Developmental Regulation of DNA Replication Initiation
in Drosophila
by
Fang Xie
B.S. in Biology (2001) Beijing University, Beijing, China Submitted to the Department of Biologyin Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biology
at the
Massachusetts Institute of Technology August, 2007
© 2007 Fang Xie. All rights reserved.
The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part
in any medium now known or hereafter created.
Signature of Author……… Department of Biology August 17, 2007 Certified by………...………. Terry L. Orr-Weaver Professor of Biology Thesis Supervisor Accepted by………..………. Stephen P. Bell Chair, Committee of Graduate Students Department of Biology
Developmental Regulation of DNA Replication Initiation
in Drosophila
by Fang Xie
Submitted to the Department of Biology
on August 17, 2007 in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biology
ABSTRACT
Developmental gene amplification in the ovarian follicle cells of Drosophila provides a powerful system for the study of metazoan DNA replication. Amplification produces 100kb gradients of amplified DNA through repeated rounds of origin firing and bidirectional movement of replication forks from these origins. The Drosophila Follicle Cell Amplicon at the cytological location 62D, DAFC-62D, is uniquely regulated, with two separate rounds of amplification in developmental stages 10 and 13 of egg chamber development. We investigated mechanisms that control the unusual timing of DAFC-62D origin activation. We first defined origin sequences in DAFC-62D by analyzing the amount of nascent replicative DNA across this amplicon. Surprisingly, the origin coincides with the coding region of a gene named yellow-g2. ORC2 localizes to the origin, as well as two other sites that do not confer origin activity. Both ORC2 and MCM2-7 display differential association with these sequences, corresponding to the two rounds of amplification. All three elements, dispersed in a 7kb central amplified region, are required for either round of DAFC-62D amplification, because deleting any one completely abolished amplification in transposon experiments. Preceded by transcription
yellow-g2 in stage 12, the late round of origin firing was ablated by the RNAPII inhibitor
α-amanitin. This effect was absent from other amplicons and insulated transposons, and was stage-13 specific for amplification at either the endogenous DAFC-62D or
heterologous transposons that did not have functional insulators. Therefore amplification at DAFC-62D in late follicle cell differentiation depends on transcription in cis.
Molecularly, blocking RNAPII transcription compromises MCM2-7 recruitment. Additional transposon and histone modification analyses confirmed the involvement of RNAPII in amplification control, which may be facilitated by favorable chromatin structure. This work provides insights in developmental regulation of origin firing, revealing one mechanism for initiation of metazoan DNA replication: recruitment of MCM2-7 by RNA polymerase II transcription.
Thesis Suporvisor: Terry L. Orr-Weaver Title: Professor of Biology
Dedicated to Lin Li 李 林 Xingui Liang 梁新桂 and Shangfa Xie 谢尚发
Acknowledgements
This thesis work would have been impossible without the full support of my advisor Terry Orr-Weaver. She accepted me (and my ideas) with an open mind, her guidance throughout the years has made graduate school less obscure, and she always inspires me to achieve more. I thank her for being the best advisor I could ever have asked for.
I am thankful to all past and current Orr-Weaver lab members with whom I shared numerous memorable moments, scientifically and non-scientifically. Julie Claycomb made the initial observations and lent tremendous help in establishing the DAFC-62D project. Discussions with Eugenia Park and Jane Kim, the replication subgroup people, have been inspiring. Everyone else, especially Tama Resnick, Jillian Pesin, David Doroquez, Yingdee Unhavaithaya, Lena Kashevsky and Raissa Formina, have made the TOW zone such a fun workplace.
My current thesis committee members, Steve Bell, Jianzhu Chen, and Troy Littleton, have been fantastic mentors and incredibly supportive. I am grateful for Professor Nick Dyson’s tremendous help as the outside member of my defense
committee. I also thank Ilaria Rebay and Paul Garrity for their advice in past committee meetings. The regular Drosophila replication meetings with David MacAlpine, Cary Lai and Steve Bell have been a constant driving force of my research.
I would like to express my gratitude to my husband, Lin Li, and my parents, Xingui Liang and Shangfa Xie, for their unconditional love. I never said “thank you” enough, and could never thank you enough. You are my rock. You will stay my rock. In the pursuit of my dreams.
TABLE OF CONTENTS Chapter One
Introduction: Activation Control of Replication and Amplification Origins 6
Gene amplification as a model for DNA replication 7
Origins of DNA replication 11
Origins of developmental gene amplification 15
Amplification control elements 23
The involvement of transcription factors in gene amplification 27
Chromatin context and amplification activity 34
A general link between replication and transcription 36
Summary of thesis 40
Chapter Two
Identification of a Drosophila Replication Origin
Developmentally Controlled by Transcription 50
Summary 51
Introduction 52
Results 55
Identification of the replication origin and ORC binding sites in DAFC-62D 55
Differential pre-RC binding in DAFC-62D 59
ORC-binding sequences are required for amplification 62 The two rounds of origin firing are interspersed by transcription 66 α-amanitin specifically inhibits DAFC-62D stage 13 amplification 70 Inhibition of transcription affects MCM2-7 localization 75
Discussion 78
Experimental procedures 86
Chapter Three
Conclusions and Future Directions 101
Differential localization of pre-RC 103
Transcriptional regulation of replication initiation 106
Distinct mechanisms of replication regulation 109
Insulators and their insensitivity to α-amanitin 112
Transcription factories 114
Appendix One: Analyses of the ACE3-ori62 Transposon 120
Appendix Two: Histone Acetylation and Amplification Activity 136
Chapter One
Introduction:
Gene amplification as a model for DNA replication
Developmental gene amplification is a process that increases the number of DNA molecules as template for transcription at specific developmental points. It has been reported in a variety of organisms as an alternate strategy to produce large quantities of transcripts over relatively short periods of time. Among the first observed examples is the amplification (about a thousand fold) of the genes that code for ribosomal RNA (rRNA) during the development of Xenopus oocytes, in order to stockpile the egg with the translational machinery necessary for rapid embryonic development (Brown and Dawid, 1968; Gall, 1968). Electron microscopy studies suggest that the Amphibian rDNA is amplified via a rolling-circle mechanism (Hourcade et al., 1973). Another example of extrachromosomal gene amplification is the rDNA in the transcriptionally active macronucleus of the protist Tetrahymena (Gall, 1974; Yao et al., 1979). The 21kb minichromosome in the form of a palindrome comprises two copies of the rDNA and is amplified up to 10,000 copies. This differs from the Amphibian oocyte rDNA however, because these palindromic minichromosomes are not produced by a rolling-circle
mechanism, but rather by bidirectional movement of the replication forks initiated from a defined origin (Figure 1A) (Kapler et al., 1996; Prescott, 1994).
Dipteran flies, including Rhynchosciara americana (Glover et al., 1982),
Bradysia hygida (Laicine et al., 1984), and Sciara coprophila (Wu et al., 1993) all utilize
gene amplification at multiple loci throughout the genome in the larval salivary glands, presumably for the production of large quantities of the structural proteins for the
construction of the cocoon. Note that unlike Tetrahymena rDNA amplification, in these organisms the gene clusters are replicated above the copy number of surrounding
sequences without forming extrachromosomal molecules. The same strategy is employed by another Dipteran fly, Drosophila melanogaster, to amplify at least four groups of genes in the ovarian follicle cells (Claycomb et al., 2004; Spradling, 1981; Spradling et al., 1980). Two of these gene clusters contain genes that encode the major structural proteins of the chorion (eggshell) (Spradling et al., 1980). It is possible that in Dipteran flies the intrachromosomal structures generated by the amplification process may be tolerated, as both the larval salivary gland and ovarian follicle cells are terminally differentiated tissues and are lost during further development. Because these cell types are nondividing, genomic aberrations accumulated during developmental gene
amplification would not be passed on to daughter cells.
Developmental gene amplification in both Tetrahymena and Dipteran flies has been consistently shown to utilize the normal replication machinery to repeatedly initiate DNA replication from dominant origins, resulting in an “onionskin” structure of nested replication bubbles/forks (Figure 1B) (Claycomb and Orr-Weaver, 2005; Tower, 2004). This provides an advantageous model for studying metazoan DNA replication for several reasons. First, amplified regions (amplicons) are relatively well defined especially given the recent employment of Comparative Genomic Hybridization (CGH) arrays (Claycomb et al., 2004). Second, the repeated firing generates a gradient of DNA copy number with the central origin(s) being the most abundant (Claycomb et al., 2002). This allows
Figure 1. Models of bidirectional replication versus amplification.
(A) Replication origin fires once and only once per cell cycle, followed by bidirectional movement (elongation) of replication forks. (B) During developmental amplification, the origin is activated multiple times consecutively. The resulting multiple replication forks form an “onionskin” structure, with the highest DNA copy number at the amplification origin.
and provides insights into the mechanisms by which the usual once-per-cell-cycle control of DNA replication can be thwarted. Third, developmental gene amplification occurs at strategic developmental times, providing the opportunity to study how DNA replication responds to developmental cues. Finally, a range of molecular and genetic tools are available in these model organisms.
Origins of DNA replication
The best-studied eukaryotic origins are those in the yeast Saccharomyces
cerevisiae. Specific, well-defined origins of DNA replication have been revealed
primarily through genetic analyses (Newlon and Theis, 1993). Consisting of an 11bp A-T-rich autonomously replicating sequence (ARS) consensus sequence (ACS) and other elements (B1 and B2) (Figure 2A), the yeast replication origins first recruit a variety of factors known as the pre-replication complex (pre-RC) (Figure 2A) to initiate DNA replication. As a component of the pre-RC, the six-subunit ORC specifically recognizes the ACS and the B1 element. Following loading of ORC, the other pre-RC factors and additional replication factors are recruited and origins are subsequently activated (Bell and Dutta, 2002). Although the protein factors appear to be highly conserved from yeasts to higher eukaryotes, the DNA sequences that define origin activity in different
organisms are not (Cvetic and Walter, 2005). Furthermore, in vitro studies in higher eukaryotes suggest that the metazoan ORC does not rely on sequence specificity to bind DNA (Remus et al., 2004; Vashee et al., 2003).
Figure 2. Classes of eukaryotic replication origins and composition of the pre-RC. (A) S. cerevisiae ARS1 is a well-defined replication origin. The 11bp ACS (and the B1 element) is specifically recognized by ORC, which together with Cdc6, Cdt1 and MCM2-7 constitute the pre-RC. The Origin of Bidirectional Replication (OBR) has been mapped immediately adjacent to the ORC-binding site. (B) The CHO DHFR locus contains a broad initiation zone, spanning the entire 55kb intergenic region between the DHFR and
2BE212 genes. Three sites show higher initiation activity. (C) The human lamin B2
replicon is markedly less complicated. The exact Transition Point (TP) from
discontinuous to continuous DNA synthesis has been determined by RIP mapping at the nucleotide resolution. See text for references. Blue pointed bars represent the coding frame of genes.
Physical mapping techniques have been developed to locate origins of DNA replication. Two-Dimensional (2D) gels separate replicating from nonreplicating molecules and allows the analysis of replication intermediates. This method is of relatively low resolution, revealing origins of replication as small as 2kb (DePamphilis, 1999). More sensitive approaches such as nascent strand analysis that employ PCR to determine the abundance of nascent strands improved the resolution of initiation sites to a few hundred basepairs (Giacca et al., 1994; Kobayashi et al., 1998a). Recently developed Replication Initiation Point (RIP) mapping has achieved nucleotide resolution by accurately defining the Transition Point (TP) from discontinuous to continuous DNA synthesis (Bielinsky and Gerbi, 1998; Gerbi and Bielinsky, 1997), and positioned the origin of bidirectional replication (OBR) immediately adjacent to the ORC-binding site in yeast (Figure 2A) (Bielinsky and Gerbi, 1998; Bielinsky and Gerbi, 1999).
In contrast to our knowledge of yeast origins, what constitutes a replication origin in higher eukaryotes is poorly understood (Bielinsky et al., 2001; DePamphilis et al., 2006; Gilbert, 2004). A handful of metazoan model replicons have been studied in detail in tissue culture cells (Cvetic and Walter, 2005; Gerbi, 2005). The fact that these systems lack convenient genetic assays has restricted metazoan origin studies to physical
biochemical mapping methods. It has been shown by 2D gels that replication of the Chinese hamster DHFR (Burhans et al., 1990; Vaughn et al., 1990) and human rDNA loci (Little et al., 1993; Yoon et al., 1995) initiates in broad regions. These data suggest the existence of large initiation zones (Gilbert, 2001), although in the DHFR locus two to three specific sites are preferred over other initiation sites spread throughout a 55kb
region (Figure 2B) (Dijkwel et al., 2002; Kobayashi et al., 1998a). On the other hand, studies of human lamin B2 (Figure 2C) and β-globin origins have identified much more defined sites of replication initiation, consistent with the classic replicon/replicator model (Gilbert, 2004; Jacob and Brenner, 1963). Thus, there seem to be two classes of
mammalian origins depending on the locus.
Origins of developmental gene amplification
As previously discussed, developmental gene amplification provides a powerful system for the study of metazoan DNA replication in vivo. In the development of the
Tetrahymena macronucleus, the 10.3kb rDNA locus is specifically excised from the
genome, converted to a ~21kb head-to-head palindrome, and telomeres are added to generate stable linear minichromosomes (Figure 3A). Then over the course of twelve hours, the rDNA minichromosomes are preferentially amplified up to 10,000-fold (Kapler et al., 1996; Prescott, 1994). Amplification initiates from two 430bp sites in
Tetrahymena thermophila (Figure 3A) (MacAlpine et al., 1997), or a single 900bp region
in T. pyriformis (Yue et al., 1998), all located within the 5’ Nontranscribed Spacer region (5’NTS) that is in the center of the palindrome. It appears that some of the amplified molecules separate from each other, as FISH studies demonstrate the presence of several hundred rDNA loci in nucleoli throughout the macronucleus (Ward et al., 1997). Given the small size (21 kb) of these extrachromosomal molecules, it is conceivable that at least some minichromosomes are fully replicated and some portion of the onionskin structures resolve. In contrast, amplification in Dipteran flies only represents a small portion of the
Figure 3. Origins of developmental gene amplification.
(A) Tetrahymena thermophila rDNA minichromosome. T represents the telomere at either end. Red ovals are nucleosome-free regions within the 5’ NTS (Nontranscribed Spacer) of the rRNA genes that show initiation activity. (B) Sciara coprophila salivary gland DNA puff II/9A. A 1kb origin (ORI) has been mapped upstream of II/9A genes by 2D and 3D gel analyses. ORI contains an ORC-binding site, immediately adjacent to the Transition Point (TP) from discontinuous to continuous DNA synthesis. (C) Drosophila
melanogaster DAFC-66D. The majority of origin activity resides in the intergenic oriβ. ACE3 is an amplification control element necessary for amplification, and it is
functionally separable from the Cp18 promoter. See text for references. Blue pointed bars represent the coding frame of genes.
giant polytene chromosomes in which the amplicons (each about 100kb in size) reside. Furthermore, suggested by FISH (Calvi et al., 1998) the onionskin structures remain held together without subsequent rearrangements.
In the Sciara salivary puff II/9A, 2D and 3D gel analyses indicate that initiation occurs over a 5.5kb region, within which a preferred 1kb portion (ORI) accounts for the majority of the origin firing (Figure 3B) (Liang and Gerbi, 1994; Liang et al., 1993). The precise nucleotide (Transition Point, TP) within the 1kb region at which DNA synthesis initiates has been determined by RIP mapping, and both recombinant ORC2 protein from Drosophila and endogenous Sciara ORC2 have been shown to bind to an 80bp segment adjacent to this initiation site (Bielinsky et al., 2001). In the related Sciarid fly,
Rhynchosciara, 2D gel analyses demonstrate that replication initiates in the salivary puff
C3 from at most 3 sites in a zone of about 6kb, and that this zone resides approximately 2kb upstream of the amplified gene C3-22 (Bielinsky et al., 2001).
In Drosophila, gene amplification of four genomic loci in the somatic follicle cells of the ovary occurs at specific stages of egg chamber development (stages 10 to 13, Figure 4A). During stages 9 and 10, the follicle cells surrounding the developing oocyte cease genomic DNA replication and begin to amplify four clusters of genes, which can be visualized as four foci by immunofluorescence following BrdU (bromodeoxyuridine, a thymidine analog) incorporation (Calvi et al., 1998). These Drosophila Amplicons in Follicle Cells (DAFCs) are named according to their cytological locations. Two of the amplicons are the X chromosome (at 7F, DAFC-7F) and the 3rd
chromosome (at 66D,
demand for the rapid synthesis of chorion proteins (Orr-Weaver, 1991). The other two loci of amplification, DAFC-30B and DAFC-62D, were recently identified by CGH array studies (Claycomb et al., 2004). These amplicons contain genes encoding a variety of proteins, including transporters, proteases, chitin-binding proteins and two putative enzymes Yellow-g and Yellow-g2, thought to be necessary for crosslinking proteins of the vitelline membrane or eggshell (Claycomb et al., 2004).
The third chromosome chorion amplicon DAFC-66D is the best studied (Figure 3C). 2D gel analysis has identified three potential replication origins within the peak amplified region, with one of these, oriβ, the 884bp sequence downstream of the Cp18 chorion protein gene being the preferred site of initiation that contains 70-80% of the origin activity (Delidakis and Kafatos, 1989; Heck and Spradling, 1990). Oriβ has ten out of eleven base pair matches to the Saccharomyces cerevisiae ARS consensus sequence that serves as an essential part of the origin of replication in yeast (Levine and Spradling, 1985). However, the significance of this sequence similarity is not clear, and notably, S. cerevisiae ARS1 origin sequences can not substitute for oriβ, thereby
confirming the sequence specificity of oriβ (Zhang and Tower, 2004). Deletion mapping of oriβ identified a 140 bp 5' element and a 226 bp A/T-rich 3' element called the β region that are necessary and sufficient to induce amplification of transposons (Zhang and Tower, 2004). The high A/T content of the β region might be important, because ORC has been shown to preferentially bind to A/T rich sequences in many species (for review see Bell, 2002). The replication initiation protein ORC2 binds
directly to oriβ during gene amplification (Austin et al., 1999). However, despite its tight association with oriβ in vivo, ORC does not preferentially bind DAFC amplification origin sequences in in vitro assays (Remus et al., 2004). This is similar to observations in other metazoan systems (Vashee et al., 2003), although contrasting Saccharomyces
cerevisiae in which specific sequences (the ARS Consensus Sequence or ACS) within the
origins are recognized (Newlon and Theis, 1993).
The developmental timing of amplification initiation appears to be highly
regulated and specific to particular amplicons. Real-time PCR suggested that DAFC-62D behaves distinctly from the other three amplicons in the timing of origin firing (Figure 4B) (Claycomb et al., 2004). In DAFC-66D, -7F and -30B, origin firing occurs only in stages 10 and 11. Afterwards there is no more increase in copy number at the central initiation sites. However, for DAFC-62D, an additional round of origin firing is observed in stage 13 within a defined 4kb region. Therefore, DAFC-62D provides a distinct model for studying not only the mechanisms of origin selection and activation, but also its developmental regulation. By identifying cis-regulatory elements in DAFCs that direct amplification as origin(s), and those that regulate amplification as enhancers by sensing differential developmental signals in different stages, we have gained important insights in understanding metazoan DNA replication in vivo.
Figure 4. Developmental timing of DAFC amplification.
(A) DAPI staining of egg chambers in stages 10 to 13. Follicle cells surround the developing oocytes. Adapted from A. C. Spradling, 1993. (B) Schematic drawing of the developmental timing of DAFC-66D and DAFC-62D amplification. About 30 fold of amplification in the center of DAFC-66D indicates 5 rounds of origin firing, all taking place in stages 10-11. By contrast, amplification of DAFC-62D is activated in two distinct stages, 10 and 13, separated by an elongation-only phase.
Amplification control elements
The relative ease of genetically manipulating Drosophila has greatly facilitated the mapping of cis-regulatory elements in DAFCs. In the P-element mediated
transformation systems, transposons that contain proper cis elements are able to amplify at ectopic genomic loci, although levels of amplification are dramatically affected by chromosomal position effects (de Cicco and Spradling, 1984; Orr-Weaver and Spradling, 1986). The introduction of insulators (Suppressor of Hairy-Wing Binding Sites
(SHWBS), Figure 5A) helps to remove position effects (Lu and Tower, 1997). The SHWBS insulators recruit proteins including Su(Hw) (Suppressor of Hairy Wing) that has been suggested to function as barriers between heterochromatin and open chromatin (Figure 5B) (Capelson and Corces, 2004; Gerasimova and Corces, 2001).
A number of studies have delineated in DAFC-66D the 320bp Amplification Control Element on the third chromosome, ACE3, required for high levels of
amplification (Figure 3C) (de Cicco and Spradling, 1984; Delidakis and Kafatos, 1989; Orr-Weaver et al., 1989; Orr-Weaver and Spradling, 1986). ACE3 is evolutionarily well conserved, located approximately 1.5kb upstream of oriβ and at the 5’ end of the s18 chorion gene. Demonstrated by 2D gel analyses (Delidakis and Kafatos, 1989; Heck and Spradling, 1990), ACE3 itself does not function as an origin of DNA replication, as it is not sufficient to support amplification in transposons protected by SHWBS insulators (Lu et al., 2001). Similarly, DAFC-7F contains an ACE element (ACE1) that is important for the amplification of this gene cluster (Spradling et al., 1987).
Figure 5. The SHWBS insulators remove position effects in P-element mediated transposon systems.
(A) Structure of transposon within the P-element sequences (black boxes). DNA sequence of interest (light-blue box) is flanked by SHWBS insulators. Arrows indicate orientation of SHWBS. mini-white (stippled box) is a reporter gene for the selection of transformation lines. (B) SHWBS binds Su(Hw) (Supressor of Hairy Wing) and additional proteins to form a barrier against surrounding heterochromatin (dark-blue boxes), so that the open chromatin structure (light-blue box) of the transposon is not affected by chromosomal position effects. See text for references.
It has been proposed that ACE serves as a developmental control element by stimulating replication initiation at nearby origins (Carminati et al., 1992; Delidakis and Kafatos, 1989; Heck and Spradling, 1990). A 142bp highly evolutionarily conserved “core” region of ACE3 has been determined responsible for the majority of ACE3’s replication stimulatory activity by deletion studies (Zhang and Tower, 2004).
Furthermore, ACE3 is necessary in cooperation with oriβ to achieve high levels of gene amplification, as a SHWBS insulator placed between ACE3 and oriβ in transposons nearly eliminates amplification. Removal of this insulator element by FLP/FRT-mediated recombination then restores amplification (Lu et al., 2001). Additionally, elimination of either ACE3 or oriβ from transposon constructs dramatically reduces amplification levels, indicating that together, ACE3 and oriβ are necessary and sufficient to drive developmental amplification (Lu et al., 2001).
Recent molecular studies provide some clues of how ACE3 might function as an amplification enhancer. ORC binds directly not only to oriβ but also to ACE3 and ACE1 in a site-specific manner, by either in vivo Chromatin Immunoprecipitation (ChIP) or in
vitro binding assays (Figure 6A) (Austin et al., 1999; Royzman et al., 1999). It has hence
been suggested that ACE3 serves as a nucleating site for ORC to spread along the
chromatin, thus influencing the ability of the region to replicate (Austin et al., 1999; Lu et al., 2001). By immunofluorescence, transposons of ACE3 multimers are capable of recruiting ORC2 in vivo (Austin et al., 1999), and support amplification presumably
initiated from proximal origins (Carminati et al., 1992). The amplification of a minimal transposon buffered by SHWBS containing only ACE3, Cp18, and oriβ is dependent on the orc2 gene product by mutant analysis, though without detection of ORC2 in
immunofluorescence (Lu et al., 2001). Therefore it appears that a certain threshold amount of ORC2 must be recruited. It may not always be detectable by staining methods, but in more sensitive assays such as ChIP ORC2 clearly associates with amplification origins and enhancers (Austin et al., 1999). Cumulatively, these data indicate that ACE3 and oriβ are functionally separable, but act cooperatively to drive gene amplification. The current working model is that ACE3 may nucleate ORC that then spreads along the chromatin to initiate replication at oriβ.
The involvement of transcription factors in gene amplification
Genetic, cytological, and biochemical approaches have also contributed to the understanding of the trans factors involved in developmental gene amplification. It has been clearly demonstrated that the proteins involved in DNA replication during a normal cell cycle are also involved in replication during gene amplification (Claycomb and Orr-Weaver, 2005; Tower, 2004). Hypomorphic mutations in Drosophila genes encoding essential components of the replication machinery lead to female sterility, disrupted eggshells, and severely decreased DAFC amplification, as measured by incorporation of BrdU or Quantitative Southern blotting (Henderson et al., 2000; Landis et al., 1997b; Landis and Tower, 1999; Tower, 2004; Underwood et al., 1990; Whittaker et al., 2000).
The archetypal DNA replication machinery includes first the formation of the pre-RC at the origins (Bell and Dutta, 2002). The ORC and DUP/Cdt1 proteins are sequentially recruited, which in turn load the putative replication fork helicase complex, MCM2-7 (Aparicio et al., 1997; Bell and Dutta, 2002; Labib et al., 2001).
In addition to conserved replication proteins, the components known to play a role in Drosophila chorion gene amplification include transcription factors. While chromatin immunoprecipitation (ChIP) experiments have shown that ORC binds directly to ACE3 and to oriβ (Austin et al., 1999; Bosco et al., 2001), additional ChIP and binding studies have localized transcription factors E2F1/DP/Rb to ACE3 during amplification stages in a complex containing ORC (Figure 6A) (Bosco et al., 2001). In the normal cell cycle, the E2F1/Rb complex acts as a transcriptional repressor until at the G1 to S phase transition phosphorylation of Rb converts E2F1 into a transcriptional activator for the expression of multiple genes required for S phase entry (Dyson, 1998; Zhu et al., 2005). E2F1 is required for chorion gene amplification because E2f1 mutants in which the DNA-binding domain is disrupted display decreased amplification and no ORC localization; a
hypomorphic Rb mutation or a mutation in E2f1 that removes the Rb binding site results in overamplification and inappropriate genomic replication (Royzman et al., 1999). These data support a model in which E2F1/Rb binds at and/or around ACE3 and represses replication until amplification stages, during which E2F1 positively regulates amplification initiation, with hyperphosphorylated Rb (pRb).
There are two E2f genes in Drosophila, E2f1 and E2f2 (Frolov et al., 2001). E2F1 is a potent activator of transcription, whereas E2F2 has been shown to repress
transcription (Dyson, 1998). In null E2f2 mutants BrdU incorporation occurs throughout the nucleus during DAFC amplification stages, failing to confine DNA synthesis to
DAFC sites (Cayirlioglu et al., 2001). In parallel, the distribution of pre-RC components
changes from being restricted to DAFC foci into being nuclear in these mutants, suggesting a repressive role of E2F2 in genomic DNA replication (Cayirlioglu et al., 2001). Although in E2f2 mutants there is a mild increase in pre-RC transcript level (Cayirlioglu et al., 2001; Cayirlioglu et al., 2003), it does not exclude the possibility that E2F2 functions directly at genomic origins to repress replication (see below).
Another transcription factor that associates with ACE3 is the Myb oncoprotein. A complex containing Myb, Mip120 (Myb Interacting Protein 120, formerly p120),
Mip130, Mip40, and Caf1 p55 interacts with ORC (Figure 6A) (Beall et al., 2002; Korenjak et al., 2004). Both the Myb and Mip120 subunits exhibit specific binding. Within ACE3, binding sites for Myb and Mip120 have been identified, and deletion of these sites from transposons nearly abolishes amplification compared to the non-deleted control (Beall et al., 2002). These results indicate that the Myb and at least one of the Mip120 binding sites are necessary for amplification. Myb is essential for viability, as
Myb mutants are lethal. Myb mutant follicle cell clones are defective in BrdU
incorporation at DAFCs, showing that Myb is necessary for gene amplification (Beall et al., 2002). The fact that by immunofluorescence ORC2 and DUP/Cdt1 are properly localized to DAFCs in Myb mutant clones indicates that Myb is required for initiation at a later step (Beall et al., 2002). Mip130 mutant females are sterile and have BrdU
Figure 6. Transcription factors involved in DAFC-66D amplification and origin specification.
(A) E2F1/DP/Rb and a complex containing Myb specifically associate with ACE3. The Rb and Myb proteins may be activated through phosphorylation. ORC is site-specifically recruited and spreads along the chromatin to initiate replication from oriβ. The
Ultraspiracle/ Ecdysone Receptor (USP/EcR) transcription factor also may regulate amplification via an interaction with ACE3. (B) During amplification stages, genomic replication is inhibited by the E2F2-containing dREAM complexes, which excludes ORC from inactive non-DAFC origins.
From these data, Mip130 appears to interact with the other Mips in a complex to repress genome-wide replication. At specific chromosomal loci Myb becomes activated in some way, perhaps by phosphorylation (Beall et al., 2004), to initiate replication or
amplification. Such a switch from repressive to active state might be important for Myb to specifically allow the initiation of amplification at the appropriate developmental time at amplification origins.
Strikingly, the Myb and Mip130 mutant phenotypes are very similar to those of
E2f1 and E2f2, respectively. In addition to genetic evidence, molecular and biochemical
studies strongly suggest the E2F and Myb proteins co-regulate replication. E2F1 and the Myb-containing complex, both localized to ACE3, may act coordinately to activate
DAFC-66D amplification (Figure 6A). Although there is no report of a physical
interaction between E2F1 and Myb, a complex containing E2F2, Myb and Mips has been purified from Drosophila embryo extracts (Korenjak et al., 2004; Lewis et al., 2004). These dREAM complexes (Drosophila Rb, E2F and Myb-interacting proteins) bind to repressed chromatin (Korenjak et al., 2004). Based on the mutant phenotypes of Myb and
E2f, it has been proposed that dREAM inhibits genome-wide replication (Korenjak et al.,
2004; Lewis et al., 2004), possibly by excluding pre-RC from genomic origins (Figure 6B); at DAFC-66D this repressive effect is reversed by E2F1 and activated Myb to achieve site-specific amplification (Figure 6A).
E2F and Myb appear to directly regulate amplification without affecting transcription of DAFC-66D genes. The transcription factor Ultraspiracle (USP) on the other hand, has been shown to bind to the promoter of the Cp18 chorion gene of
DAFC-66D (Shea et al., 1990). USP is a zinc finger protein differentially enriched in the follicle
cells, and it is a developmentally important member of the family of nuclear steroid hormone receptors (Oro et al., 1992; Shea et al., 1990). It heterodimerizes with another member of the nuclear receptor superfamily, ecdysteroid receptor protein (EcR), to function as a receptor for the steroid hormone ecdysone (Christianson et al., 1992; Yao et al., 1992). Ecdysone governs egg chamber development, and maternal EcR is required for normal oogenesis (Buszczak et al., 1999; Carney and Bender, 2000). EcR displays increased activity in follicle cells during amplification stages (Hackney et al., 2007). Dominant negative mutants of EcR (DNEcR) can dimerize with USP and bind DNA, but they do not activate target gene expression (Cherbas et al., 2003; Hackney et al., 2007). Interestingly, introduction of DNEcR into follicle cells not only reduces chorion gene expression, but also results in significantly decreased amplification, and the eggs laid by mutant mother display thin eggshells and shortened dorsal appendages (Hackney et al., 2007). Taken together, these results indicate that the USP/EcR heterodimer mediates ecdysone regulation of chorion gene amplification and transcription (Figure 6A). These two events may be separable from each other because ACE3, discrete from sequences controlling transcription (Orr-Weaver et al., 1989), harbors a good match to Ecdysone Response Element (Hackney et al., 2007).
In Sciara coprophila the amplification of salivary gland DNA puff II/9A maybe similarly influenced by ecdysone, the master regulator of insect development (Crouse, 1968; Foulk et al., 2006). Ecdysone induces transcription of the II/9A genes (Bienz-Tadmor et al., 1991; DiBartolomeis and Gerbi, 1989; Wu et al., 1993). In vitro
incubation of pre-amplification stage salivary glands with ecdysone induces premature amplification, and injection of ecdysone into pre-amplification stage larvae results in amplification in vivo (Foulk et al., 2006). A putative EcRE is found directly adjacent to the ORC-binding site in the II/9A origin (Bielinsky et al., 2001) and is efficiently bound by the Sciara EcR in in vitro binding assays (Foulk et al., 2006). The Sciara and
Drosophila results indicate that the ecdysone and EcR transcription factor control of developmental gene amplification may be conserved in insects.
Recently proteins TIF1-4 (Type I interacting Factor) that are necessary for rDNA amplification in Tetrahymena have been purified as complexes with differential DNA binding activities within the initiation zone (Mohammad et al., 2000). Notably TIF1 possesses limited homology to a transcription factor in plants, p24 (Mohammad et al., 2000). Together with data from Drosophila and Sciara, it is clear that developmental gene amplification is under complex control that involves a number of transcription factors, acting to modulate the use of origins for amplification. These factors may play repressive or active roles, depending on the developmental stage, the genomic locus and the chromatin context.
Chromatin context and amplification activity
It is not surprising that the replication and amplification machinery requires a favorable chromatin context to access DNA (Groth et al., 2007). Eukaryotic DNA is packaged into an organized, higher-order chromatin structure by histone proteins (Loden and van Steensel, 2005). Post-translational modifications of histones such as acetylation
of histone N-terminal lysine residues induces chromosomal changes, resulting in the loss of chromosomal repression to allow successful transcription of the underlying genes, as well as replication of DNA molecules (Fukuda et al., 2006). Recent studies in yeast have provided evidence that posttranslational chromatin modification can control the
efficiency and/or timing of chromosomal origin activity (Aparicio et al., 2004; Vogelauer et al., 2002).
Using the DAFC systems, independent groups have demonstrated that histones H3 and H4 at and around ACE3 are hyperacetylated during gene amplification (Aggarwal and Calvi, 2004; Hartl et al., 2007), and the lysine residues that are acetylated are
associated with replication and not transcription (Hartl et al., 2007). Furthermore, the acetylation of H3 and H4 is not the result of histone deposition after replication, as the hyperacetylation is confined to the origins of DAFC-66D and not associated with the replication forks (Hartl et al., 2007). Hyperacetylation of histone H4 leads to
redistribution of ORC2 from amplification foci to a genome-wide staining pattern; tethering histone acetyl transferase (HAT) increases amplification levels of a transposon with ACE3 and oriβ (Aggarwal and Calvi, 2004). Conversely, tethering of a histone deacetylase (HDAC) or a chromatin repressor to ACE3 reduces amplification (Aggarwal and Calvi, 2004). These observations suggest chromatin structure may have a definite role in amplification origin activity and that origin firing may be facilitated by a modification of the chromatin state.
Such modifications may be conducted through recruitment of histone-modifying enzymes and/or chromatin-remodeling proteins by transcription factors (Kohzaki and
Murakami, 2005). In X. laevis eggs, injected plasmid DNA undergoes site-specific initiation of replication in the presence of a transcription factor that is known to recruit a chromatin-remodeling complex (Danis et al., 2004). This effect does not require active transcription, but rather correlates with the acetylation level of histone H3 at the initiation sites (Danis et al., 2004). The E2F/Rb and Myb complexes are good candidates that may function at DAFC amplification origins to recruit HATs or HDACs to modulate the accessibility of the chromatin at the origin (Beall et al., 2002; Bosco et al., 2001). For example, Rb has been shown in a number of organisms to repress transcription by
remodeling chromatin structure through interaction with proteins involved in nucleosome remodeling, histone acetylation/deacetylation and methylation (Giacinti and Giordano, 2006). Finally, chromatin state and nucleosomal positioning may also play a role in gene amplification in Sciara and Tetrahymena (Clever and Ellgaard, 1970; Giacinti and
Giordano, 2006; MacAlpine et al., 1997; Mok et al., 2001).
A general link between replication and transcription
Transcription factors appear to function by several means at the amplification origins to modulate their activity. Both E2F1 and Myb have been shown to interact with ORC (Beall et al., 2002; Bosco et al., 2001). Possibly with some degree of redundancy, they recruit ORC through direct interaction. Transcription factors may also directly recruit proteins to modify chromatin structure to facilitate the assembly of the replication machinery. It is probably not a pure coincidence, however, that some common chromatin
features are shared by active transcription and replication, considering the ultimate goal of gene amplification is to augment transcript level.
There is mounting evidence for a general link between transcription and replication. Replication origins are frequently found upstream of transcription units (Mechali, 2001), and there are several examples in which they coincide with promoter sequences (Kohzaki and Murakami, 2005). In the human β-globin and c-myc replicons, transcription regulatory elements have been shown to be essential for replication
initiation (Aladjem et al., 1995; Liu et al., 2003). In addition to studies of specific replication sites, genome-wide mapping of replication origins in eukaryotes has been facilitated by recent advances in DNA microarray technology, and has begun to establish the spatial and temporal program of replication initiation (MacAlpine and Bell, 2005). Microarray analyses of genomic replication in Drosophila and human cells show a correlation between regions undergoing active transcription and early replication (Jeon et al., 2005; MacAlpine et al., 2004; Schubeler et al., 2002; Woodfine et al., 2004). A more extensive study of Drosophila chromosome 2L in Kc cells uncovered a frequent
colocalization of ORC and RNA polymerase II (RNAPII) binding sites, implying a connection between transcription and ORC localization (MacAlpine et al., 2004).
A number of studies report positive effects of transcription factors on DNA replication (Kohzaki and Murakami, 2005). The recruitment of transcription factors alters origin activity on episomal plasmids in both S. cerevisiae and X. laevis eggs (Cheng et al., 1992; Danis et al., 2004). Similarly, expression of a CREB-GAL4 fusion protein restores replication origin activity of the mutant c-myc locus where a GAL4p
binding cassette replaces all regulatory sequences of the c-myc gene (Ghosh et al., 2004). These results suggest that transcription factor binding can enhance replication origin activity. In Chinese hamster ovary (CHO) cells the dihydrofolate reductase (DHFR) gene locus contains a 55-kb zone of potential initiation sites of replication upstream of the gene (Burhans et al., 1990; Vaughn et al., 1990). In mutants with parts or all of the DHFR promoter deleted such that transcription is undetectable, initiation in the intergenic space is markedly suppressed (but not eliminated); restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restores origin activity to the wild-type pattern (Saha et al., 2004).
However, 2D gel analysis of the promoterless DHFR mutants reveals that initiation occurrs at a low level not only in the intergenic region, but also in the body of the DHFR gene, which had never been observed in the wild-type locus (Saha et al., 2004). Thus transcription seems to suppress replication initiation in the body of the gene, and help define the boundaries of the downstream origin (Saha et al., 2004). In a mutant human c-myc locus with the c-myc promoter replaced by inducible GFP-encoding genes, replication initiation is repressed upon induction of transcription (Ghosh et al., 2004). When basal or induced transcription complexes is slowed by the presence of α-amanitin, origin activity depends on the orientation of the transcription unit (Ghosh et al., 2004). These data suggest that high levels of transcription or the persistence of transcription complexes can repress replication initiation. Taken together, the seemingly dual role of transcription on origin firing may be important to ensure high activity of intergenic
origins, while suppressing initiation within the gene body to avoid disruption of pre-RCs by the passage of the transcriptional machinery.
Another theme of transcriptional control of origin firing is the involvement of RNAPII. Transcription factors mediate the enhancer --> activator--> mediator -->
RNAPII --> promoter pathway to initiate mRNA transcription via RNAPII in virtually all eukaryotes (Kornberg, 2005). In Chinese hamster ovary cells it has been reported that inhibition of RNAPII transcription by α-amanitin results in deregulation of replication initiation at the DHFR locus (Kornberg, 2005). In Sciara salivary puff II/9A, although transcription of the II-9-1 gene does not begin until amplification is complete, the promoter of II-9-1 is occupied by RNAPII during amplification stages, and it is this presence that is thought to limit the right-hand boundary of the initiation zone during amplification (Sasaki et al., 2006). Furthermore, a direct physical interaction has been reported between RNAPII and MCM2-7 in yeast (Gauthier et al., 2002; Holland et al., 2002), raising the possibility that the transcriptional machinery serves to load the MCM complex to origins in some developmental contexts. Recently the hyperphosphorylated form of RNAPII implicated in transcriptional elongation has been shown to
co-immunoprecipitate with DNA polymerase ε (Rytkonen et al., 2006).
In addition to direct interactions with replication proteins, RNAPII has been shown to be required for histone displacement within the transcriptionally activated gene’s coding region preceding RNAPII (Brown and Kingston, 1997; Lee et al., 2004; Schwabish and Struhl, 2004; Zhao et al., 2005). In the human hsp70 gene, transcription activation leads to nucleosomal disassembly in the first 400 bp coding sequence in front
of RNAPII (Brown and Kingston, 1997). More recently, it has been demonstrated that histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with RNAPII association and transcriptional activity, suggesting efficient eviction of core histones from the DNA by transcription (Schwabish and Struhl, 2004). Additionally, MCM2-7 associated DNA is more susceptible to nuclease digestion, indicating that these chromatin domains may be less tightly compacted, although the causal and consequence relation is not clear (Forsburg, 2004; Holthoff et al., 1998; Richter et al., 1998). Finally, the elongating form of RNAPII is in association with chromatin remodeling and histone-modifying factors (Sims et al., 2004). All together, these physical interactions between promoters, the transcriptional machinery, factors regulating chromatin structures, replication proteins and finally replication and
amplification origins suggest a complex picture of transcription and replication regulation in the chromatin context.
Summary of thesis
This thesis work investigated mechanisms that control the unique timing of
DAFC-62D origin activation using cytological, molecular and genetic methods. We first
defined the origin sequences in DAFC-62D by analyzing the amount of nascent
replicative DNA across this amplicon. Surprisingly the origin coincided with the coding region of a 62D gene. ORC2 localized to the origin, as well as two other sites that did not confer origin activity. Both ORC2 and MCM2-7 displayed differential association with these sequences, corresponding to the two rounds of amplification in two separate
developmental stages (10 and 13). All three elements, dispersed in a 7kb central amplified region, were required for either round of DAFC-62D amplification, because deleting any one completely abolished amplification in transposon experiments. Preceded by transcription of the 62D gene in stage 12, the late round of origin firing was ablated by the RNAPII inhibitor α-amanitin. This effect was absent from other amplicons and insulated transposons, and specific to the stage 13 amplification at DAFC-62D and transposons that did not have functional insulators. Finally, blocking RNAPII transcription compromised MCM2-7 recruitment as suggested by ChIP analysis.
Our studies of the regulation of DAFC-62D yield several unexpected findings. We find that the positioning of ORC and MCM2-7 can be affected by differentiation stage. Transcription via RNAPII in cis controls localization of replication factors and origin activation. The comparative analyses of DAFC-62D and -66D demonstrate that there are distinct mechanisms for differential regulation of amplification origins during Drosophila follicle cell development. Transposon experiments suggest their distinct behavior than the endogenous amplicon may be accounted for by the insulators’ unique properties.
Together our findings provide critical insights into how metazoan DNA replication is controlled in response to developmental cues.
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