Transcription factors (TFs) have been a focal point of molecular biology research for decades, with evolving methodologies offering progressively deeper insights into their critical roles in gene regulation. Recent advancements in experimental and computational techniques have significantly enhanced our understanding of TF functionality, yet this depth of knowledge varies widely across the spectrum of known TFs — from extensively characterized ones with quantitative binding affinity data to those scarcely studied or understood. In this work, we systematically carried out binding and expression experiments on all Escherichia coli TFs using a standardized computational pipeline to identify direct and indirect regulatory targets. We further leveraged our binding data to develop a novel biophysically motivated neural network capable of predicting TF-DNA binding affinity from DNA sequence. This approach allowed us to design binding sites with specified affinities, including those stronger than any sequence observed in nature, which we validate experimentally using an in vitro binding assay. We further optimized this assay to provide insight into complex TF binding regimes, where chemical signals can modulate TF binding affinity. Finally, we demonstrate the utility of systematically mapping TF binding sites through a case study on a previously thought dormant TF acquired from viral infection, revealing an unexpected phenotype where it can hijack the host cell. This work not only offers broad insights into the determinants of TF binding and regulation, but also provides a means to predictively engineer binding sites with desired affinity, while demonstrating the power of efficient data processing in uncovering intricate biological processes. / 2025-05-23T00:00:00Z

No / Eczema is widely considered to be an exacerbation of alkaline stress to the skin. Epidermal barrier dysfunction is a feature of eczema pathology, which predisposes affected individuals to distressing morbid symptoms. At least two serine proteases, stratum corneum chymotryptic enzyme (kallikrein 7 [KLK7]) and stratum corneum tryptic enzyme (kallikrien 5 [KLK5]), have increased activity levels in eczematous lesions and both have been implicated in the destruction of corneodesomosomes, which are crucial to epidermal integrity. The present in vitro study investigated whether transcriptional gene silencing after siRNA transfection could influence the activity of these signature enzymes in an in vitro model of eczema induced by alkaline shock. HaCaT epithelial cells were subjected to alkaline stress by the addition of 1,1,3,3-tetramethyl guanidine “superbase” (TMG) to the culture media. The culture media were subsequently tested for chymotryspin, trypsin, plasmin, and urokinase activity using colorimetric peptide assays and for reactive oxygen species using WST1 cell viability reagent. Cells that had been transfected with small interfering ribonucleic acid (siRNA) against KLK5 and KLK7 for 24 h before alkaline shock did not exhibit the increase in serine protease levels observed in untreated controls. Moreover, an endpoint MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) confirmed that detachment of cells from the culture substrate observed in alkaline-stressed cells did not occur in siRNA-treated cells. This in vitro study has established the proof-of-principle that siRNA therapy appears to mitigate the consequences of alkaline shock to the serine protease-associated fragility of epithelial cells that is characteristic of eczema.

Yes / Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

Les différents mécanismes de régulation posttranscriptionnelle de l’expression des gènes sont de plus en plus reconnus comme des processus essentiels dans divers phénomènes physiologiques importants, comme la prolifération cellulaire et la réponse aux dommages à l’ADN. Deux des protéines impliquées dans ce type de régulation sont Staufen1 (Stau1) et Staufen2 (Stau2). Elles sont des protéines de liaison à l’ARN double brin qui contribuent au transport de l’ARN messager (ARNm), au contrôle de la traduction, à l’épissage alternatif et sont responsables de la dégradation de certains ARNm spécifiques. Les protéines Staufen peuvent en effet s’associer à des ARNm bien précis, d’autant plus que, majoritairement, Stau1 et Stau2 ne se retrouvent pas en complexe avec les mêmes cibles. De nombreuses évidences récentes montrent l’implication de divers mécanismes de régulation posttranscriptionnelle dans la réponse aux dommages à l’ADN, plusieurs protéines de liaison à l’ARN y participant d’ailleurs. De façon importante, cette réponse dicte un ou plusieurs destin(s) à la cellule qui doit réagir à la suite de dommages à l’intégrité de son ADN: réparation de l’ADN, arrêt de la prolifération cellulaire, apoptose. Nous avons donc fait l’hypothèse que l’expression de Stau1 et/ou de Stau2 pourrait être affectée en réponse à un stress génotoxique, ce qui pourrait avoir comme conséquence de moduler l’expression et/ou la stabilité de leurs ARNm cibles. De même, notre laboratoire a récemment observé que l’expression de Stau1 varie pendant le cycle cellulaire, celle-ci étant plus élevée jusqu’au début de la mitose (prométaphase), puis elle diminue alors que les cellules complètent leur division. Par conséquent, nous avons fait l’hypothèse que Stau1 pourrait lier des ARNm de façon différentielle dans des cellules bloquées en prométaphase et dans des cellules asynchrones. D’un côté, en employant la camptothécine (CPT), une drogue causant des dommages à l’ADN, pour traiter des cellules de la lignée de cancer colorectal HCT116, nous avons observé que seule l’expression de Stau2 est réduite de façon considérable, tant au niveau de la protéine que de l’ARNm. L’utilisation d’autres agents cytotoxiques a permis de confirmer cette observation initiale. De plus, nous avons constaté que l’expression de Stau2 est touchée même dans des conditions n’engendrant pas une réponse apoptotique, ce qui suggère que cette déplétion de Stau2 est possiblement importante pour la mise en place d’une réponse appropriée aux dommages à l’ADN. D’ailleurs, la surexpression de Stau2 conjointement avec le traitement à la CPT entraîne un retard dans l’induction de l’apoptose dans les cellules HCT116. Nous avons aussi montré que la diminution de l’expression de Stau2 est due à une régulation de sa transcription en réponse au stress génotoxique, ce pourquoi une région minimale du promoteur putatif de Stau2 est nécessaire. Également, nous avons identifié que le facteur de transcription E2F1, couramment impliqué dans la réponse aux dommages à l’ADN, peut contrôler l’expression de Stau2. Ainsi, E2F1 permet une augmentation de l’expression de Stau2 dans des cellules non traitées, mais cette hausse est abolie dans des cellules traitées à la CPT, ce qui suggère que la CPT pourrait agir en inhibant l’activation transcriptionnelle de Stau2 par E2F1. Enfin, nous avons observé que certains ARNm associés à Stau2, et codant pour des protéines impliquées dans la réponse aux dommages à l’ADN et l’apoptose, sont exprimés différemment dans des cellules traitées à la CPT et des cellules non traitées. D’un autre côté, nous avons identifié les ARNm associés à Stau1 lors de la prométaphase, alors que l’expression de Stau1 est à son niveau le plus élevé pendant le cycle cellulaire, grâce à une étude à grande échelle de micropuces d’ADN dans des cellules HEK293T. Nous avons par la suite confirmé l’association entre Stau1 et certains ARNm d’intérêts, donc codant pour des protéines impliquées dans la régulation de la prolifération cellulaire et/ou le déroulement de la mitose. Une comparaison de la liaison de ces ARNm à Stau1 dans des cellules bloquées en prométaphase par rapport à des cellules asynchrones nous a permis de constater une association préférentielle dans les cellules en prométaphase. Ceci suggère une augmentation potentielle de la régulation de ces ARNm par Stau1 à ce moment du cycle cellulaire. Les données présentées dans cette thèse indiquent vraisemblablement que la régulation posttranscriptionnelle de l’expression génique contrôlée par les protéines Staufen se fait en partie grâce à la modulation de l’expression de Stau1 et de Stau2 en fonction des conditions cellulaires. Nous envisageons alors que cette variation de l’expression des protéines Staufen ait des conséquences sur des sous-ensembles d’ARNm auxquels elles sont liées et que de cette façon, elles jouent un rôle pour réguler des processus physiologiques essentiels comme la réponse aux dommages à l’ADN et la progression dans le cycle cellulaire. / The various mecanisms of post-transcriptional regulation of gene expression are more and more recognized as essential processes in diverse important physiological phenomenons, like cell proliferation and the DNA damage response (DDR). Two of the proteins implicated in this type of regulation are Staufen1 (Stau1) and Staufen2 (Stau2). They are double-stranded RNA binding proteins contributing to messenger RNA (mRNA) transport, translation control, alternative splicing and are responsible for the degradation of some specific mRNAs. The Staufen proteins are indeed able to associate with particular mRNAs. Interestingly, Stau1 and Stau2 predominantly form complexes with different targets. Recent evidences show the implication of various post-transcriptional regulation mecanisms in the DDR, moreover several RNA binding proteins are involved. Importantly, this response dictates one or several cell fates following damage to the integrity of the cell’s DNA: DNA repair, cell proliferation arrest, apoptosis. We hypothesized that Stau1 and/or Stau2 expression could be affected in response to genotoxic stress, which could consequently modulate the expression and/or the stability of their mRNA targets. Also, our laboratory has recently observed that Stau1 expression varies during the cell cycle. It is elevated up to the beginning of mitosis (prometaphase) and it decreases as cells complete their division. We therefore hypothesized that Stau1 could differentially bind mRNAs in cells blocked in prometaphasis and in asynchronous cells. On the one hand, by using camptothecin (CPT), a DNA damaging agent, to treat cells from the colorectal cancer cell line HCT116, we observed that only the expression of Stau2 is considerably reduced, both at the level of the protein and that of the mRNA. The use of other cytotoxic agents allowed us to confirm this initial observation. We also noted that Stau2 expression is down-regulated even in conditions that do not induce apoptosis, suggesting that the decrease in Stau2 expression may be required for a proper DDR. Indeed, Stau2 overexpression together with the CPT treatment causes a delay in apoptosis induction in HCT116 cells. We also showed that Stau2 down-regulation is due to the regulation of its transcription in response to the genotoxic stress, which necessitates a minimal region in Stau2’s putative promoter. Besides, we identified the E2F1 transcription factor, commonly implicated in the DDR, as a regulator of Stau2 expression. E2F1 thus stimulates an increase in Stau2 expression in non-treated cells, but this up-regulation is abolished in CPT-treated cells, which suggests that CPT could act by inhibiting Stau2 transcriptional activation by E2F1. Finally, we observed that some Stau2-associated mRNAs, which code for proteins implicated in the DDR and apoptosis, are differentially expressed in CPT-treated cells compared to non-treated cells. On the other hand, we identified Stau1-associated mRNAs during prometaphase, when Stau1 expression is at its highest level in the cell cycle, by performing a large-scale study using DNA microarrays in HEK293T cells. We subsequently confirmed the association between Stau1 and some mRNAs of interest, mainly coding for proteins involved in the regulation of cell proliferation and/or mitosis progression. A comparison of the association between Stau1 and these mRNAs in prometaphase-blocked cells with that in asynchronous cells allowed us to notice a preferential association in prometaphase-blocked cells. This suggests a potential increase of the regulation of these mRNAs by Stau1 at that point of the cell cycle. The data presented in this thesis indicate that in all likelihood the post-transcriptional regulation of gene expression controlled by the Staufen proteins happens in part thanks to the modulation of Stau1 and Stau2 expression according to the cellular conditions. We then contemplate that this fluctuation in Staufen proteins expression has consequences on mRNA subsets with which they associate, and that this may mean they have an important role to play in regulating essential physiological processes like DDR and cell cycle progression.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Σκοπός της παρούσης διατριβής είναι να προτείνει ένα τυπικό πλαίσιο για μια μεθοδολογία ανάλυσης που να ενσωματώνει ποσοτικά, και λειτουργικά δεδομένα και στοχεύει στο σχεδιασμό πειραμάτων για την επιβέβαιωση μεταγραφικών παραγόντων που δεσμεύονται σε ένα λειτουργικά ενεργό μοτίβο στον υποκινητή γονιδίων. Το πλαίσιο αυτό που βασίζεται στη Bayesian πιθανοκρατική θεωρία όπως αυτή θεμελιώνεται στη θεωρία λήψης αποφάσεων, εφαρμόζεται σε ένα πρόβλημα από το ερευνητικό πεδίο της ανοσοβιολογίας. Συγκεκριμένα μελετούμε την ανίχνευση μεταγραφικών παραγόντων που ενέχονται στην αρνητική ρύθμιση της γονιδιακής έκφρασης κατά τη διαδικασία ενεργοποίσης των Τ λεμφοκυττάτων. Η υιοθέτηση του προτεινούμενου πλαισίου σμιλεύει μαι αυστηρή διαδοχή διεξαγωγής in vitro και in silico πειραμάτων που ξεκινά από τεχνικές μαζικής ανάλυσης γονιδιακής έκφρασης, περνά μέσα από βάσεις δεδομένων μεταγραφικών παραγόντων και μέσω πειραμάτων ηλεκτροφορητικής κινητικότητας (Electromobility Shift Assays) στοχεύεται στην επιβεβαίωση που προσφέρουν τα πειράματα διαμόλυνσης (transfection and reporter assays). Κατά την τυποποίηση της λογικοφανούς αυτής προσέγγισης ανακύπτει ένα από τα γνωστότερα προβλήματα της εφαρμοσμένης στατιστικής, το πρόβλημα των "δύο μέσων όρων" ή πρόβλημα Behrens-Fisher. Για τη λύση αυτού του προβλήματος προτείνονται νέα μαθηματικά εργαλεία τα οποία αξιοποίηθηκαν για την κατασκευή αντίστοιχου λογισμικού. Με την εφαρμογή αυτών των εργαλείων στο εφαρμοσμένο ανοσοβιολογικό πρόβλημα προέκυψε μια μη αναμενόμενη σχέση μεταξύ δυο φαινομενικά μη συνδεόμενων συστημάτων¨των γονιδίων των κυτταροκινών και του ιού HIV. Μέσω της περιγραφόμενης μεθοδολογίας κατέστη εφικτή μια υποθεσο-εξαρτώμενη προσέγγιση σε ένα σημαντικό πρόβλημα το οποίο δεν ήταν δυνατό να λύθέί με κλασσικές βιοχημικές τεχνικές λόγω τεχνικών δυσκολιών / The current disertation concerns the description of a formal framework and an analytic methodology which aims to validate transcription factors controlling gene expression through functional and quantitative data. This Bayesian decision theory inspired framework is applied to a specific immunobiological problem. The problem targetted was the discovery of transcriptional repressors implicated in the negative control of T cell activation. Adopting the proposed framework leads one to a staged experimental strategy which starts from high-throughput gene expression data and transcription factor databases and through Electrophoretic Mobility Shift Assays targets the design of transfection and reporter gene assays. The formalization of the proposed approach, led to one of the famous applied statistics problems i.e. the two means or Behrens - Fisher problem. In order to deal with the computational aspects of this problem, we applied a novel integral transformations and ported them to software. The application of these tools to the immunobiological problem led to an unexpected connection between two seemingly unrelated systems: cytokine gene protomers and HIV LTR. The proposed methodology enabled a hypothesis-driven approach to an important basic immunobiological problem which could not be solved by standard biochemical techniques.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.

Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.