Advanced imaging methods unveil new mechanisms transcriptional regulation in the context of nuclear organization

It has long been appreciated that organization of macromolecules within the cell is paramount to proper cellular function. As the number of tools in the molecular and cell biologist’s toolbox grows, the list of ways cells achieve organization also grows. This is especially true for the process of transcription and its regulation, where the fundamental questions remain a challenge to address, but where a convergence of genetic, genomic, structural and imaging techniques offer the possibility of answers. The proper loading of an RNA polymerase on a gene requires the coordinated action of hundreds of proteins and other macro molecules, as well as cellular genome to be maintained in a topologically permissive state; yet as a system, gene regulation requires interactions to be dynamic enough to respond to the changing needs of the cell in response to internal and external ques. How a cell constructs a system that is, on the one hand robust, and on the other hand flexible remains a challenge to answer.In Chapter 1 of this thesis, we will introduce the study of transcription regulation as a general topic, and discuss a number of fluorescence imaging techniques that have fundamentally changed the our understanding of transcription regulation. Chapter 2 will focus on one particular aspect of cellular organization which has recently come into vogue—that of membraneless compartment formation through liquid-liquid phase separation. A thorough investigation of the literature, particularly in light of the experiments which we foreshadow and introduce in more depth in Chapter 3, suggests that much more work is needed before phase separation as a means of biological organization can become a general paradigm. Chapter 3 presents a particularly poignant example of the issues raised in Chapter 2. Using Herpes Simplex Virus as a model system because of its ability to form compartments in the nucleus, we demonstrate that recruitment of Pol II and other proteins can be explained through the availability of nonspecific binding sites rather than phase separation. Lastly, Chapter 4 will present technical findings on the optimal fluorescent dyes to use for in vivo labeling of proteins for imaging applications.In summary, this dissertation underscores the way in which new imaging techniques reveal new biological principles and challenge old models. It will be of great interest to watch these ideas and techniques mature beyond the work presented in this dissertation

The role of Ultrabithorax negative autoregulation in Drosophila melanogaster

One of the more striking features of animal development is that a limited set of developmental control genes is used repeatedly, in different contexts (within an organism and between species), to form different structures. To achieve this, gene regulatory networks must be versatile. Transcription factors regulate target genes by acting combinatorially, and must be deployed with spatial, temporal, and quantitative precision. In addition to being versatile, gene regulatory networks are robust, enabling animal development to yield reproducible outcomes despite environmental and genetic variation. Focusing on the D. melanogaster Hox gene Ultrabithorax (Ubx), I explore how cis-regulatory elements of developmental control genes contribute to these two hallmarks of developmental biology: versatility and robustness. Ubx specifies the identity of the third thoracic (T3) segment along the anterior-posterior axis of the developing fly. It is required for the development of T3 appendages including the haltere - a dorsal appendage that helps the fly balance during flight. Not only is Ubx presence required, but its levels are also important: Ubx levels are inversely correlated with haltere size. In Chapter 2, we describe how Ubx negative autoregulation establishes different Ubx levels in two different spatial domains of the developing haltere: the proximal haltere (which forms the joint and body wall in the adult) and the distal haltere (which forms the capitellum - the appendage proper). Ubx directly represses its own transcription with the aid of Homothorax (Hth) and Extradenticle (Exd) in the developing proximal haltere. Distally, Hth is absent, Exd is cytoplasmic, and Ubx levels are high. We identify an enhancer that captures this regulation and identify a binding site for Ubx/Exd/Hth. In Chapter 3, we describe another function for Ubx negative autoregulation: promoting developmental robustness by buffering haltere size against changes in Ubx levels. Haltere size is inversely correlated with Ubx levels, but neither haltere size nor Ubx levels change in step with changes in Ubx copy number, suggesting the possibility of phenotypic buffering. Consistently, certain Ubx enhancer traps are silenced in response to increases in Ubx gene dose. Here, we show that functional Ubx protein must exceed a certain threshold to silence Ubx enhancer traps, confirming the idea that it reflects Ubx negative autoregulation at work. Together with the results from Chapter 2, this shows that a single gene can employ the same mechanism to achieve two seemingly opposing purposes: conferring variation and robustness to its expression. Finally, we investigate Ubx enhancer trap silencing in response to naturally occurring genetic variation. We previously described that the same Ubx enhancer traps that are silenced by increases in Ubx copy number are also silenced in F1 offspring of outcrosses to certain wild populations of D. melanogaster. Although it is unclear if this is due to Ubx negative autoregulation or an independent mechanism, our data argue that the Ubx locus, and not the P-element insertions themselves, are being silenced. Interestingly, we find that i) silencing is suppressed by a gain-of-function mutation in a gene that opposes the spread of heterochromatin and ii) the expression of Position Effect Variegation reporters also changes when outcrossed to certain wild populations of D. melanogaster. Together, these results suggest that there are considerable fluctuations in the transcriptional landscape between different populations of a given species

The emulation of fatty add synthetase genes in B.napus

The main aim of this work was to analyse a cloned ACP gene promoter, which had been shown to contain regulatory information that directed both spatial and temporal expression. Gel retardation assays were used initially to define protein binding sites within the ACP05 promoter. Following such analysis, a DNA motif that interacted with a sequence specific binding factor was identified. This factor was detected in embryo extracts and was not present in leaf extracts. The stability and binding characteristics of the ACP promoter binding protein were studied after heparin agarose chromatography, using gel retardation assays to follow the protein. A binding site oligonucleotide was synthesised and used as a probe to screen an expression library, in a "South Western" cloning experiment. A single positive clone, λBS2xi, was isolated. The DNA binding specificity of the recombinant protein was determined by gel retardation. The clone was confirmed to encode a functional sequence specific DNA binding domain. Northern hybridisations demonstrated the mRNA was expressed and in a tissue specific manner: levels of message were high in seed, low in root and not detected in leaf tissue. Endonuclease restriction of the lambda clone with EcoRI excised a 3.0kbp fragment that corresponded to the entire cDNA. The fragment was subcloned in pSK(^+) and several strategies were used to characterise and sequence it. A set of nested deletions was generated, but reliable sequence data was not obtained from the first half of the cDNA. A section of readable sequence data was obtained approximately 1.5Kbp from the 5' end of the deleted cDNA. Exhaustive databank searches using the sequence data demonstrated that it corresponded to β- galactosidase. Southern analysis further demonstrated that this sequence was present in λBS2xi. Further work required to characterise XBS2xi is discussed. A second related subject of this thesis concerns a second member of the FAS complex, enoyl- ACP reductase (ER). Prior to this work it was shown that there were four ER isoforms, expressed in both leaf and seed. The levels of expression of individual isoforms were different, there being two major forms and two minor forms. One leaf expressed clone, pERL8 had been isolated and characterised. A DNA probe that encompassed the 3' untranslated region (3'UTR) was generated from pERL8 and used to screen a cDNA library generated from embryo. Eleven positives were isolated and ten were successfully subcloned by plasmid rescue. The clones were sequenced with internal primers. Exhaustive searches of databanks using the sequence data demonstrated that five clones corresponded to seed storage proteins and five were confirmed as ER. The ER clones were divided into two groups on the basis of sequence differences. One clone was identical to pERL8 and represented an isoform expressed in both leaf and seed. The sequences of the clones within a group was identical, apart from an "insert" present in the 3' UTR of one clone from both groups. Whether these inserts were real or artifacts could not be demonstrated conclusively as further sequencing showed both clones with inserts in the 3' UTR were 5' truncated. In order to determine whether all four ER isoforms had been isolated further work would-be required. This is also discussed

Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand

EP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ-mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246-260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy

Advanced gene expression control in therapeutic human cells using synthetic transcriptional programs

An enduring goal of synthetic biology is to engineer cells to perform increasingly sophisticated therapeutic functions. Inspired by natural gene expression programs that elegantly govern diverse cellular behaviors, numerous technological advances have employed synthetic transcriptional programs to coordinate therapeutic cellular activities. These programs are mediated by heterologous or engineered transcription factors that orthogonally regulate the expression of therapeutic agents. However, key features of existing transcriptional components and programs render them unsuitable for therapeutic applications, including high potential for immunogenic or off-target effects and challenging delivery of large genetic payloads. There remains a need for well-designed elements that overcome these fundamental barriers to translation and ultimately enable customizable, tunable, and effective therapeutic responses. Here, we describe an advanced platform for synthetic transcriptional control poised for diverse therapeutic applications. We first engineered a class of programmable transcriptional regulators based upon zinc finger DNA-binding domains, which are highly advantageous due to their compact size and derivation from native mammalian transcriptional systems. We constructed a library of humanized synthetic transcription factors with binding motifs that were unique and putatively orthogonal to the human genome. Our evaluation of cellular transcriptome response when these synthetic components were expressed in cell lines revealed highly specific on-target and low off-target regulation. Furthermore, we developed two highly useful classes of regulatable gene expression programs, by connecting our synthetic transcription factors to domains responsive to favorable exogenous or endogenous signals. We first generated synthetic transcription programs responsive to safe and FDA-approved small molecules, and demonstrated how these programs could tunably and dynamically regulate the expression of therapeutic agents in relevant in vitro and in vivo contexts. Moreover, we constructed synthetic transcription programs responsive to desirable endogenous signals using customizable synthetic Notch receptors. Taken together, we envision that our advanced platform for synthetic transcriptional regulation will facilitate the design of sophisticated therapeutic programs for a wide range of gene and cell therapy applications.2022-01-18T00:00:00

Protein-protein interactions mediated by Cys2His2 zinc-fingers

The C2H2 zinc finger motif is a compact ~ 30 amino acid molecular recognition domain that comprises a beta-hairpin followed by an alpha-helix. These domains are typically found as tandem arrays that mediate specific interactions with various macromolecules including DNA, RNA and other proteins. Although very well characterized as a DNA-binding domain, relatively little is currently understood about the molecular details of protein-protein interactions mediated by C2H2 ZFs. The Ikaros and Hunchback transcription factor family provides an ideal model system for studying ZF mediated protein-protein interactions. Ikaros, the founding member of this family is defined as a classical C2H2 ZF protein composed of a cluster of four C2H2 ZFs at the N-terminus and two additional C2H2 ZFs at the C-terminus. While the N-terminal ZFs are involved in specific DNA recognition, the C-terminal domain (termed as Dimerization Zinc Finger or DZF domain) has been shown to mediate the homo- and hetero-typic interactions. In this thesis, the DZF domains found in the Ikaros and Hunchback transcription factor family have been examined using a combination of genetic, biochemical and functional assays. To test if protein-interacting C2H2 ZFs can be used to create novel protein-protein interaction specificities, libraries of synthetic DZFs were constructed by shuffling individual C2H2 ZFs from DZF domains found in the human Ikaros and other related transcription factors. Using a bacterial-based selection system, we identified synthetic heterodimeric DZFs that can mediate activation of a reporter gene in bacterial cells. These protein-protein interaction domains can also be used to reconstitute a synthetic bi-partite activator in the nucleus of a human cell, which results in transcriptional activation of the endogenous VEGF-A gene. In addition, these synthetic two-finger domains can be linked together to create more extended protein-protein interaction interfaces. Analysis of the interaction specificities of these domains led to the discovery of a novel anti-parallel interaction mode for the DZF domain. The homo-typic interaction mediated by different DZF domains was examined in greater detail using mutational analysis. These studies narrowed down residues that are likely to be important for dimerization mediated by the Hunchback DZF domain. To obtain further information about the physical and chemical interaction surface we attempted to purify active peptides consisting of different DZF domains for X-ray crystallography. Although highly purified DZF peptides were obtained, various attempts to refold these peptides into active domains resulted in the formation of aggregates consisting of the various DZFs. Based on findings in the bacterial and cell culture systems, we started exploring if Hunchback dimerizes in Drosophila melanogaster using its DZF domain and if dimerization is essential for the function of the protein. Constructs encoding the full-length Hunchback protein harboring various natural and modified DZF domains were generated and expressed in transgenic flies. These transgenics were used to perform functional in vivo studies of the Hunchback DZF domain in Neuroblast specification during Drosophila melanogaster development. We confirmed previous studies that the C-terminal domain in Hunchback is important for maintaining the function of Hunchback in specifying early-born temporal identity in Drosophila neural stem cell lineages. Importantly, our results indicate that this domain can be functionally replaced with a heterologous (i.e., non fly) DZF domain, suggesting that the importance of the DZF domain is due to its ability to mediate dimerization

Targeting therapeutic vulnerabilities associated with EWS fusion proteins in Ewing sarcoma

Ewing sarcoma (ES) is a are and aggressive bone tumour affecting children and young adults and that requires better therapeutic options to improve patient outcomes. ES is characterised by chromosomal rearrangements producing a fusion gene, the most predominant occurring between EWSR1 and FLI1 (85%). Recent evidence shows that the chimeric oncoprotein EWS-FLI recruits chromatin remodellers that epigenetically rewire transcription to establish its oncogenic programme. Additionally, transcriptional dysregulation is known to induce replication stress (RS) and genomic instability. To mitigate potential genotoxic damage, ES cells are particularly dependent on the replication stress response (RSR). Based on these EWS-FLI1-specific molecular effects, this thesis investigates two separate therapeutic strategies: (i) inhibition of the epigenetic modifier KDM1A, and (ii) exploiting the dependency on the RSR. Catalytic inhibition of histone demethylase KDM1A is demonstrated to be insufficient as a therapeutic strategy for ES, although roles beyond its demethylase function remain a possibility. To identify therapeutic combinations targeting the dependency on the RSR, clinically available drugs inhibiting the ATR-CHK1-WEE1 axis were tested in 3D spheroids of ES cell lines. Each drug candidate was combined at clinically relevant doses with SN-38, the active metabolite of topoisomerase I inhibitor irinotecan, currently used to treat relapsed ES. Combinations revealed cytotoxicity and decreased growth in ES spheroids following WEE1 and ATR inhibition, both concurrent with SN-38. Based on the strength of responses, further investigations prioritised the effects of the WEE1 inhibitor AZD1775 combined with SN-38 in additional ES cell lines and a model ectopically expressing EWS-FLI1. DNA damage, apoptosis, and cell cycle analysis uncovered two responses in ES cell lines, one characterised by cell death, the other resembling growth arrest. These may be dependent on the cell lines' mutational background and could act as a predictive biomarker. Taken together these findings identify a promising novel therapeutic strategy for ES

The Role of ZBP-89 in Globin Regulation

The ameliorating effect of increased γ‐globin gene expression in β‐thalassemia and Sickle cell disease, favored γ‐globin gene reactivation as the most attractive treatment approach. This has led to intense research efforts to elucidate the mechanism of γ‐globin gene switching with the aim of reversing the switching process. Many DNA cis elements (e.g. LCR, ‐117 HPFH mutation, BGL3 region) as well as trans regulatory protein such as BCL11a, LDB1, and KLF1 has been identified that are important for developmental globin genes regulation. This and other evidences from literature suggest the involvement of other protein factors, as yet unknown, on γ‐globin promoter or LCR that work remotely with previously known protein factors. We recently described an unbiased proteomics approach; Targeted Chromatin Purification (TChP), to identify other possible proteins involved in γ‐globin silencing. ZBP‐89 was identified as one of the proteins in γ‐globin chromatin purification. Here we report functional study on the role of ZBP‐89 in globin regulation

Detection and analysis of LIM domain-mediated interactions between transcription factors

LIM-homeodomain (LIM-HD) proteins are a class of transcription factors involved in tissue specification and cell determination during development and are important in adult gene regulation. Six families of LIM-HD proteins, with two close paralogues in each family, are commonly found in tetrapods. They bind DNA via HDs, whereas their interactions with other proteins are mediated mainly by a pair of closely spaced LIM-domains (LIMs) in each protein. These proteins take part in various transcriptional complexes with Ldb1 and other cofactors that contain LIM-interaction domains (LIDs). In this thesis, protein-protein interactions of LIM-HD proteins were analysed in order to better understand the molecular mechanisms of transcriptional complex formation. Based on previous research that showed LIM-LID mediated interactions between Lhx3 and Isl1, yeast two-hybrid mating arrays were used to investigate how widespread protein-protein interactions are amongst the 12 mammalian LIM-HD proteins. Due to high levels of background growth in experiments with full-length proteins in pGBT9 vectors, the mating arrays focused on LIM-domain mediated interactions with full-length LIM-HDs or known LIDs. The arrays revealed a relatively strong interaction between Lhx3 (or Lhx4) and Isl1 (or Isl2), and detected weaker interactions between Lmx1a or Lmx1b and the LIM-binding domain of Isl1. The contribution of separate LIM-domains to the overall interaction with Ldb1 for each of the proteins was analysed by the same method. In most cases one of the LIM domains in each protein was able to independently interact with the LID domain of Ldb1 by yeast two-hybrid analysis indicating a dominant binder: LIM1 in Isl1 and Isl2, or LIM2 in other proteins. The exceptions were paralogues Lhx1 and Lhx5, for which no separate domain showed interaction with Ldb1LID by this approach. All tandem LIM-domain constructs showed a much stronger interaction with Ldb1LID than any isolated LIM domain supporting the idea that both domains are required for high affinity binding to Ldb1. Bimolecular Fluorescence Complementation experiments in yeast were designed and conducted as an alternative approach to test interactions between full-length LIM-HD proteins in the hope that a non-transcription based assay would lead to no or less background signal compared to yeast two-hybrid analysis. A plasmid system was developed based on existing yeast two-hybrid vectors using split green fluorescent proteins in place of domains from the GAL4 transcription factor. The assay was able to detect interactions between different LIMs and their partners but unfortunately interactions between full-length proteins were still difficult to detect due to low fluorescence, self-complementation in the controls and localization effects. LIM domains from LIM-HD proteins cannot be used in standard bimolecular binding assays because they tend to be insoluble and/or aggregate in the absence of a binding partner. Stable, soluble intramolecular ‘tethered complexes’ can be generated in which LIMs are tethered to Ldb1LID via a flexible linker. Introduction of a specific protease site into the tether allows the formation of intermolecular cut complexes, which have previously been used in homologous competition ELISA experiments. In this thesis attempts were made to develop more robust biophysical binding assays that could be used to assess the binding affinities of different LIMs for Ldb1LID. Several different labelling approaches were used to generate proteins with fluorescent tags for use in fluorescence anisotropy assays. In one of these approaches expressed protein ligation was applied to generate proteins with an N-terminal fluorescein. Although this labelling strategy was of low efficiency for LIMs-Ldb1LID tethered constructs, some preliminary fluorescence anisotropy experiments were carried out, which indicated that this could be a useful strategy providing a more efficient labelling strategy can be found. GFP-tagged tethered complexes were easier to generate, but could not be used in anisotropy experiments because of the intrinsically high anisotropy of GFP proteins. However, preliminary experiments indicated that these proteins can be used in clear native gel shift competition assays to compare binding affinities of different tandem LIM domains to Ldb1LID. Data presented in this thesis provide valuable insight into protein-protein interactions of LIM-HD transcription factors and the advantages, as well as disadvantages, of applied experimental approaches. The results and their implications are discussed, raising questions that can be resolved in future studies