Browsing by Subject "Transcription factors"
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Item Open Access Deciphering the Quantitative Effects of Cooperativity and Mutations on Transcription Factor Binding(2022) Martin, VincentiusTranscription factor (TF) proteins bind to DNA in a sequence specific manner to regulate gene expression. The binding affinity of TFs for individual sites is well characterized and can be represented using DNA motif models such as position weight matrices. However, there are many factors influencing TF-DNA recognition in the cell, leading to complexities than cannot be captured by motif models alone. Here, we present our studies on two factors: cooperative TF binding and alterations in TF binding due to DNA mutations. Both factors require quantitative and rigorous approaches to distinguish real effects from random noise.
First, we present a new method for characterizing cooperative binding of TFs to DNA. This method addresses the issue that TF binding sites located in close proximity, which occurs frequently across the human genome, are not necessarily bound cooperatively. To distinguish between cooperative and independent binding, we developed a high-throughput on-chip binding assay designed specifically to measure TF binding to neighboring sites. Using the experimental data from our assay, we trained machine learning models to differentiate between cooperative and independent binding of TFs. This method enabled us to reveal molecular mechanisms used by TFs to bind DNA cooperatively.
Second, we introduce QBiC-Pred (Quantitative Predictions of TF Binding Changes Due to Sequence Variants), an ordinary least squares based method to predict the magnitude of the effect of DNA mutations on TF-DNA recognition. We implemented QBiC-Pred as a web service: qbic.genome.duke.edu, which allows users to run our models through a user-friendly web interface. We used this method to identify non-recurring putative regulatory driver mutations in cancer. Our approach is novel because we prioritize mutations based on their effects on transcription factor (TF) binding, instead of relying on the recurrence of the mutations among tumor samples---which is often difficult to perform as individual non-coding mutations are rarely seen in more than one donor. Focusing on the functional effects of non-coding mutations across regulatory regions, we identified dozens of genes whose regulation in tumor cells is likely to be significantly perturbed by non-coding mutations.
Item Open Access Mechanisms of specificity in neuronal activity-regulated gene transcription.(2012) Lyons, Michelle RenéeIn the nervous system, activity-regulated gene transcription is one of the fundamental processes responsible for orchestrating proper brain development–a process that in humans takes over 20 years. Moreover, activity-dependent regulation of gene expression continues to be important for normal brain function throughout life; for example, some forms of synaptic plasticity important for learning and memory are known to rely on alterations in gene transcription elicited by sensory input. In the last two decades, increasingly comprehensive studies have described complex patterns of gene transcription induced and/or repressed following different kinds of stimuli that act in concert to effect changes in neuronal and synaptic physiology. A key theme to emerge from these studies is that of specificity, meaning that different kinds of stimuli up- and down regulate distinct sets of genes. The importance of such signaling specificity in synapse-to-nucleus communication becomes readily apparent in studies examining the physiological effects of the loss of one or more forms of transcriptional specificity – often, such genetic manipulations result in aberrant synapse formation, neuronal cell death, and/or cognitive impairment in mutant mice. The two primary loci at which mechanisms of signaling specificity typically act are 1) at the synapse – in the form of calcium channel number, localization, and subunit composition – and 2) in the nucleus – in the form of transcription factor expression, localization, and post-translational modification. My dissertation research has focused on the mechanisms of specificity that govern the activity-regulated transcription of the gene encoding Brain-derived Neurotrophic Factor(Bdnf). BDNF is a secreted protein that has numerous important functions in nervous system development and plasticity, including neuronal survival, neurite outgrowth, synapse formation, and long-term potentiation. Due to Bdnf’s complex transcriptional regulation by various forms of neural stimuli, it is well positioned to function as a transducer through which altered neural activity states can lead to changes in neuronal physiology and synaptic function. In this dissertation, I show that different families of transcription factors, and even different isoforms or splice variants within a single family, can specifically regulate Bdnf transcription in an age- and stimulus-dependent manner. Additionally, I characterize another mechanism of synapse-to-nucleus signaling specificity that is dependent upon NMDA-type glutamate receptor subunit composition, and provide evidence that the effect this signaling pathway has on gene transcription is important for normal GABAergic synapse formation. Taken together, my dissertation research sheds light on several novel signaling mechanisms that could lend specificity to the activity-dependent transcription of Bdnf exon IV. My data indicate that distinct neuronal stimuli can differentially regulate the Calcium-Response Element CaRE1 within Bdnf promoter IV through activation of two distinct transcription factors: Calcium-Response Factor (CaRF) and Myocyte Enhancer Factor 2 (MEF2). Furthermore, individual members of the MEF2 family of transcription factors differentially regulate the expression of Bdnf, and different MEF2C splice variants are unequally responsive to L-type voltage-gated calcium channel activation. Additionally, I show here for the first time that the NMDA-type glutamate receptor subunit NR3A (also known as GluN3A) is capable of exerting an effect on NMDA receptor-dependent Bdnf exon IV transcription, and that changes in the expression levels of NR3A may function to regulate the threshold for activation of synaptic plasticity-inducing transcriptional programs during brain development. Finally, I provide evidence that the transcription factor CaRF might function in the regulation of homeostatic programs of gene transcription in an age- and stimulus-specific manner. Together, these data describe multiple novel mechanisms of specificity in neuronal activity-regulated gene transcription, some of which function at the synapse, others of which function in the nucleus.Item Open Access Modeling Multi-factor Binding of the Genome(2010) Wasson, Todd StevenHundreds of different factors adorn the eukaryotic genome, binding to it in large number. These DNA binding factors (DBFs) include nucleosomes, transcription factors (TFs), and other proteins and protein complexes, such as the origin recognition complex (ORC). DBFs compete with one another for binding along the genome, yet many current models of genome binding do not consider different types of DBFs together simultaneously. Additionally, binding is a stochastic process that results in a continuum of binding probabilities at any position along the genome, but many current models tend to consider positions as being either binding sites or not.
Here, we present a model that allows a multitude of DBFs, each at different concentrations, to compete with one another for binding sites along the genome. The result is an 'occupancy profile', a probabilistic description of the DNA occupancy of each factor at each position. We implement our model efficiently as the software package COMPETE. We demonstrate genome-wide and at specific loci how modeling nucleosome binding alters TF binding, and vice versa, and illustrate how factor concentration influences binding occupancy. Binding cooperativity between nearby TFs arises implicitly via mutual competition with nucleosomes. Our method applies not only to TFs, but also recapitulates known occupancy profiles of a well-studied replication origin with and without ORC binding.
We then develop a statistical framework for tuning our model concentrations to further improve its predictions. Importantly, this tuning optimizes with respect to actual biological data. We take steps to ensure that our tuned parameters are biologically plausible.
Finally, we discuss novel extensions and applications of our model, suggesting next steps in its development and deployment.
Item Open Access Modeling Nuclease Digestion Data to Predict the Dynamics of Genome-wide Transcription Factor Occupancy(2016) Luo, KaixuanIdentifying and deciphering the complex regulatory information embedded in the genome is critical to our understanding of biology and the etiology of complex diseases. The regulation of gene expression is governed largely by the occupancy of transcription factors (TFs) at various cognate binding sites. Characterizing TF binding is particularly challenging since TF occupancy is not just complex but also dynamic. Current genome-wide surveys of TF binding sites typically use chromatin immunoprecipitation (ChIP), which is limited to measuring one TF at a time, thus less scalable in profiling the dynamics of TF occupancy across cell types or conditions. This dissertation develops novel computational frameworks to model sequencing data from DNase and/or MNase nuclease digestion assays that allows multiple TFs to be surveyed in a single experiment, in both human and yeast. We predicted occupancy landscapes and constructed a cell-type specificity map for many TFs across human cell types, revealed novel relationships between TF occupancy and TF expression, and monitored the occupancy dynamics of various TFs in response to androgen and estrogen hormone simulations. The TF/cell type occupancy matrix generated from our model expands the total output of the ENCODE ChIP-seq efforts by a factor of nearly 200 times. These computational frameworks serve as an innovative and cost effective strategy which enables efficient profiling of TF occupancy landscapes across different cell types or dynamic conditions in a high-throughput manner.
Item Open Access Molecular Control of Morphogenesis in the Sea Urchin Embryo(2015) Martik, Megan LeeGene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development.
The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.
This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control.
In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.
In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.
The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.
Item Open Access Protein-DNA Binding: Discovering Motifs and Distinguishing Direct from Indirect Interactions(2009) Gordan, Raluca MihaelaThe initiation of two major processes in the eukaryotic cell, gene transcription and DNA replication, is regulated largely through interactions between proteins or protein complexes and DNA. Although a lot is known about the interacting proteins and their role in regulating transcription and replication, the specific DNA binding motifs of many regulatory proteins and complexes are still to be determined. For this purpose, many computational tools for DNA motif discovery have been developed in the last two decades. These tools employ a variety of strategies, from exhaustive search to sampling techniques, with the hope of finding over-represented motifs in sets of co-regulated or co-bound sequences. Despite the variety of computational tools aimed at solving the problem of motif discovery, their ability to correctly detect known DNA motifs is still limited. The motifs are usually short and many times degenerate, which makes them difficult to distinguish from genomic background. We believe the most efficient strategy for improving the performance of motif discovery is not to use increasingly complex computational and statistical methods and models, but to incorporate more of the biology into the computational techniques, in a principled manner. To this end, we propose a novel motif discovery algorithm: PRIORITY. Based on a general Gibbs sampling framework, PRIORITY has a major advantage over other motif discovery tools: it can incorporate different types of biological information (e.g., nucleosome positioning information) to guide the search for DNA binding sites toward regions where these sites are more likely to occur (e.g., nucleosome-free regions).
We use transcription factor (TF) binding data from yeast chromatin immunoprecipitation (ChIP-chip) experiments to test the performance of our motif discovery algorithm when incorporating three types of biological information: information about nucleosome positioning, information about DNA double-helical stability, and evolutionary conservation information. In each case, incorporating additional biological information has proven very useful in increasing the accuracy of motif finding, with the number of correctly identified motifs increasing with up to 52%. PRIORITY is not restricted to TF binding data. In this work, we also analyze origin recognition complex (ORC) binding data and show that PRIORITY can utilize DNA structural information to predict the binding specificity of the yeast ORC.
Despite the improvement obtained using additional biological information, the success of motif discovery algorithms in identifying known motifs is still limited, especially when applied to sequences bound in vivo (such as those of ChIP-chip) because the observed protein-DNA interactions are not necessarily direct. Some TFs associate with DNA only indirectly via protein partners, while others exhibit both direct and indirect binding. We propose a novel method to distinguish between direct and indirect TF-DNA interactions, integrating in vivo TF binding data, in vivo nucleosome occupancy data, and in vitro motifs from protein binding microarrays. When applied to yeast ChIP-chip data, our method reveals that only 48% of the ChIP-chip data sets can be readily explained by direct binding of the profiled TF, while 16% can be explained by indirect DNA binding. In the remaining 36%, we found that none of the motifs used in our analysis was able to explain the ChIP-chip data, either because the data was too noisy or because the set of motifs was incomplete. As more in vitro motifs become available, our method can be used to build a complete catalog of direct and indirect TF-DNA interactions.
Item Open Access The Regulation of Type 3 ILC and γδ T Cell Plasticity(2022) Parker, Morgan ELymphocytes take on effector programs coordinated by lineage-defining transcription factors (LDTF), resulting in the production of cytokines that fight specific types of pathogens. Therefore, both adaptive and innate lymphocyte lineages can take on specialized effector programs; the type 1 program mediated by T-bet for killing intracellular pathogens and tumors, the type 2 program controlled by GATA3 for protection against helminths, and the type 3 program mediated by RORγt for fighting extracellular bacteria and fungi. While each program can be defined by a single LDTF, many context-dependent situations arise that lead to more than one LDTF being expressed in a cell at a given time. The dual expression of LDTFs can result in the switching of effector programs within a differentiated cell. Nevertheless, LDTFs work in a cooperative manner with signal-dependent TFs and other TFs that sense environmental cues to ultimately control effector fates.
Environmental signals can be sensed by various classes of cell-surface receptors that modulate the downstream signaling effectors and subsequent transcriptional output of a cell for differentiation, proliferation, maintenance, and effector function. Surface receptors, such as the T cell receptor (TCR), cytokine receptors, and costimulatory receptors, translate the environmental cues into downstream signaling cascades that act in concert to promote the differentiation of lymphocyte subsets. Cytokines fine-tune the activation and repression of lymphocytes through phosphorylation of signal transducer and activator of transcription (STAT) TFs that translocate into the nucleus, bind DNA, and regulate gene expression at key loci. Acting alongside STAT TFs, AP-1 TFs are basic leucine zipper (bZIP) TFs that help translate environmental cues into effector programming through binding to key TF and effector cytokine loci.
The ability of a differentiated cell to switch to an alternative fate is referred to as plasticity. Innate lymphoid cells (ILCs) are remarkably plastic at steady state and fate-mapping studies in the mouse intestine revealed that RORγt+ ILCs (ILC3s) can upregulate T-bet and shut down RORγt expression for full conversion to a type 1 ILC (ILC1). ILC3s help maintain healthy mucosal barriers through the production of IL-22 that promotes the release of antimicrobial peptides from epithelial cells. ILC3 to ILC1 plasticity therefore results in a shift from IL-22 to IFNγ production. While increased IFNγ production can be protective against viruses and intracellular pathogens, it can result in many autoimmune and inflammatory diseases when dysregulated. Notably, ILC3 plasticity is implicated in Crohn's disease.
Although the environmental cues regulating ILC3 plasticity were somewhat known, the molecular mechanisms governing ILC3 plasticity were undefined. Here, we identified the AP-1 TF c-Maf as an essential regulator of ILC3 homeostasis and plasticity that limits physiological ILC1 conversion. Phenotypic analysis of effector status in Maf-deficient CCR6- ILC3s using flow cytometry revealed a skewing towards T-bet and IFNγ production. To determine the molecular mechanisms by which c-Maf supported the type 3 program, we evaluated the global changes in transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and transcription factor motif enrichment. We found that c-Maf promoted ILC3 accessibility and supported RORγt activity and expression of type 3 effector genes. Conversely, c-Maf restrained T-bet expression and function, thereby antagonizing the type 1 program. We performed ATAC-seq on transitioning subsets in the CCR6- ILC3 compartment all the way through conversion to ILC1s to understand the chromatin landscape changes taking place during ILC3 plasticity. These results solidified c-Maf as a gatekeeper of type 1 regulatory transformation and a controller of ILC3 fate.
Item Open Access Transcription Factors as Competitors in Gene Regulation and DNA Damage Repair(2022) Zhang, YuningTranscription factors (TFs) bind genomic DNA to regulate gene expression. In the cell, the genome is decorated with numerous proteins, including nucleosomes and proteins involved in processes such as DNA repair and replication, which could compete with TFs. While the competition with nucleosomes is well studied, TFs can also compete with other DNA-binding proteins (e.g. other TFs, DNA repair enzymes, polymerases). The rules and the impact of such competition remain largely unknown. Here, we investigate how TFs compete with each other and with repair enzymes, and we reveal the significant role TFs play as competitors in multiple pathways.To capture the binding profiles of competing TFs, we designed a quantitative cell-free assay that we applied to study Cbf1-Pho4 competition in yeast and MYC-MAD competition in human. We found that TFs greatly influence each other’s occupancy, in a way that is dictated by the proteins’ divergence in DNA-binding specificity. Analyses of ChIP-seq data confirmed that the patterns of TF-TF competition, as observed in vitro, are preserved in the nuclear environment. Furthermore, gene expression data suggests that Cbf1-Pho4 competition plays a critical role in the specific activation of target genes in the cell. In the MYC-MAD system, we found that quantitative in vitro knowledge facilitates the interpretation of in vivo ChIP-seq data and reveals subtle signals in gene regulatory networks, demonstrating the advantage of combining in vitro quantification with in vivo detection. Next, we adapted our assay to study the competition between TFs and DNA repair enzymes. Recently (Afek et al. 2020) we showed that TFs bind with high affinity to mismatches, which can result from replication errors. We thus hypothesized that TFs can compete with TDG, the glycosylase that recognize T-G mismatch and initiates base excision repair, and MutS, the mismatch-binding enzyme that initiates mismatch repair. Our high-throughput competition assay showed that, as predicted, the binding of both repair enzymes to DNA decreases significantly in the presence of TFs. In addition, the magnitude of the decreases in repair enzyme binding correlates well with the TF binding levels, indicating specific competition. This suggests that, in the cell, TFs bound to mismatches may affect repair and lead to increased mutagenesis at regulatory sites. Overall, our study proposes an approach for studying competition between DNA-binding proteins in a quantitative and high-throughput manner, and highlights the significance of this competition not only for gene regulation (where TFs are known to play an important role), but also in DNA repair.