclusions. In particular, noise in the data can cause objects to fall into the same cluster by chance rather than due to meaningful association. In the first part of this thesis I demonstrate approaches to clustering data robustly in the presence of noise and apply robust clustering to analyze the transcriptional response to injury in a neuron cell. In the second part of this thesis I describe identifying hidden specificity determining residues (SDPs) from alignments of protein sequences descended through gene duplication from a common ancestor (paralogs) and apply the approach to identify numerous putative SDPs in bacterial transcription factors in the LacI family. Finally, I describe and demonstrate a new algorithm for reconstructing the history of duplications by which paralogs descended from their common ancestor. This algorithm addresses the complexity of such reconstruction due to indeterminate or erroneous homology assignments made by sequence alignment algorithms and to the vast prevalence of divergence through speciation over divergence through gene duplication in protein evolution

Transcriptional Regulation by Competing Transcription Factor Modules

Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input–output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits

Targets of Heterochromatin Assembly in Drosophila melanogaster

Heterochromatin is classically defined as densely staining regions of the genome; these domains are typically late replicating and show little recombination. Correct assembly of heterochromatin is critical for chromosome stability. Assembly begins with histone deacetylation and H3 lysine 9 di- and trimethylation: H3K9me2/3); the methylated H3 is typically bound by Heterochromatin Protein 1a: HP1a). Heterochromatin predominates at pericentric and telomeric domains --regions abundant in transposable elements: TEs) and satellite repeats. Transcription of these TEs has been found to generate a platform for assembly of heterochromatin through RNAi in S. pombe and A. thaliana, and may play a critical role in Drosophila melanogaster. However, the precise role of RNAi in heterochromatin assembly for a metazoan system such as flies remains unclear. However, 1360, a DNA transposable element in D. melanogaster, has been found to be sufficient to promote heterochromatin assembly in a repeat-rich region, as shown by a variegating phenotype of a hsp70-white reporter. RNAi components and heterochromatin factors such as HP1a were both implicated in this 1360-sensitive variegation, a form of position effect variegation: PEV). Here, I sought to determine the extent and mechanism of TE-sensitive PEV. A collection of 1360-sensitive landing pad insertion lines containing the hsp70-w reporter was generated. This tool allows for the repeated sampling of altered 1360 constructs in a variety of chromatin contexts, a useful a platform to study the attributes of 1360-sensitive variegation as well as PEV generally. We found 1360-sensitive PEV to extend to sites outside of annotated heterochromatin, although most sensitive sites lie within or proximal to heterochromatic masses. I used biochemical approaches to show that 1360-sensitive PEV corresponds to HP1a accumulation over the hsp70-w promoter region, confirming that the silencing is due to heterochromatin assembly. The deletion of sites within the 1360 element with homology to the PIWI-interacting RNAs: piRNAs) in 1360 suppressed PEV, as did dominant mutations in PIWI domain proteins. Similar results were obtained using Invader4, a retrotransposon, in the same landing pad site. The results support a mechanism that uses piRNAs for transposon-sensitive HP1a-silencing, likely early in development, with persistent effects observed in the adult somatic tissue of the eye. To determine if the sequence determinants required for 1360-sensitive silencing in a euchromatic region: as seen above) also operate in a repetitious sequence environment, where interspersed signals may operate cooperatively, I investigated a 1360-sensitive site in the piRNA generating locus 42AB. We find that mutations in piwi, along with many prototypical Su(var) mutations, result in weak suppression of variegation at this site, while an ago2 mutation enhances variegation. Tests of various fragments of the TEs do not reveal a strong dependency on piRNA matching sequences, contrary to the euchromatic site driven to a heterochromatic form by the added TE. These findings indicate that suppression of PEV by mutations in the genes for RNAi components occurs in a limited number of heterochromatic domains, predominantly those near gene clusters - sites typically found at the border between euchromatin and heterochromatin. Thus chromosomal context appears to be an important determinant for RNAi-dependent 1360-sensitive PEV. This finding helps to reconcile reports of inconsistent PEV effects from mutations in RNAi components that have been carried out using reporters in different domains. Collectively, these results indicate the TEs can act as sequence determinants of heterochromatin assembly at a subset of genomic sites using an RNAi-mediated targeting mechanism

Transcriptional Silencing and Anti-Silencing of Virulence Genes in the Bacterial Pathogen Shigella Flexneri: VIRB, DNA Supercoiling, and the Histone-Like Nucleoid Structuring Protein

Transcriptional silencing and anti-silencing affect many aspects of bacterial physiology, including virulence in bacterial pathogens. In Shigella species, a group of gram-negative pathogens that cause bacillary dysentery in humans, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences virulence genes found on the large virulence plasmid while VirB anti-silences these genes. However, the mechanistic details of their interplay are not fully understood. To elucidate their regulatory mechanisms, I use the icsP virulence locus, which shares a long intergenic region with the divergently transcribed ospZ gene (1535 bp from TSS to TSS). Prior to this work, two discrete H-NS binding regions had been identified, suggesting H-NS-mediated bridging of these two regions as the mechanism of silencing. However, I show that changes to the spacing and helical phasing designed to disrupt the potential bridging were tolerated, suggesting an alternate mechanism of silencing is at play. In addition to H-NS, two other H-NS homologs found in S. flexneri, StpA and Sfh, can also silence the icsP promoter. Interestingly, VirB counters transcriptional silencing mediated by these other H-NS homologs. The site required for VirB-dependent anti-silencing of the icsP promoter is located over 1 kb upstream of the TSS, and nearly 500 bp upstream of the ospZ promoter, but exactly how VirB accomplishes this long-range regulation is not known. I show that VirB docks to this recognition site in vitro and has a high specificity for this site in vivo. Using a combination of 1D and 2D chloroquine-based agarose gel electrophoresis, I demonstrate that, upon docking to its recognition site, VirB triggers a loss of negative supercoiling of our VirB-dependent PicsP-lacZ reporter; importantly, this phenomenon occurs with native VirB levels in S. flexneri. Because H-NS is sensitive to DNA topology at some promoters, it is tantalizing to envision that VirBmediated changes in supercoiling alleviate H-NS-mediated silencing of virulence genes in Shigella. Although anti-silencing proteins in other bacteria, including related pathogens, bear little sequence homology to VirB, the possibility that changes to DNA supercoiling mechanistically unite this group of proteins requires further consideration when studying transcriptional silencing and anti-silencing processes in bacteria