Bcl11b is at the Nexus of Wnt and MAP Kinase Signaling Pathways in Two Stages of Thymocyte Development

Leukemogenesis, uncontrolled proliferation of dysfunctional, transformed immature lymphocytes, appears, especially in children, to be associated with abrogation of normal lymphopoiesis, and thymopoiesis in the case of T cell leukemias. Sequence specific transcription factors (SSTFs) are nuclear control switches that respond to cellular signals to alter cell state and cell fate by controlling gene transcription. Their activities are highly and precisely regulated by diverse means such as alterations in protein stability, post-translational modifications (PTMs) and/or protein-protein interactions. Several SSTFs have been identified as “T cell lineage-determining factors” essential for proper maturation of thymocytes into T cells in immune system development. Mutations or dysregulation of these T cell lineage-specifying SSTFs are associated with leukemogenesis. SSTFs act in coordination to regulate gene transcription in multi-protein complexes. There are millions of potential combinations of SSTFs available to fine-tune repression or activation of individual genes in response to multiple, complex extracellular and intracellular signaling inputs. To narrow our studies to SSTFs most likely to be of consequence in leukemogenesis, we focused on modifications and interactions between lineage-determining SSTFs in the thymocyte maturation process. Tcf1 is a T cell lineage-determining SSTF and has been identified as the nuclear effector of Wnt/Gsk3/β-catenin -dependent signaling. Bcl11b is a nuclear target of the T-cell receptor (TCR)-mediated MAP Kinase (MAPK) signaling cascade, but there is no indication from the literature that an interaction between Tcf1 and Bcl11b has been investigated. As an impetus for this investigation, another group demonstrated the ability of Bcl11b to regulate Wnt target genes in a different system, but a molecular link between the Wnt signaling pathway and Bcl11b was not established. Through our studies in primary murine thymocytes (mostly double positive (DP) cells), we identified a previously unknown physical interaction between Bcl11b and Tcf1, and among Bcl11B, TCF1 and β-catenin in Jurkat cells, a human T cell leukemia cell line. Our further studies in mutant Jurkat cells revealed that neither sumoylation nor up to 285 C terminal amino acids in Bcl11b are required for BCL11B to complex with TCF1 or β-catenin. We identified several thousand gene promoter regions that are co-occupied by the two factors, suggesting that the Bcl11b-Tcf1 interaction may coordinately regulate many genes required for thymocyte maturation. Using a gene reporter assay, we identified that the Bcl2l1 gene, encoding for the anti-apoptotic protein Bcl-xl, is co-regulated by BCL11B and TCF1. Treatment of thymocytes to mimic stimulation of the Wnt/Gsk3 signaling pathway also resulted in significant changes in the PTMs of Bcl11b, including increased sumoylation and decreased phosphorylation. Previous studies identified two kinases that are involved in modification of Bcl11b, Erk1/2 and p38 MAPK; in this study we identified a third candidate kinase, Gsk3, that may be responsible for basal phosphorylation. Inhibition of Gsk3 in thymocytes, mimicking canonical Wnt pathway activation, reduced the Tcf1-Bcl11b interaction, suggesting a potential mechanism by which Bcl11b regulates Wnt target genes. By investigating DN3-like P2C2 cells, a cell line from an earlier stage in thymocyte development relative to DP cells, we observed different patterns of PTM kinetics in response to signaling pathway activation, as compared to primary thymocytes. Treatment to mimic pre-TCR/MAP kinase pathway activation in P2C2 cells mostly replicated findings in primary thymocytes in terms of BCL11B PTM kinetic changes, with the P2C2 cells having a slightly delayed time course overall. However, this delay creates a situation in which desumoylation and dephosphorylation occurs simultaneously, in contrast to the opposing modifications by sumoylation and phosphorylation observed in DP thymocytes in response to MAPK pathway activation. Nevertheless, the opposite regulation of phosphorylation and sumoylation that we observed in primary thymocytes is mimicked under other conditions in the DN-like cells. Hyper-phosphorylation of Bcl11b after treatment with a broad phosphatase inhibitor coincided with near complete desumoylation. Additionally, activation of Wnt/Gsk3-dependent signaling in P2C2 cells resulted in composite dephosphorylation and sumoylation of Bcl11b. P2C2 cells displayed a higher basal level of β-catenin than primary thymocytes, and a higher ratio of long (β-catenin responsive) to short (β-catenin independent) Tcf1 isoforms. Unlike in DP thymocytes, we did not observe an interaction between Bcl11b and Tcf1 in the DN-like cells. Our results demonstrate that PTMs and specific interacting partners of Bcl11b in P2C2 cells are distinct from those previously characterized in DP thymocytes. Our results contribute to the existing knowledge regarding dynamic regulation of Bcl11b PTMs, and the interaction of Bcl11b with specific transcriptional complexes during distinct stages of T cell development. These findings may contribute to a better understanding of the molecular mechanisms that regulate Bcl11b transcriptional activity in different cell types, potentially leading to novel treatments for T cell leukemias

A Systems Biology Approach to Epigenetic Gene Regulation

The ability to control when, and how much of the genetic code is being expressed is the underlying principle behind gene regulation. Control of gene production is able to influence a cell's phenotype by determining which structural components of the cell's observable traits (shape, growth, and behavior) are made. In multicellular organism’s different cell types are able to arise from the same genetic code due to a difference in the patterns of genes being expressed. Essentially anywhere in the process of gene expression from transcription, RNA processing, translation, and post-translational modifications of the protein is subject to regulation. As transcription is the first step in the process of gene expression, it is the first level of regulation for influencing the cell phenotype. The actions of transcription factors, histone modifiers, and other proteins work together to influence RNA polymerase's ability to complete the process of transcription. The actions of transcription factors are able to influence transcription by controlling the ability of RNA polymerase to be recruited to the start of a protein coding region and histone modifiers can rearrange the histones of the chromatin causing entire regions of a chromosome to become exposed or sequestered. These transcriptional regulators are able to work in a combinatorial fashion with one another to either activate and/or repress wide repertoires of transcriptional targets. Mapping out a network of interactions between these transcriptional regulators in gene expression programs allows researchers to understand how each protein is able to influence the phenotype of the cell, and how mutations to any of these transcriptional regulators are able to drive the cell into a diseased state. In the case of cancer, changes in the mechanisms of gene regulation brought on by mutations to these transcriptional regulators may drive the cell's hyper proliferative state. With the creation of next generation sequencing researchers are now better able to define where regulation is taking place in the genome, and how much it is able to influence gene expression. This gives researchers the ability to build these gene regulatory networks and evaluate their impact on gene expression. The subsequent chapters of this dissertation are a reflection of my published work investigating the contribution of oncogenic processes to gene regulatory networks in cancer through the study of hyperactivating somatic mutation of a histone modifier, changes in transcription factor response element specificity, epigenetic regulation of transcription factor signaling, and a transcription factor coactivation network

The Role of the MLL-HOXA9 Axis in Normal and Malignant Hematopoiesis.

The MLL-HOXA9 axis plays a critical role in the regulation of development and hematopoiesis. Chromosome translocations of MLL are closely associated with human leukemia and constitutive activation of HOXA9 is required for the leukemogenesis. Although being studied for decades, important questions remain to be answered in this field. For example, how is the activity of MLL regulated during hematopoiesis? What are the mechanisms of MLL fusion-mediated transformation? And how does HOXA9 regulate gene expression and mediate leukemogenesis? The work described in this dissertation touches every aspect of the above questions. Chapter 2 and Chapter 3 address the first two questions by studying the function of the PHD/Bromo region of MLL. This region is invariably disrupted or deleted in oncogenic MLL fusion proteins and is incompatible to MLL fusion mediated transformation. We found that this region mediates MLL ubiquitination in multiple ways. The Bromodomain recruits ECS(ASB2) E3 ligase complex through interaction with ASB2. ECS(ASB2) mediates ubiquitination and degradation of MLL during hematopoietic differentiation, thus contributing to the down-regulation of MLL target genes, including HOXA9 and MEIS1. On the other hand, the second PHD finger (PHD2) of MLL has intrinsic E3 ligase activity in the presence of the E2 conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4. Further, mutation of PHD2 affects MLL stability and transactivation ability. Notably, the oncogenic MLL fusion proteins do not have the PHD/Bromo region, and are resistant to both degradation pathways. The increased protein stability may contribute to MLL fusion mediated transformation. In Chapter 4, the cooperation between Hoxa9 and its potential cofactors Cebpa and Pu.1 was studied using high throughput sequencing technologies. Analysis of ChIP-seq and RNA-seq data reveals that genome-wide binding of Cebpa and Pu.1 significantly overlaps with the binding of Hoxa9. Co-bound regions are enriched with the enhancer mark H3K4me1, and are enriched for hematopoiesis related pathways. Further, co-binding of Cebpa and Pu.1 associates with Hoxa9 regulated genes but does not predict activation or repression activity of Hoxa9.PhDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/100097/1/jywang_1.pd

Post-translational regulation of HD-Zip IV transcription factors in Arabidopsis

Doctor of PhilosophyDepartment of BiologyKathrin SchrickClass IV homeodomain leucine-zipper (HD-Zip IV) transcription factors (TFs) are master regulators of epidermal cell fate during embryonic and post-embryonic development in plants. GLABRA2 (GL2), the founding member of the HD-Zip IV TFs, plays critical roles in trichome and root hair development as well as in biosynthesis of anthocyanin, seed coat mucilage and seed oil. It has four defined domains including a DNA-binding homeodomain (HD), a leucine-zipper dimerization domain termed Zipper Loop Zipper (ZLZ), a Steroidogenic Acute Regulatory (StAR) protein-related lipid Transfer (START) domain, and a START-associated domain. Previous work demonstrated that the START domain of GL2 is involved in ligand binding and is required for TF dimerization, stability and activity. However, a detailed mechanism controlling the level of HD-Zip IV TFs in plants remains unknown. The overall goal of this project is to identify and characterize post-translational regulatory mechanisms affecting the turnover and activity of HD-Zip IV TFs in Arabidopsis. To determine protein half-lives, in vivo cycloheximide chase experiments were performed using Arabidopsis seedlings, uncovering a role for the ZLZ domain in destabilizing GL2. In addition, the Ubiquitin/26S proteasome system was implicated in the degradation of unstable GL2 protein resulting from START domain mutation. Next, bioinformatics tools predicted multiple, high confidence conserved ubiquitination and SUMOylation sites in the ZLZ domain of GL2 and the modifications were verified in vivo by performing immunoprecipitation experiments. Site-directed mutagenesis of the candidate amino acids resulted in a gain-of-function phenotype characterized by an increased frequency of abaxial leaf curling in lysine-to-arginine (K-R) mutants. In contrast to EYFP-tagged protein, plants expressing GL2 with a smaller 6x His 3x FLAG tag did not display leaf curling, suggesting that this phenotype is associated with increased protein stability. Ongoing experiments focus on RNA-Seq experiments to characterize the molecular mechanisms of abaxial leaf curling. In addition to ubiquitin and SUMO (small ubiquitin-like modifier) sites, two high confidence SUMO-interacting motifs (SIMs) were bioinformatically predicted in the GL2 START domain from Arabidopsis. SIMs are short hydrophobic sequences present in many proteins that can non-covalently bind to SUMO-conjugated proteins. Mutational analysis with confocal microscopy revealed defects in trichome branching and nuclear localization of the protein in SIM mutants. Yeast two-hybrid experiments failed to detect GL2-SUMO interactions as most of the Arabidopsis thaliana SUMO isoforms were not expressed in yeast. However, unlike wild-type protein, SIM mutants displayed impaired dimerization. These results indicate the role of SIMs in the subcellular localization and TF activity of GL2. Further experiments will be required to identify proteins that associate with GL2 through SUMO-SIM interactions. Overall, the findings suggest that regulatory mechanisms involving ubiquitin and/or SUMO contribute to GL2 levels and activity in plants. This work provides new insight into how the protein levels of critical transcriptional regulators of epidermal cell differentiation are fine-tuned in plants. The new knowledge will ultimately guide molecular toolkits for engineering HD-Zip IV TFs to establish more resilient and high yielding cultivars

From global to targeted chromatin proteomics: mapping the control unit of cellular identity

Cellular identity is established and maintained by the chromatome, which consists of transcriptional, epigenetic and structural regulators of the chromatin proteome. Serving as a control hub, the chromatome processes incoming signaling cues and modifies the transcriptional program, resulting in a specific cellular phenotype. To fully understand cell-type specific gene regulation, multi-level chromatome analysis is necessary. Chromatin-associated proteins can be explored using mass spectrometry (MS)-based proteomic methods to assess (i) total protein abundances, (ii) chromatin-associated individual complexes, (iii) global or (iv) locus-specific chromatin compositions, and (v) nucleotide and histone (post-translational) modifications. Global chromatin proteomics trails behind other areas of proteomics in terms of data comprehensiveness, accuracy, and throughput. The main aim of this work was the development of an MS-based proteomic method, Chromatin Aggregation Capture followed by Data Independent Acquisition (ChAC-DIA), which enables the comprehensive identification and accurate quantification of chromatin regulators, including those present in low quantities, across different pluripotency stages. ChAC-DIA identified 2-3 times more chromatin-associated proteins with enhanced accuracy and efficiency, required 100-fold less sample material, and halved the MS data acquisition time compared to prior methods. By applying ChAC-DIA an extensive atlas was constructed that encompasses proteomes, chromatomes, and chromatin affinities across the three key phases of pluripotency. The data served not only to verify bona fide pluripotency regulators such as REX1, OCT6 and SOX1, but also to identify new phase-specific factors like JADE1/2/3, QSER1, SUV39H1/2 and FLYWCH1. Moreover, this study offers a straightforward strategy for distinguishing between translation-driven changes in chromatin binding and alterations in nuclear localization or chromatin affinity. Using this approach, we observed that certain heterochromatic proteins, such as HP1β, KAP1, and SUV39H1, exhibited enhanced chromatin affinities towards the exit from pluripotency, which we could demonstrate to be a conserved feature in both mouse and human. In subsequent collaborative endeavors, chromatin proteomics was applied to study epigenetic regulations in several biological contexts. In three distinct projects, chromatin immunoprecipitation combined with MS was employed to analyze the interaction networks of the naive pluripotency marker DPPA3, the histone H3 lysine 9 trimethyl (H3K9me3) reader HP1β and the methylcytosine dioxygenase TET1. Moreover, the KAP1-dependent ubiquitinome was investigated, the composition of HP1β-driven phase-separated droplets was studied, and a proteomic workflow was developed to screen for the efficient incorporation of non-canonical amino acids into target proteins. This work also provided a detailed protocol for probing locus-specific chromatin composition as well as full proteome analyses upon genetic perturbations targeting epigenetic modifiers in an acute myeloid leukemia cell culture model and embryonic stem cells at various pluripotency stages. In a last collaboration, it was tested whether the histone methyltransferases SUV39H1/2 primarily contribute to H3K9me3 formation in visceral endoderm descendants. In summary, this work provides a powerful method to study the global chromatome of any model in development and disease, sheds new light on dynamic rearrangements of pluripotency governing regulatory complexes and contributes to a broad range of epigenetic research by harnessing multi-level chromatome analyses

Proteomics based approaches to identify DNA binding transcriptional regulators and phosphoproteins of ERG

Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-cell acute lymphoblastic leukaemia (T- ALL) and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remain poorly understood. In order to discover transcriptional regulators of ERG, I employed a quantitative mass spectrometry (MS)-based method to identify factors binding the ERG +85 stem cell enhancer region in MOLT-4 and KG-1 cells. Using this approach, I identified a number of known transcription factors (TF) bound to the ERG +85 enhancer in leukaemic cells along with previously unknown binders, ETV6 and IKZF1. I confirmed that ETV6 and IKZF1 were also bound in vivo at the ERG +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells (HSPCs). Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators of ERG and a number of genes regulated by a densely interconnected network of the heptad TF. Furthermore, I showed that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis. Protein phosphorylation is known to regulate the transcriptional activity of genes. Aberrant protein phosphorylation can lead to numerous haematological malignancies, but few studies have focused on phosphorylations of DNA bound TF at regulatory DNA. By adapting the reverseChIP method I develop an approach to identify and quantify of site-specific phosphorylation if TF on DNA in vitro using MOLT-4 and KG-1 cells. I have identified phosphorylated DNA bound TFs and 28 novel phosphorylation sites that are uniquely enriched at the ERG+85 enhancer under a leukaemia context. Oncoprotein ETS1 was identified as exhibiting changes in phosphorylation when bound to DNA compared to the nuclear input, this finding was also validated by western blot. This work significantly expands existing knowledge of the regulation of ERG over-expression driven by the ERG+85 enhancer in leukaemic and HSPCs cells. In addition, both reverseChIP methods can be utilised to study other regulatory regions of interest

Interplay Between P53 and Epigenetic Pathways in Cancer

The human TP53 gene encodes the most potent tumor suppressor protein p53. More than half of all human cancers contain mutations in the TP53 gene, while the majority of the remaining cases involve other mechanisms to inactivate wild-type p53 function. In the first part of my dissertation research, I have explored the mechanism of suppressed wild-type p53 activity in teratocarcinoma. In the teratocarcinoma cell line NTera2, we show that wild-type p53 is mono-methylated at Lysine 370 and Lysine 382. These post-translational modifications contribute to the compromised tumor suppressive activity of p53 despite a high level of wild-type protein in NTera2 cells. This study provides evidence for an epigenetic mechanism that cancer cells can exploit to inactivate p53 wild-type function. The paradigm provides insight into understanding the modes of p53 regulation, and can likely be applied to other cancer types with wild-type p53 proteins. On the other hand, cancers with TP53 mutations are mostly found to contain missense substitutions of the TP53 gene, resulting in expression of full length, but mutant forms of p53 that confer tumor-promoting “gain-of-function” (GOF) to cancer. In the second section of my dissertation, I have investigated the mechanism of this GOF property by examining genome-wide p53 binding profiles in multiple cancer cell lines bearing p53 mutations. This reveals an epigenetic mechanism underlying mutant p53 GOF. Various GOF p53 mutants bind to and upregulate genes including MLL1, MLL2, and MOZ, leading to genome-wide changes of histone modifications. These studies also demonstrate a critical functional role of the MLL pathway in mediating mutant p53 GOF cancer phenotypes, and that genetic or pharmacological perturbation of MLL function can achieve specific inhibition of GOF p53 cell proliferation. Overall, studies described in this dissertation demonstrate the crosstalk between p53 signaling and chromatin regulatory pathways, contribute to our knowledge of p53 cancer biology with respect to epigenetic regulation, and in the long term suggest new therapeutic opportunities in targeting cancers according to their p53 mutational status

Expanding the mass spectrometry toolkit for interrogating chromatin proteomics

Chromatin is comprised of DNA and a vast network of proteins, which help structure and regulate a cell’s genetic material through processes including cell differentiation, regulation of genes, and DNA repair. At the core of chromatin structure lays a nucleosome, consisting of DNA wrapped around an octameric protein complex made of histones. Histones can undergo post translational modifications (PTMs) which govern protein-protein interactions (PPIs) and ultimately control gene activation and suppression. Histone PTMs can already be quantitated using existing well defined methods such as liquid chromatography mass spectrometry (LC-MS). However with the increasing popularity of techniques such as chromatin immunoprecipitation aimed at identifying and understanding the role of PTMs under very specific circumstances, ever smaller amounts of histones are being produced and push heavily on the limits of LC-MS detection. We were able to reduce the number of cells required for a typical histone PTM LC-MS analysis from 10^6 cells to 10^5 cells and permitting technical replicates at this level. Chemical cross-linking mass spectrometry is another useful tool in characterising PPIs whilst simultaneously providing limited structural information, even from native cellular environments and cell lysates making it highly promising for chromatin. Much development has been made in this technology, however data analysis for this technique can still be difficult and laborious. We proposed to address this by simplifying the complexity of the data by altering the functional chemistry of the cross-linker, with limited levels success. Finally, some molecules have great therapeutic potential in addressing erroneous chromatin regulations that are implicated in a number of cancers. In order for more efficient therapeutics to be developed, it is important to identify how and where they bind to proteins. We were able to address this issue for a ligand-protein pair, for a protein known to be implicated in cancer and modify histones.Open Acces

Genetic tool development and metabolic engineering in model and non-model yeasts

The use of microbial cell factories such as yeast to convert plant-derived sugars and biomass into biofuels and other products offers a route towards a sustainable, carbon-neutral economy. Saccharomyces cerevisiae has been extensively engineered in efforts produce many classes of compounds. However, much of the genome of this best-studied eukaryote remains poorly characterized for fermentation-relevant phenotypes and therefore inaccessible to rational engineering. Genome-scale engineering involves the creation vast numbers of mutants, targeting all genes in the host genome and then screening the resultant library to identify novel genetic determinants of phenotypes of interest. Building upon previously developed genome-scale engineering strategies and CRISPR-based tools, we created a tri-functional CRISPR mutant library covering overexpression, downregulation, and deletion of each ORF in the BY4742 genome with 4-6 gRNAs per gene using CRISPR activation, interference, and deletion, respectively. The library was screened to identify genetic determinants of furfural tolerance, protein display, and increased production of S-adenosylmethionine (SAM) using next-generation sequencing, flow cytometry, and a fluorescent biosensor. Downregulation of SIZ1 combined with overexpression of NAT1 and downregulation of PDR1 was found to dramatically improve furfural tolerance and display of Trichoderma reesei endoglucanase II was improved by deletion of HOC1 and repression of NUP157. For SAM overproduction, FACS screening using a SAM-responsive biosensor identified the combination of upregulation of SNZ3, RFC4, and RPS18B to improve SAM productivity by 2.2-fold and 1.6-fold in laboratory and industrial yeast strains, respectively. Transcriptomic and metabolomic analyses of the engineered SAM-producing strains indicated that upregulation of vitamin B6 metabolism greatly increases the activity the transsulfuration pathway used to produce SAM. Using genome-scale engineering of laboratory yeast strains to inform and guide industrial yeast strain engineering presents an effective approach to design microbial cell factories for industrial applications. While S. cerevisiae has proven to be a highly effective platform for conversion of glucose to ethanol, its inability to grow natively on lignocellulosic sugars such as xylose and arabinose, and its preference for utilizing carbon flux for anaerobic fermentation over aerobic respiration limit its capability to process lignocellulosic biomass, and to produce acetyl-CoA-derived bioproducts such as terpenoids and fatty acids and their derivatives. Although S. cerevisiae has been extensively engineered to improve its capabilities in these roles, an alternative increasingly investigated by the scientific community is the exploration of other, “non-model” organisms with different metabolic properties. The oleaginous, red yeast Rhodosporidium toruloides, which can grow natively on lignocellulosic sugars and can naturally produce high levels of lipids and isoprenoids is one such organism. However, its large phylogenetic distance from other yeast species means that genetic tools and understanding of the metabolism of R. toruloides remain limited. We have sought to develop tools facilitating the genetic manipulation of R. toruloides and to apply them in the metabolic engineering of this non-model yeast. Firstly, a high-efficiency CRISPR knockout system was developed in the strain NP11. Cas9 expression was optimized using codon optimization and screening of various nuclear localization sequences and constitutive promoters of different strengths. Due to the lack of a suitable RNA polymerase III promoter for gRNA expression, a novel self-splicing fusion 5S rRNA-tRNA promoter was developed, allowing greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78% by expressing two tandem gRNA cassettes. This CRISPR knockout tool was applied in combination with overexpression of native and heterologous genes to optimize the titer of a fatty alcohol-producing strain expressing a fatty acyl-CoA reductase gene from Marinobacter aqueolei. Following co-expression of this gene and a Cas9 gene to facilitate creation of knockout mutants, a panel of metabolic engineering targets were explored. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The DNA-protein mapping technique CUT&RUN was used to identify the centromeric regions of R. toruloides IFO0880, an essential genetic element for the creation of a stable episomal plasmid (in combination with a replication origin). CUT&RUN was used to map genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and several were tested for, but did not show activity as replication origins in their own right. These centromeric sequences contained below average GC content, below average but variable gene density, and in most cases low to moderate sequence conservation. Future efforts to identify a replication origin in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative origin replication elements. Finally, two ongoing efforts in genetic tool development in R. toruloides are described. CRISPR activation and interference were explored through the fusion of dSpCas9 with various trans-activation and trans-repression elements. Efficient CRISPRi was obtained by fusing Cas9 with two tandem repression domains from S. cerevisiae transcription factors, while the commonly used VP64, VP64-p65, and VPR activation domains were not functional. Two Cas orthologs, SaCas9 and LbCpf1 were also tested for gene deletion in R. toruloides and were found to be functional following codon optimization. Efforts were also made to develop an inducible Cre/lox selection marker recovery system in the strain IFO0880. A Cre gene was codon optimized and found to be functional under constitutive expression. However, the inducible promoters tested so far based on reports in different R. toruloides strains, pLAD1, pCTR3, and pNAR1 were found to be either too leaky or nonfunctional in this strain. Characterization of additional promoters with tight but strongly inducible expression will enable the completion of this system in the future

Engineering and Delivery of Synthetic Chromatin Effectors

abstract: Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.Dissertation/ThesisDoctoral Dissertation Biological Design 201