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Thesis advisor: Michelle Meyer / Prokaryotic ribosomes are key to cell viability and an important area of study in model bacterial organisms. Some ribosomal proteins negatively regulate their own synthesis and that of the polycistronic operons they occur within. If levels of an autoregulatory ribosomal protein are higher than necessary for normal ribosome assembly, it binds to the 5’-untranslated region of its own mRNA transcript, preventing further translation of itself and any other proteins on its operon. We and others have shown bacteria growth defects when overexpressing ribosomal proteins (e.g. L20 and S6:S18); therefore, we hypothesized that an overabundance of autoregulatory proteins would negatively affect cell fitness due to decreased expression of the operon gene products, many of which are essential components of the ribosome. The regulation of ribosomal proteins is best described in E. coli, so we decided to use it as a model organism to investigate how overexpression of specific ribosomal proteins would affect cell growth. We examined the effects of overexpressing ribosomal proteins S15, S20, S2, S6:S18, S8, L20, L10, S1, L25, L7 and L1 on cell growth. We find the most severe growth defect in response to L20 overexpression. We performed rescue experiments for L20, L10, and S6:S18 by synthetically overexpressing the entire operon rather than just the regulatory protein. We find that this rescues the fitness of S6:S18 overexpression slightly, and L20 and L10 overexpression to a high degree. We also examined whether homologs of L20, L10, and S7 from B. subtilis and T. thermophilus induce the same changes in growth to deduce the regulatory interrelationships between different bacterial phyla. Bacillus L20 and L10 overexpression both showed drastic fitness defects. As our arsenal of effective antibiotics dwindles, our results suggest that targeting the ribosomal protein operons may be an effective area for pharmaceutical development. / Thesis (BS) — Boston College, 2017. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Arts and Sciences Honors Program. / Discipline: Biology.

Pax6 is a highly conserved developmental regulator with a complex temporal, spatial and quantitative expression pattern, that is crucial for correct development of the central nervous system, the eye, and pancreas. Accordingly, the Pax6 gene resides in a complex genomic locus containing a large array of long-range tissue-specific cis-regulatory elements primarily identified through multispecies sequence conservation and reporter studies. I have set out to understand how the chromatin architecture of the locus contributes to the mechanism and specificity of cis-regulatory interactions. As well as addressing whether the DNA looping model for regulatory interactions applies to the mouse Pax6 locus, I will identify which elements facilitate such interactions and if they vary between cell types. Utilising ChIP-array technology the distribution and variability of key regulatory histone modifications and factors were assessed in a set of Pax6 expressing and non-expressing mouse cell lines, acting as models for different regulatory states of the locus. Work in other loci suggests a key role for CTCF and cohesin (subunit Rad21) in chromatin organisation and long distance regulatory interactions. ChIP-chip for CTCF/Rad21 across the Pax6 locus identified numerous sites within the gene and at distal regulatory locations. The majority of these sites are cell type invariable. The active enhancer modification H3K27ac identified both known and several novel putative enhancer elements distributed through the locus that are highly cell type specific. A subset of CTCF/Rad21 sites also acquire the active enhancer modification H3K27ac in a cell type dependent manor, suggesting that CTCF/Rad21 may facilitate looping to the target gene from these sites. Using reporter based assays, putative regulatory elements marked by the looping factors and active histone modifications showed a diverse range of functional activities. Unexpectedly only 3 of the 7 CTCF sites tested showed classical insulator activity in an enhancer blocking reporter assay. Surprisingly the strongest insulator tested resided within intron 7 the Pax6 gene. Other CTCF/Rad21 sites were neutral or enhancers in the insulator assay. This reveals the disparity between predicting regulatory properties using ChIP binding profiles alone and the actual outcome of functional reporter experiments. A novel element, CTCF6 showed a ChIP signature of CTCF/Rad21/H3K27ac in all Pax6 expressing tissues, and functioned as a strong enhancer in transient transfection and stable LacZ reporter assays. CTCF6 recapitulated a broad range of Pax6 expression patterns, at multiple embryonic stages, including the brain, neural tube and pancreas. A second novel element, E-120 identified in the pancreatic derived cell line, drove stable embryonic reporter expression in the embryonic pancreas and sub set of brain regions. Together this has expanded the repertoire and size of Pax6’s regulatory landscape particular in the upstream region. Chromatin conformation capture (3C) was used to characterise the dynamic chromatin architecture of the locus and identify the interaction profiles from three CTCF/Rad21 binding regulatory locations within the Pax6 locus. This revealed a core set of regulatory interactions with the Pax6 gene, while individual elements showed a more variable set of cell type specific interactions. The CTCF6 enhancer showed highly cell type specific promoter interactions throughout the Pax6 gene, indicative of enhancer-promoter looping not detected in the non-expressing cells. While the downstream site CTCF5 at the edge of a cluster of regulatory elements known as the DRR (differentially regulated region), interacted with both the gene and an upstream element CTCF7 300 kb away only in the Pax6 expressing locus. Together these results reveal Pax6 has a chromatin hub structure with regulatory loops from upstream and downstream bringing distant yet variable active elements in to the vicinity of the Pax6 promoters where they can act. This work has revealed new roles for CTCF/cohesin sites in transcriptional regulation of Pax6 and how the cis-regulatory activity and structure of the locus varies across different cell types.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

Caenorhabditis Elegans genome maintains active H3K36me3 chromatin domains interspersed with repressive H3K27me3 domains on the autosomes’ distal ends. The mechanisms stabilizing these domains and the prevention of position-effect variegation remains unknown as no insulator elements have been identified in C. elegans. De-novo motif discovery applied on mes-4 binding sites links the H3K36me3-specific methyltransferase to a class of non-coding DNA known as Periodic An/Tn Clusters (PATCs). PATCs display characteristics of insulator elements such as local nucleosome depletion and their restriction to genes with specific expression profiles and chromatin marks. Finally, I describe a set of experiments to further investigate the role of PATCs and mes-4 in the maintenance of stable chromatin domains using a synthetic biology approach.