Drosophila E2F and DP genes : their role in the regulation of G1-S progression and the activity of DNA replication origins

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1998.Includes bibliographical references.Late in Drosophila embryogenesis the cells giving rise to most of the larval tissues switch from a mitotic cycle to the endo cycle. The endo cycle consists of only S phase and a gap phase, leading to polyteny. It is preceded by the first detectable G1 period in Drosophila embryogenesis and developmentally regulated induction of S phase genes in the pattern of DNA synthesis. My analysis of the regulation of G1-S progression began with a genetic screen for mutations that disrupt the transcription pattern of S phase genes in mitotic and polytene cells of the embryo. A number of mutations in known and novel genes were isolated. Further analysis and phenotypic characterization has focused on the mutations that nearly eliminate the transcription of S phase genes in mitotic and polytene cells. These mutations are in the Drosophila homologs to the mammalian E2F and DP genes. The E2F transcription factor, a heterodimer of E2F and DP subunits, is linked to the G1-S transition in mammalian cells. Five mutations in the Drosophila DP gene (dDP) were isolated and provide the first opportunity to examine the in vivo role of this gene. Despite a pronounced effect on the G1-S transcription of S phase genes in dDP and dE2F mutant embryos, a block to replication was not observed. Null mutations in dDP and dE2F cause lethality late in development with some mitotic and polytene tissues being underdeveloped or absent. The mutant phenotypes reveal a positive role for E2F/DP in cell cycle progression in mitotic and endo cycle cells. Weak alleles of dE2F and dDP develop to adulthood and exhibit defects in oogenesis. Analyses of these mutants showed that E2F/DP controls differential regulation of replication origins within polyploid S phase. In ovarian nurse cells, E2F/DP limits replication of heterochromatic sequences. In follicle cells, E2F/DP is required to shut off genomic replication and activate the amplification of chorion loci. In addition to the positive and negative effects on the activity of replication origins, E2F/DP is necessary for nuclear lamin breakdown in nurse cells and subsequent nurse cell apoptosis.by Irena Royzman.Ph.D

Purification and Characterization of Retinoblastoma like Factor-containing Protein Complexes from Drosophila melanogaster

The Retinoblastoma protein (pRb) was the first tumor suppressor protein to be identified. It is the founding member of the so called pRb or pocket protein family, comprising two additional members (p107 and p130) in mammalian cells, and its best characterized function is the regulation of the E2F family of transcription factors. Today, the pRb-E2F network represents one of the best understood pathways implicated in cell cycle regulation and differentiation. Pocket proteins negatively regulate the transactivation properties of E2F proteins by two mechanisms: First, binding of pocket proteins to E2F masks the E2F transactivation domain and thereby impairs transcriptional activation. Second, pocket proteins interact with several chromatin modifying and chromatin binding proteins and recruit these proteins to E2F target genes, where they help to establish a repressive chromatin conformation. In this work, advantage was taken of the relative simplicity of the Drosophila melanogaster pRb-E2F network to purify and functionally characterize native pRb repressor complexes. Two related multisubunit complexes that only differ in their pocket protein subunit (RBF1 or RBF2) have been purified from Drosophila embryo nuclear extract. These complexes contain several novel pocket protein-associated polypeptides and localize to transcriptionally silent regions on Drosophila polytene chromosomes. Moreover, they specifically associate with deacetylated histone tails, which are a hallmark of transcriptionally silent chromatin. In cycling Drosophila S2 cells, the purified complexes redundantly repress the expression of a certain class of E2F target genes implicated in differentiation and development, whereas they do not control the expression of cell cycle-regulated E2F targets. Interestingly, the isolated complexes seem to be highly conserved between different organisms. Genes encoding the Caenorhabditis elegans homologs of the complex subunits act within the same genetic pathway involved in vulval cell fate determination and they functionally cooperate in different developmental processes. Furthermore, a complex with striking homology to the Drosophila complexes also exists in human cells. In the light of the specific repression of developmentally regulated E2F target genes in cycling Drosophila cells, it is conceivable that the complexes prevent the uncontrolled expression of genes important during differentiation. Since the C. elegans homologs of the complex subunits are also involved in cell fate determination, this might be a highly conserved feature of the isolated complexes

The Human and Drosophila ERH are Functionally Equivalent: Evidence from Transgenic Studies

The enhancer of rudimentary, e(r), gene encodes a small highly conserved protein, enhancer of rudimentary homolog (ERH), which has been shown to have a regulatory function in cell division, Notch signaling, and cancer progression. Human and Drosophila ERH, both 104 amino acids in length, are 76% identical and 84% similar. The high sequence identity translates into nearly identical tertiary structures. Previous studies on the expression of the human and Drosophila e(r) genes reveal that the two genes are similarly regulated. Data in the present study using an e(r)-eGFP reporter gene confirm these results, showing a high expression of the reporter in the ovaries, testes, and brain. The high structural and regulatory conservation of e(r) and ERH argue that human and Drosophila ERH may be biochemically and functionally equivalent. To test this hypothesis, a chimeric transgene containing the Drosophila e(r) non-coding regions and the human e(r) coding region was constructed and used to establish transgenic Drosophila stocks. This transgene can rescue all of the mutant phenotypes of an e(r) deletion, and Drosophila stocks in which the fly ERH has been replaced with the human ERH are fully healthy and viable. These studies demonstrate that the human and Drosophila ERH are functionally equivalent, suggesting that studies on the activity of the human ERH can be done in Drosophila, where a multitude of genetic and developmental tools are available

A role for the DP subunit of the E2F transcription factor in axis determination during Drosophila oogenesis

The E2F family of transcription factors contributes to cell cycle control by regulating the transcription of DNA replication factors. Functional 'E2F' is a DNA-binding heterodimer composed of E2F and DP proteins. Drosophila contains two E2F genes (dE2F, dE2F2) and one DP gene (dDP). Mutation of either dE2F or dDP eliminates G(1)-S transcription of known replication factors during embryogenesis and compromises DNA replication. However, the analysis of these mutant phenotypes is complicated by the perdurance of maternally supplied gene function. To address this and to further analyze the role of E2F transcription factors in development we have phenotypically characterized mitotic clones of dDP mutant cells in the female germline. Our analysis indicates that dDP is required for several essential processes during oogenesis. In a fraction of the mutant egg chambers the germ cells execute one extra round of mitosis, suggesting that in this tissue dDP is uniquely utilized for cell cycle arrest rather than cell cycle progression. Mutation of dDP in the germline also prevents nurse cell cytoplasm transfer to the oocyte, resulting in a 'dumpless' phenotype that blocks oocyte development. This phenotype likely results from both disruption of the actin cytoskeleton and a failure of nurse cell apoptosis, each of which are required for normal cytoplasmic transfer. Lastly, we found that dDP is required for the establishment of the dorsal-ventral axis, as loss of dDP function prevents the localized expression of the EGFR ligand Gurken in the oocyte, which initiates dorsal-ventral polarity in the egg chamber. Thus we have uncovered new functions for E2F transcription factors during development, including an unexpected role in pattern formation

DNA-binding specificity of the E2F gene family, and cloning and characterization of a novel family mamber

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.by Brian A. Fairchild.Ph.D

Purification and Characterization of Retinoblastoma like Factor-containing Protein Complexes from Drosophila melanogaster

The Retinoblastoma protein (pRb) was the first tumor suppressor protein to be identified. It is the founding member of the so called pRb or pocket protein family, comprising two additional members (p107 and p130) in mammalian cells, and its best characterized function is the regulation of the E2F family of transcription factors. Today, the pRb-E2F network represents one of the best understood pathways implicated in cell cycle regulation and differentiation. Pocket proteins negatively regulate the transactivation properties of E2F proteins by two mechanisms: First, binding of pocket proteins to E2F masks the E2F transactivation domain and thereby impairs transcriptional activation. Second, pocket proteins interact with several chromatin modifying and chromatin binding proteins and recruit these proteins to E2F target genes, where they help to establish a repressive chromatin conformation. In this work, advantage was taken of the relative simplicity of the Drosophila melanogaster pRb-E2F network to purify and functionally characterize native pRb repressor complexes. Two related multisubunit complexes that only differ in their pocket protein subunit (RBF1 or RBF2) have been purified from Drosophila embryo nuclear extract. These complexes contain several novel pocket protein-associated polypeptides and localize to transcriptionally silent regions on Drosophila polytene chromosomes. Moreover, they specifically associate with deacetylated histone tails, which are a hallmark of transcriptionally silent chromatin. In cycling Drosophila S2 cells, the purified complexes redundantly repress the expression of a certain class of E2F target genes implicated in differentiation and development, whereas they do not control the expression of cell cycle-regulated E2F targets. Interestingly, the isolated complexes seem to be highly conserved between different organisms. Genes encoding the Caenorhabditis elegans homologs of the complex subunits act within the same genetic pathway involved in vulval cell fate determination and they functionally cooperate in different developmental processes. Furthermore, a complex with striking homology to the Drosophila complexes also exists in human cells. In the light of the specific repression of developmentally regulated E2F target genes in cycling Drosophila cells, it is conceivable that the complexes prevent the uncontrolled expression of genes important during differentiation. Since the C. elegans homologs of the complex subunits are also involved in cell fate determination, this might be a highly conserved feature of the isolated complexes

The regulation of E2F

The cellular transcription factor E2F plays a critical role in co-ordinating the transcription of target genes necessary for cell cycle progression. E2F interacts with important regulators of the cell cycle, such as the Rb tumour suppresser protein, related proteins p107 and p130 and cyclins and cyclin- dependent kinases. The cellular E2F activity is a heterodimer consisting of a DP family member, of which three members have been characterised and an E2F family member, of which six family members have been isolated. Murine DP-3 differs from the other DP proteins due to striking complexity at the RNA level. RNA analysis has shown that extensive processing gives rise to four different DP-3 proteins, alpha, beta, gamma? and sigma. These variants arise via two open reading frame changes, including the insertion of the E region, a 16 residue sequence within the DNA binding domain of DP-3 alpha and sigma, which forms part of a nuclear localisation signal. Additionally, the insertion of single glutamine codon has been noted in the DP-3gamma isoform. Extensive splicing within the 5'UTR of DP-3 results in translation initiation at two different methionine codons. Since DP-1 is the major component of the E2F DNA binding activity in mammalian cells and the role of DP-3 remains unclear, the aim of this study was to investigate the expression of DP-3 at the RNA and protein level, in an attempt to understand the role of DP-3 in the E2F heterodimer. In an effort to understand the post-transcriptional control of DP-3, the murine DP-3 gene was isolated. Analysis of the exon/intron arrangement of the DP-3 gene and comparison of the DP-3 genomic and cDNA sequences provided insight into the post-transcriptional regulation of DP-3, in particular at sites such as the E region. Additionally, comparison with the murine DP-1 gene revealed striking conservation in genomic organisation, suggesting that they are ancestrally related. Analysis of DP-3 RNA via Northern blotting was performed to study the expression pattern of the different DP-3 RNAs. A range of different mouse tissues and tissue culture cell lines were tested for the presence of DP-3 RNA and DP-3 RNA abundance was analysed during cellular processes such as differentiation. An investigation into the effects of the different DP-3 5'UTRs on translational regulation of DP-3 protein expression was performed using chimeric DP-3 5'UTR-luciferase reporter constructs. The translational potential of each 5'UTR was analysed by transient transfection in a range of mammalian cells and was found to be different for each 5'UTR. The translational ability of each 5'UTR was also analysed in vitro. The effect of the tumour suppresser p53 on the translational ability of the DP-3 5'UTRs was also assessed. p53 is known to influence the translation of both its own RNA and that of cdk4. Analysis suggested that p53 might influence the translation of specific DP-3 isoforms. To aid the detection of DP-3 protein, anti-peptide polyclonal antibodies were made. These were used to study DP-3 expression by western blotting in a range of different mammalian cells and by immunostaining. These results imply that the expression of DP-3 is highly regulated at the post-transcriptional level. Although a definite role for DP-3 in E2F mediated processes has yet to be assigned, these results provide insight into the control of DP-3 expression, which may ultimately be linked to the role of DP-3 in cells

Functional characterization of the role of Imp, a Drosophila mRNA binding protein, during oogenesis

textEstablishment of cell polarity requires the involvement of several posttranscriptional regulatory mechanisms, including mRNA localization and translational control. A family of highly conserved RNA binding proteins in vertebrates, VICKZ (V̲g1RBP/V̲era, I̲MP-1, 2, 3, C̲RD-BP, K̲OC, Z̲BP-1) proteins, has been shown to act in these two processes. Previous studies of the posttranscriptional mechanisms mediated by VICKZ family members have been largely limited by the lack of genetic approaches in certain vertebrate systems. Identification of Imp, the Drosophila member of the VICKZ family, opened the possibility to use genetic approaches to investigate the roles of a VICKZ family member in mRNA localization and translational control. In this dissertation, we show that Imp is associated with Squid and Hrp48, two heterogeneous proteins (hnRNP) that complex with one another to regulate localized expression of gurken (grk). In addition, Imp binds grk mRNA with high affinity in vitro and is concentrated at the site of grk localization in midstage oocytes. Mutation of the Imp gene does not substantially alter grk expression, but does partially suppress the grk mis-expression phenotype of fs(1)k10 mutants. In contrast, overexpression of Imp in germ line cells results in mislocalization of grk mRNA and protein. The opposing effects of reduced and elevated Imp activities on grk expression suggest that Imp acts in regulation of grk expression, but in a redundant way. To further explore the mechanisms by which localized expression of grk is regulated by Imp, a deficiency screen was conducted to search for dominant modifiers of the dorsalized phenotype resulting from Imp overexpression. Twelve genomic regions were identified to contain dominant modifiers of the Imp overexpression phenotype. Further characterization of mutants of genes within these genomic regions led to identification of five modifiers, including cyclin E (cycE), E2f transcriptional factor 1 (E2f1), lingerer (lig), snail (sna) and mushroom body expressed (mub). E2f1 encodes a transcriptional factor that is involved in regulating the G1 to S phase transition during mitosis. Mutation of E2f1 results in altered grk mRNA and protein distribution within oocyte, revealing a role for this gene in regulation of grk expression.Cellular and Molecular Biolog

Der Drosophila-Nephrozyt als Werkzeug zur Validierung von Genen, die eine Rolle in der Regulation der glomerulären Filtration spielen

Im Zuge dieser Arbeit wurden die in genomweiten Assoziationsstudien (GWAS) mit einer veränderten glomerulären Filtrationsrate (GFR) assoziierten Gene AMPKγ, CG9413, CG16717, CG32702, Dachshund, DP Transkriptionsfaktor und PIP5K59B, sowie die microRNA 210 (mir210) hinsichtlich ihres Einflusses auf die Morphologie und Funktion der Filtrationsbarriere der Niere untersucht. Dies geschah am Modell der Drosophila melanogaster, deren Nephrozyten bezüglich Entwicklung, Morphologie und Funktion stark den menschlichen Podozyten ähneln (Weavers et al. 2009). Meine Untersuchungen konnten einen Einfluss der Kandidatengene auf die Funktion und teilweise auch auf die Morphologie der Nephrozyten zeigen. Für einen Zusammenhang zwischen mir210 und der Nierenfunktion konnte ich in meinen Untersuchungen keinen Hinweis finden, da weder die Morphologie der für die Filtration wichtigen kortikalen Randzone noch die Nephrozytenfunktion beeinträchtigt waren. Drosophila-Nephrozyten stellen damit ein einfaches und schnelles Werkzeug dar, um die Funktion konservierter Gene für die Filtrationsbarriere zu analyiseren und mögliche Kandidatengene zu validieren