Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

RNA Localization and Translational Regulation on the Endoplasmic Reticulum

mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.

Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.

UNLABELLED: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

Targeted Gene Repression Technologies for Regenerative Medicine, Genomics, and Gene Therapy

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Exploring Mechanisms of Bacterial Adaptation to Seasonal Temperature Change

This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

The Role of TET Proteins in the Epigenetic Regulation of Neural Gene Expression and Behavior

Understanding how genes affect behavior is critical to develop precise therapies for human behavioral disorders. The ability to investigate the relationship between genes and behavior has been greatly advanced over the last few decades due to progress in gene-targeting technology. Recently, the Tet gene family was discovered and implicated in epigenetic modification of DNA methylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). 5hmC and its catalysts, the TET proteins, are highly abundant in the postnatal brain but with unclear functions. To investigate their neural functions, we generated new lines of Tet1 and Tet3 mutant mice using a gene targeting approach. We designed both mutations to cause a frameshift by deleting the largest coding exon of Tet1 (Tet1Δe4) and the catalytic domain of Tet3 (Tet3Δe7-9). As Tet1 is also highly expressed in embryonic stem cells (ESCs), we generated Tet1 homozygous deleted ESCs through sequential targeting to compare the function of Tet1 in the brain to its role in ESCs. To test our hypothesis that TET proteins epigenetically regulate transcription of key neural genes important for normal brain function, we examined transcriptional and epigenetic differences in the Tet1Δe4 mouse brain. The oxytocin receptor (OXTR), a neural gene implicated in social behaviors, is suggested to be epigenetically regulated by an unknown mechanism. Interestingly, several human studies have found associations between OXTR DNA hypermethylation and a wide spectrum of behavioral traits and neuropsychiatric disorders including autism spectrum disorders. Here we report the first evidence for an epigenetic mechanism of Oxtr transcription as expression of Oxtr is reduced in the brains of Tet1Δe4-/- mice. Likewise, the CpG island overlapping the promoter of Oxtr is hypermethylated during early embryonic development and persists into adulthood. We also discovered altered histone modifications at the hypermethylated regions, indicating the loss of TET1 has broad effects on the chromatin structure at Oxtr. Unexpectedly, we discovered an array of novel mRNA isoforms of Oxtr that are selectively reduced in Tet1Δe4-/- mice. Additionally, Tet1Δe4-/- mice display increased agonistic behaviors and impaired maternal care and short-term memory. Our findings support a novel role for TET1 in regulating Oxtr expression by preventing DNA hypermethylation and implicate TET1 in social behaviors, offering novel insight into Oxtr epigenetic regulation and its role in neuropsychiatric disorders.