No description available.

In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.

Transcription, the first step in gene expression, is a highly regulated process which relies on a multi-protein complex to occur. Among these proteins are transcription factors, including initiation and elongation factors, which play differing roles in early and late stages of transcription. The mechanisms of transition from transcription initiation to elongation are not well understood in archaea, nor are the structures of the transcription factors involved. For transcription to occur in vitro, transcription factors TATA binding protein (TBP) and Transcription Factor B (TFB) are sufficient to allow RNA polymerase (RNAP) to synthesize RNA from template DNA. Another factor, Transcription Factor E (TFE), can also join the initiation complex and is likely to be essential in vivo. TFE is known to contribute to initiation by enhancing promoter opening, and while it has been shown to persist in elongation complexes, its role after initiation is unknown. Spt4/5, the archaeal homolog of the only universally conserved RNAP-associated factor, is known to join complexes in elongation steps and enhance processivity of the polymerase. However, if Spt4/5 joins pre-initiated complexes, it has been shown to inhibit transcription activity. The experiments in this thesis show that TFE and Spt4/5 participate in a crucial interchange at the upstream fork of the transcription bubble that helps define the timing of Spt4/5 binding. Using unnatural amino acid crosslinking techniques, the points of proximity between specific regions of these two factors and the template DNA have been mapped to identify possible sites of interaction. Competitive crosslinking assays indicate the exact timing of the shift in affinity between TFE and Spt4/5 for their shared binding site on RNAP. These data, combined with transcription assays, suggest a new role for TFE in preventing premature Spt4/5 binding, corresponding with a unique localized mobility within the winged helix of TFE.

During neural development, axons rely on extracellular cues to reach their target regions. Although extracellular signaling is one of the principal determinants for the growth of developing axons, only a small handful of known signaling cues has been identified. The existence of some 86 billion neurons of different subtypes, which ultimately form numerous functional circuits in the human nervous system, means an enormous number of extracellular cues would be required during development. Current views hold that even if more extracellular cues were to be discovered, they would never number large enough to account for the complexity of the human nervous system. Rather, intracellular signaling pathways and other cell-intrinsic mechanisms expand the ways in which a neuron can respond to extracellular cues by tuning the degree of responsiveness to them. Cell-intrinsic signaling pathways also give axons the ability to actively control their own development. These pathways can operate independently of the extracellular environment or even independently of the cell body, where the majority of protein synthesis takes place. For example, the local translation of proteins in the axon gives it autonomous control to immediately respond to changing demands in the environment. Local translation also occurs in other cell types, but the compartmentalized control over growth is especially important for neurons since the axon can extend up to a meter away from the cell body. In addition to local translation, axonally derived transcription factors, which can be locally synthesized in or localized to the axon, provide another means to control axon development. Axonally derived transcription factors act as physiological sensors and relay information about events happening in the periphery back to the cell body in order to effectuate a global response. It has recently been shown that transcription factors belonging to the OASIS family are activated by proteolysis in axons. Following their activation by proteolytic cleavage, the transcriptionally active N-terminus of these factors is transported to the cell body to activate global transcriptional pathways. For at least one OASIS family member, CREB3L2, this cleavage event simultaneously produces the C-terminus, which is capable of undergoing secretion. The secreted C-terminus of CREB3L2 acts as an accessory ligand for the activation of Hh pathways in chondrocytes. The generation of two bioactive proteins from one transcription factor, a transcriptionally active portion and a secreted portion, raised the question of whether there was a local function for OASIS transcription factors in axons. Through my research, I identified a mechanism in which DRG axons secrete the C-terminus of CREB3L2, which promotes axon growth in a paracrine manner. CREB3L2 is a transcription factor whose translation is induced by physiological ER stress. For CREB3L2 to be active, it must be cleaved by S2P, which I found is expressed in developing axons. Following proteolysis of CREB3L2 by S2P, the secreted C-terminus of CREB3L2 promotes the formation of Shh and Ptch1 complexes along axons. I found that upon depletion of the secreted CREB3L2 C-terminus, binding of Shh to the Ptch1 receptor is diminished. Returning the CREB3L2 C-terminus to the cultures exogenously was sufficient to rescue the formation of these complexes. These results highlight an intrinsic role for Shh signaling in developing DRG axons. Moreover, these results demonstrate how ER stress machinery is recruited to axons and promotes axon outgrowth. Finally, these results illustrate a novel, neuron-intrinsic mechanism by which developing axons actively regulate their own growth.

No description available.

Abstract not available

The transcription factor Pax5 is essential for commitment to the B lineage as the development of these cells is arrested at an early stage in the bone marrow of Pax5 deficient mice. Pax5 deficient pro-B cells display remarkable plasticity and are able to differentiate into other cell lineages both in vitro and in vivo. Several Pax5 target genes have been previously reported but none are able to explain the developmental block observed at the early to late pro-B cell stage. To determine the exact mechanisms by which Pax5 controls B cell development, I have undertaken a cDNA microarray screen with a custom generated B cell-specific cDNA library. By identifying genes that are differentially expressed between Pax5 deficient and wild type pro-B cells, I have identified a number of potential Pax5 target genes. The microarray screen identified lymphoid-restricted membrane protein (Lrmp or Jaw1) as a novel Pax5 activated transcript. Using RT-PCR and a Pax5-estrogen receptor inducible system, I confirmed that Jaw1 is a direct Pax5 target gene whose expression is confined, in resting cells, to the earliest stages of B and T cell development. The biological relevance of Jaw1 for cell fate specification has been tested by transducing bone marrow progenitor cells with murine retroviral vectors and reconstituting haemopoiesis in vivo. I have reported that over-expression of Jaw1 in these cells results in a decrease in the development of B and NK lymphocytes and a marked increase in the development of myeloid cells in the bone marrow of reconstituted mice. This result suggests that Jaw1 may play an important role in the early development of lymphocytes in the bone marrow. Whilst initial analysis of Jaw1 deficient mice has revealed no overt defects in lymphopoiesis, I postulate that Jaw1 is involved in IP3-induced calcium signaling downstream of the pre-BCR and BCR. This hypothesis has resulted from analysis of the function of IRAG, the only known Jaw1 homologue combined with data that Jaw1 is expressed in early B cells in the BM as well as in germinal centers in the spleen. Pax5 mutant mice usually die before weaning and the cause of death is currently unknown, suggesting that Pax5 is expressed in a previously unreported tissue. To investigate this hypothesis I produced a monoclonal antibody to Pax5 and screened for novel expression domains during embryonic development and also in neonate mice. These studies did not detect any new Pax5 expression domains, but did reveal that this antibody cross-reacts with Pax2 and/or Pax8 in the developing kidney and brain. Biochemical analysis of the serum from Pax5 deficient mice also did not reveal the likely cause of death in these animals, beyond the general signs of dehydration and starvation that are likely to be secondary to the underlying defect. / Doctor of Philosophy (PhD)

Gene expression and its regulation is a highly coordinated system, involved in many biological processes such as cell growth, division and differentiation. Transcriptional regions, involved in gene regulation, consist of a heterogeneous collection of smaller regulatory elements. In some cases, co-regulated genes contain a common set of transcription factor binding sites (TFBS). Analysis of promoter regions is the major approach in understanding the transcriptional regulatory mechanisms. It is also useful for interpretation of mammalian gene expression studies, where co-expressed genes may share motifs representing putative TFBS. Motif identification also has the advantage that it can predict control regions in genes that have not been measured experimentally. However, a common problem is incomplete genomic sequence for the experimental species of interest. The approach here is to identify and use orthologous gene promoter sequences from a related and well-characterised species. The primary aim of this study was to identify and predict regulatory TFBS in species where promoter sequence does not exist or is incomplete. The MEME programme was employed for the motif prediction step. The predicted elements were subsequently compared to known TFBS using TRANSFAC and JASPAR databases for identification. A methodology based on relative entropy was used. The validity of the method was confirmed as the predicted motifs in the training set were the expected sites involved in regulation of muscle development. The technique was applied to two data sets, generated from expressed sequence tag (EST) clustering analysis and microarray experiments. All data sets, software and results are available on the accompanying CD. Bovine expression data was analysed for cardiac-specific expression using two separate approaches, combining bovine library EST frequency and human gene expression ratios. For each approach, the orthologous human and bovine promoter sequences were analysed for common motifs. Across all comparisons, 37% of motifs were identified as known TFBS using the TRANSFAC and JASPAR databases. As the human comparison had more promoter sequences available, this was the main limiting factor for the corresponding bovine analysis, rather than cross-species divergence or accuracy of gene expression measurement. Results from this study demonstrate that using promoter sequences from a related species is a viable approach when studying gene expression in species with limited amount of genomic sequence. As the bovine genome becomes more complete, it can in turn serve as the reference genome for other agriculturally important ruminants, such as sheep, goat and deer. The second application concerned in silico analysis of gene regulation patterns in response to stimuli. Recently it has been shown that a mutation in the bone morphogenetic receptor IB leads to an increased ovulation rate in sheep. The objective of this study was to analyse gene expression patterns in cultured cells in response to four members of the BMP family, i.e. BMP2, BMP4, BMP6 and BMP7 and the control TGFβ. Microarray data was provided by J. Young. Twelve highly upregulated genes were stimulated by all BMPs, seven of which are known BMP target genes. Analysis of the predicted motifs identified four elements that may be involved in the regulation process. Cross-species comparison for one of the genes, ID1, showed high conservation of one of the motifs across 11 mammalian genomes. This particular motif had not been identified as a known binding site. In summary, the analysis of the expression data suggest an extension of the list of BMP targets. The proposed method is relatively robust when sufficiently co-expressed (co-regulated) sequences can be identified, whether from the same or another species.

"October, 2004" / Bibliography: leaves 118-154. / xii, 154 leaves : ill. (some col.), plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Microbiology and Immunology, 2005

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.