Anatomy and Neuroscience - Theses
Permanent URI for this collection
Search Results
1 - 4 of 4
-
ItemThe KH-domain protein Psi fine-tunes transcription of developmental genes to pattern cell and tissue growth in Drosophila( 2019)Mammalian FUBP-family proteins play roles in both transcription and RNA processing, by binding single stranded nucleic acids via their KH domains. Particularly, in vitro mammalian tissue culture studies suggest that FUBP1 drives MYC transcription in response to growth stimuli. MYC is a key regulator of growth and cell cycle progression essential for normal development. Furthermore, expression of MYC is aberrantly upregulated in approximately 70% of all cancers. Understanding the mechanisms that control MYC expression may lead to insights into how MYC dysregulated in diseases such as cancer. However, the significance of the transcriptional control by FUBP1 proteins at the MYC promoter and genome-wide has remained unclear in the context of development in whole organisms. Mammalian studies have been complicated by the fact that mammals have three FUBP-family proteins that bind overlapping genomic targets. However, in Drosophila, the three mammalian FUBP proteins are represented by one ortholog, Psi. Due to a high degree of structural similarity between FUBP1 and Psi proteins, we hypothesized that Psi will also regulate transcription of MYC and other targets. We have taken advantage of the reduced functional redundancy in Drosophila to demonstrate that an essential in vivo function of Psi is control of cell and tissue growth. Analysis of published Co-IP-mass spectrometry screens positioned Psi in an interactome predominantly comprised of RNA Polymerase II transcriptional machinery, particularly the transcriptional Mediator (MED) complex, a known sensor of developmental signalling inputs. Our work indicates that a key transcriptional target of the Psi/MED network that impacts tissue growth is Myc. During development of the wing disc epithelial tissue, Psi was required to promote Myc expression in response to activated PI3K pathway signalling. In addition to Myc, Psi binds to multiple transcriptional targets in the wing epithelium in vivo. Intersection of RNA-seq to detect differentially expressed genes following Psi depletion, and wing-specific direct binding sites identified genome-wide by Targeted DamID, revealed a developmental patterning signature for Psi including Wnt, Notch and TGF-beta cell fate determinant pathways. Moreover, analysis of direct Psi targets identified several novel growth regulators, including the TGF-beta signalling regulator tolkin (tok) and the CD36 scavenger receptor-related epithelial membrane protein (emp). Together our data suggest Psi integrates cellular signalling inputs to directly modulate transcriptional outputs and fine-tune development.
-
ItemCharacterisation of innate immune gene activities in rested and stimulated states( 2017)Monocytes and macrophages are among the first line of response to an infectious agent. The quality of this response will influence the type of T-lymphocyte recruited and may determine the outcome of an infection. While much is known about the repertoire of genes expressed by monocytes and macrophages in response to an acute challenge, little is known about the potential to diversify these responses through the generation of transcript isoforms. The role of transcriptional complexity in determining immune responses to stimulation has not been previously addressed in primary human monocytes. Transcriptional complexity is a measure of the abundance and diversity of transcript isoforms expressed in a genome. It has previously been demonstrated on a few genes in human myeloid cells that transcript isoforms can be expressed in a stimulus-specific manner and play important roles in immune responses. Although the expression of different transcript isoforms has been previously evaluated through the study of alternate splicing, the contribution of alternate transcription start sites has not been systematically addressed. We used CAGE-seq and ATAC-seq to characterise the extent of altering transcription start site usage following exposing monocytes to different stimuli. The comparison of the differences in alternate transcription start site usage between monocytes and macrophages revealed that these two cell types have a similar repertoire of transcription start sites. However, the transcriptional complexity focuses were different in these two cell types. Monocytes displayed dynamic changes in the alternate transcription start site usage in response to distinct stimuli, suggesting its plastic roles in immune responses. Although the mechanisms underlying the transcription start site selection have not been determined in this study, we proposed a model that differential recruitment of transcription factors to distinct promoters was predicted to initiate transcription from different transcription start sites. We have identified the potential transcription factors that were used to select transcription start sites in different conditions. The better understanding of the transcriptional complexity in primary human monocytes will assist gaining better insight into the involvement of monocytes in immune responses.
-
ItemFine-tuning expression of dMYC: how the single stranded DNA binding proteins Hfp and Psi regulate dMYC expression in Drosophila( 2015)MYC proteins are critical regulators of growth and cell cycle progression essential to animal development (Lee and Quinn, 2014; Lee et al., 2012; Levens, 2010). MYC is overexpressed in approximately 70% of all cancers (Dang, 2010; 2014). Understanding the molecular mechanisms that control MYC activity is crucial to understand normal development and may lead to insights into MYC dysregulation in diseases such as cancer. This thesis investigates the regulation of MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to environmental and cellular signaling pathways to coordinately regulate MYC transcription and cell growth. Based on in vitro studies we predicted that the presence of paused, but transcriptionally active, RNA polymerase II (RNA Pol II) on the MYC promoter is required for a rapid response to cell signaling. Of particular interest to this project, David Levens' ex vivo cell culture studies have shown that in response to mitogenic signals in serum the single stranded DNA binding protein Far upstream element Binding Protein (FBP) is enriched on the promoter of the MYC oncogene. FBP binding is associated with depletion of RNA Pol II from the promoter and increased MYC transcription (Liu et al., 2006). Subsequent to the peak in MYC mRNA, FBP Interacting Repressor (FIR) is detected at the FUSE and is required for the return of MYC transcription to basal levels. Moreover, the FIR protein is required for repression of MYC transcription via interaction with the general transcription factor (TFIIH) complex (Liu et al., 2006; 2001), and is dysregulated in human colorectal cancer (Matsushita et al., 2006). This mechanism of MYC repression appears to be conserved between mammals and Drosophila, as we have demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC (dMYC) in a manner dependent on the XPB helicase subunit of the TFIIH complex (Mitchell et al., 2010). Xeroderma Pigmentosum (XP) is a rare autosomal-recessive disease characterized by pigment changes, premature ageing and in a few patients malignant tumour development (Cleaver, 2005; Oh et al., 2006). Moreover, cancer progression occurs in certain patients, but not others, with identical C terminal mutations in the XPB helicase (Oh et al., 2006). The issues of compound heterozygosity and mixed genetic backgrounds have compounded problems of resolving the disease mechanism in the small number of XPB patients. Thus mechanisms driving overproliferation and cancer associated with these XPB mutations are currently unknown (Cleaver, 2005). With Drosophila we have used single alleles and, for the first time, made clear connections between genotype and phenotype. Using Drosophila models we show that C-terminally truncated XPB/Hay alleles enhance tissue overgrowth due to reduced abundance of FIR/Hfp, to further impair transcriptionally repression of dMYC. Thus, we predict defective transcriptional repression of MYC by FIR/Hfp might provide one mechanism for spontaneous cancer progression in XP patients. Dissecting the role of the FBP protein in MYC regulation has been complicated by the fact that mammals have multiple FBP family members (FBP 1, 2 and 3), which bind highly overlapping transcriptional targets (Chung et al., 2006). Originally, based on the finding that FBP is required for activated MYC transcription in vitro (He et al., 2000; Liu et al., 2001), we predicted FBP might play a role in activating RNA pol II release and MYC transcript elongation. We used Drosophila models to test whether the sole FBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that depletion of FBP/Psi reduced dMYC mRNA levels, suggesting FBP/Psi is normally required for maintaining endogenous levels of dMYC. Moreover, ChIP for poised RNA Pol II (Ser 5) proximal to the dMYC transcription start site revealed that FBP/Psi depletion reduced RNA Pol II Ser 5 enrichment. Together these data suggest FBP/Psi is required for maintaining poised RNA Pol II on the dMYC promoter and for maintaining dMYC transcription in vivo. Based on David Levens' extensive in vitro studies, we predict that FBP/Psi and FIR/Hfp are required for “fine-tuning” signal stimulated MYC transcription. We conducted a candidate screen of known mitogenic signals to identify those capable of most strongly stimulating dMYC transcription in vivo. The Ras/ERK pathway was the most robust activator of the dMYC promoter, and was able to significantly increase dMYC mRNA abundance. Importantly, we demonstrated that Ras-stimulated dMYC transcription was significantly reduced by depletion of FBP/Psi. Thus, endogenous levels of FBP/Psi are required for maximal stimulation of dMYC transcription by mitogenic signals in vivo.
-
ItemPsi regulates transcription of the MYC oncogene in Drosophila melanogaster( 2014)The MYC oncogene drives cell growth and consequently, increased MYC is associated with most human cancers. Thus tight regulation of MYC is critical and this thesis investigates one mechanism for controlling MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to the environment in order to activate MYC transcription and cell growth. Based on in vitro studies we predict that the presence of paused, but transcriptionally active, RNA polymerase II in the MYC promoter will be required for a rapid response to the cellular environment. Of particular interest to this project, human tissue culture studies from David Levens group (NIH, Bethesda) have shown that in response to serum the single stranded DNA binding proteins FUBP and FIR are recruited to the promoter of the MYC oncogene, and are associated with release of paused RNA Polymerase II. Moreover, the FIR protein is required for repression of MYC transcription, which occurs via interaction with the general transcription factor TFIIH complex. This mechanism for MYC repression appears to be conserved between mammals and Drosophila, as the Quinn lab has demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC in a manner dependent on interaction with the TFIIH complex. However, dissecting the role of the FUBP protein (FUBP1) in MYC regulation has been complicated by the fact that mammals have multiple FUBP family members (FUBP 1,2 and 3), which bind highly overlapping transcriptional targets. Originally, based on the finding that FUBP1 binds the MYC promoter prior to FIR, we predicted FUBP1 might play a role in activating RNA Polymerase II release and MYC transcript elongation. This thesis has used the Drosophila melanogaster model system to answer the question of whether the sole FUBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that Psi knockdown significantly reduced dMYC mRNA levels, which suggests Psi may be normally required for maintaining dMYC transcription in vivo. In line with a direct role in regulating MYC transcription, Chromatin Immunoprecipitation (ChIP) revealed enrichment for Psi on the dMYC promoter. Moreover, ChIP for poised RNA Pol II (Serine 5) proximal to the dMYC transcription start site, revealed that Psi knockdown results in a reduced RNA Polymerase II Serine 5 enrichment, consistent with the reduction in dMYC mRNA in the Psi knockdown. Together the experiments detailed in this thesis provide the first evidence that MYC is regulated by RNA Polymerase II pausing in vivo. Moreover, we demonstrate that Psi, the ortholog of mammalian FUBP1, is normally required for accumulation of the activated (Ser 5 phosphorylated) RNA Polymerase II on the dMYC promoter and, thus, for the regulation dMYC transcription.