Derepression of Heterochromatin Inactivation by Induction of a Nearby Promoter in Drosophila Melanogaster

Graduate Theses, Dissertations, and Problem Reports

1998

Derepression of heterochromatin inactivation by induction of a nearby promoter in Drosophila melanogaster

Daniel Renick McNeill West Virginia University

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Recommended Citation McNeill, Daniel Renick, "Derepression of heterochromatin inactivation by induction of a nearby promoter in Drosophila melanogaster" (1998). Graduate Theses, Dissertations, and Problem Reports. 921. https://researchrepository.wvu.edu/etd/921

This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Derepression of heterochromatin inactivation by induction

of a nearby promoter in Drosophila melanogaster

Daniel R. McNeill

Thesis submitted to the Faculty of West Virginia University In Partial Fulfillment of the Requirements for the degree of

Master of Science in Cellular and Molecular Biology

Clifton P. Bishop, Chair Dan Pannacione Jeff Price

December, 1998 Morgantown, West Virginia

Keywords: Silencing, Heterochromatin, Position-effect, Development

of a nearby promoter in Drosophila melanogaster

Daniel R. McNeill

Abstract Position-effect variegation (PEV) describes the compaction of a euchromatic gene placed next to a region of heterochromatin. This compaction into heterochromatin in some instances results in a mosaic phenotype in Drosophila melanogaster. Since its discovery in the 1930's the phenomenon of PEV has been extensively analyzed and many models describing the mechanics have been proposed. But, until recently a model system for analyzing the phenomenon at various developmental stages was not available. In 1996, Lu, Bishop, and Eissenberg were able to use a transgenic construct that variegates for both a heat shock 70 promoter-driven lacZ construct and a mini-white gene, to allow more in- depth characterization of the developmental changes inherent in the formation of heterochromatin. The results of their research demonstrated that heterochromatic inactivation of gene expression is subject to a developmentally programmed derepression. Using this same construct, I have further characterized the phenomenon by classically constructing several genetic lines, which were either capable or incapable of initiating a heat shock response, but otherwise genetically identical. These lines were reared in both heat shock and non-heat shock conditions and then variegation was assessed by the amount of eye pigment deposited by the derepression of the mini-white gene. Our results indicate that derepression is a progressive event and that the expression of a nearby gene, Hsp-70 in this experimentation, can facilitate the derepression of nearby genes compressed into heterochromatin. It can be reasoned that this cooperative derepression may be playing a role in the expression of genes contained within B- heterochromatin. In this region there appears to be a dispersal of euchromatic genes around heterochromatic ones. Dedication

I dedicate this work to my daughter Zoie Catherine McNeill and my fiancee Lori Paton whose love and beauty have been an inspiration to continue striving to achieve the utmost of my abilities intellectually, emotionally, and spiritually.

iii Acknowledgements

I would like to thank: Dr. Keith Garbutt for his most thorough assistance in compiling the statistics used in the analysis of this experiment. My committee members; Dr. Jeff Price and Dr. Dan Pannacione for their excellent advice and editing. Mostly, I would like to thank my mentor and chair of my committee, Dr. Clifton P. Bishop whose understanding, compassion, and scholarship was always offered freely.

iv Table of Contents

Item Page Introduction...... 1 Materials and Methods...... 16 Description and Rearing of Fly Stocks...... 16 Figure 1: P-element transgene construct...... 17 Figure 2: Classical Genetics...... 18 Classical Genetics...... 19 Temperature Shift Experiments...... 19 Eye Pigmentation Determination...... 20/21 Double Blind Study...... 21 Results...... 22 Table 1: Red Eye Pigmentation Assay for Cross 1 Males...... 23 Table 2: Red Eye Pigmentation Assay for Cross 1 Females...... 24 Table 3: Red Eye Pigmentation Assay for Control Cross 2...... 25 Discussion...... 26 References...... 32

v Introduction

The in-depth study of heterochromatin and its role in the expression of the genes of Drosophila began during the early years of Drosophila genetics. In the yearly reports from TH Morgan's laboratory in the 1920s and 1930s, the discovery of new genetic markers was described, and it was noted that only a few genes mapped to the Y chromosome, the proximal portion of the X chromosome, or the areas surrounding the attachment sites for spindles on the autosomes. So few were mapped to this region, which was latter to be called the heterochromatic portion, that it was called the "inert" region (Schultz, 1939). This reasoning was later found to be flawed as it was discovered that heterochromatin was in fact able to influence gene expression.

In 1930, Muller described the repressive effect that heterochromatin can exert on a euchromatic gene. In the course of his experimentation, he discovered several mutations of the white+ (w+) gene that resulted in variegated eye color phenotypes. Each of these mutations was found, after subsequent experimentation, to be the result of placing the euchromatic gene, via chromosomal rearrangement, in close proximity to the heterochromatic portion of the genome (Schultz, 1936). The basic properties of this occurrence were outlined via experimentation centered around the white-mottled (wm) alleles and other mosaic producing rearrangements. It resulted in the coining of the term position effect variegation (PEV) to describe the resultant phenomenon (Lewis, 1950 and Spradling, 1990). These early investigators demonstrated that novel junction-creating breakpoints between heterochromatin and euchromatin could result in the mosaic inactivation of the euchromatic genes, including genes expressed at differing developmental stages and genes which

1 would be expressed in different tissues. The position effects were further shown to be resolved by recombining the gene so that the euchromatic allele was moved away from the euchromatic-heterochromatic breakpoint or by causing a second movement of the variegating allele away from the heterochromatin which fully restored the wild type phenotype. As such, this demonstrated that it was the association with the heterochromatin, and not a mutation in the variegating allele itself, which was responsible for the observed variegating expression. It was also found that a single breakpoint could affect more than one gene, with the strength of the effect diminishing as the distance from the breakpoint was increased. Also, the degree of variegation observed was related to the dose of other heterochromatic regions present in particular, the dosage of the predominantly heterochromatic Y chromosomes present. Thus, by changing the percentage of heterochromatin, as related to euchromatin present in the nucleus, the variegating gene expression could be influenced (Weiler and Wakimoto, 1995, and Spofford, 1976).

In 1945, Heitz (1928) introduced the term heterochromatin to describe the regions of the chromosome which remain as deeply staining compacted bodies throughout the cell cycle, including interphase. Thus, the definition of heterochromatin was and still is based mainly on morphological criteria and on the temporal and spatial display of the chromosomes. This allows investigators to use light microscopy to distinguish these regions from those of euchromatin, which is seen to condense at metaphase, but appears to be diffuse during interphase. An interesting point is that the regions which are defined, perhaps erroneously, as being constitutive heterochromatin may change with time and between different tissue types, and the boundaries between heterochromatin and euchromatin are labile (John, 1988). One usually finds heterochromatin in

2 telomeric or pericentric locations (Heitz, 1928). Regions of heterochromatin have been seen to aggregate together in the various cell types, thus causing a profound influence on the three dimensional organization of chromosomes and on association of non-homologous chromosomes (Heitz, 1928).

The heterochromatin of Drosophila melanogaster has been characterized more than in any other organism (Gatti and Pimpinelli, 1992). In a typical diploid cell, 30-35% of the karyotype has been found to be heterochromatic. This includes the entire Y chromosome, approximately 40% of the X chromosome, 25% of chromosomes 2 and 3, and over half of chromosome 4. Heterochromatin has also been found to be highly enriched in repetitive DNA sequences (Lohe and Roberts, 1988). When this is examined closer it is found that certain satellite and middle repetitive sequences preferentially cluster at various chromosomal locations in heterochromatic regions. Lohe and Hilliker (1993) have estimated that approximately 80% of diploid cell heterochromatin can be accounted for by 11 different satellite sequences. When these sequences are analyzed using long-range molecular mapping, these sequences are found to be in the 100- to 900-kb range (Le and Duricka, 1995). These regions are interspersed and interrupted by regions of middle repetitive DNA, which have been shown to have sequence homology with transposable elements (TEs) (Lohe and Brutlag, 1987). These TE's show vary little variation among the wild type population of Drosophila and as such have been proposed to represent the stable structural portion of heterochromatin (Pimpinelli et al., 1995).

Further research by Heitz (1928) produced evidence that different portions of heterochromatin showed different characteristics and morphologies. He named

3 these alpha and beta. Alpha heterochromatin appears to contain very few genes and is made up of mainly repetitive satellite sequences and some middle repetitive elements located near the center of the centromere (Gall et al., 1971). Structurally this form of heterochromatin is very compact, transcriptionally inactive, and relatively under-replicated in polytene nuclei. In the diploid interphase nucleus, this portion is seen to remain quite compact and only replicates in the late S-phase portion of the cell cycle (Gall et al., 1971). It is this portion which is referred to as constitutive heterochromatin, meaning that these regions are usually heterochromatic in virtually all cells at all times. It is also this portion of heterochromatin which is mainly involved in the production of the variegating effects associated with the PEV phenomenon. Exceptions to this rule exist such as the B- heterochromatic genes light and cubitus interruptus, that are seen to variegate when they are placed next to a euchromatic portion of the chromosomes (Spofford, 1976).

Beta heterochromatin is a type of heterochromatin which is found at the heterochromatin-euchromatin junction and also comprises the fourth chromosome. It has characteristics which allow it to be distinguished from the alpha form. Morphologically, Heitz (1934) defined it as the chromocentral material that is similar to euchromatin in that it is capable of "growth" and expansion, but that differs from euchromatin in that it forms a diffuse meshwork instead of well-structured chromomeres. It has been suggested that this type of heterochromatin forms from sequences which are located throughout heterochromatin. It is thought they replicate to high levels and then loop out and aggregate together to form the majority of the chromocenter (Traverse and Pardue, 1989). It is also thought that these sequences include single copy sequences and middle repetitive elements that were located in the

4 heterochromatin of X chromosomes and autosomes (Devlin and Holm, 1990).

This is supported by the work of Miklos et al. (1988) which indicate that beta heterochromatin contains more middle repetitive elements than euchromatin. This occurrence could also be explained because beta heterochromatin regions show a low level of meiotic recombination, and as such may act as a graveyard for repetitive sequences for they appear to accumulate in this region and are subsequently compacted into heterochromatin. This renders these sequences non-functional. It has also been demonstrated that at least some beta heterochromatic sequences are actively transcribed. This has been supported by 3H-uridine incorporation studies (Lakhotia and Jacob, 1974) and by detection of the RNA produced by the transcription of specific single-copy sequences (Beissmann et al., 1981, Devlin et al., 1990). The reason for the structural differences between alpha and beta heterochromatin is functionally unknown at this time. It can however be proposed that these differences could be useful in the regulation of heterochromatic genes (Zhang and Spradling, 1995) or it may also prove to be an integral part of the PEV mechanism seen in polytene tissues.

Researchers have identified other factors which play a role in the development of heterochromatin. One environmental factor which plays a significant role is temperature. The amount of variegation may be altered by the temperature in which the flies are allowed to develop: as a rule, flies which are raised at higher temperatures exhibit a more wild-type phenotype (variegation is suppressed) when they are compared to flies raised at a lower temperature. This occurrence has been used by researchers in temperature shift experiments. In these experiments, changes in temperature and their subsequent changes in a heterochromatin-induced inactivation have been

5 used to delineate crucial developmental times during which the formation of heterochromatin seems to be maximally sensitive. It has been found in certain instances of PEV that both the phenotypic variegation for the PEV loci and the extent of the cytological manifestations of heterochromatic spreading are the most sensitive to temperature changes in the early stages of embryogenesis. In other instances of PEV, however, the major effects of temperature are seen in later stages (Kornher and Kaufman, 1986). Eissenberg (1989) interpreted this information as an indication that the inactivation of the variegating gene is a decision which occurs in the early stages of embryogenesis. It should also be noted that some of the PEV phenotypes comprise large patches of activated and inactivated cells, which supports this interpretation. It suggests that the decision to transcriptionally inactivate the variegating gene is one that occurs in the early stages of development and is propagated clonally. In contrast, some PEV phenotypes are fine grained mosaics, suggesting that the decision to inactivate the variegating gene occurs later in development or is amenable to reversal as development proceeds.

Also, in addition to the previously mentioned Y chromosomal effects, other genetic factors, including parental effects and modifier genes, have been found to play a role in the development of heterochromatin. For parental effects, both maternal and paternal effects have been found. In maternal effects, the gene product stored in the unfertilized egg from a mutant female, whether mRNAs or proteins, is altered either in quantity or quality. This alters the phenotype of the progeny of that female even though the progeny do not receive the mutant allele itself. For example, a female with mutant genes encoding proteins for chromatin components or modifying genes could produce eggs lacking products essential for wild type chromosomal conformation, thereby altering the

6 degree of PEV expression in her progeny (Krejci et al., 1989, Bishop and Jackson, 1996).

Paternally related effects are exemplified by imprinting-like effects. Genomic imprinting has been found to be wide spread among many living organisms (Krejci et al., 1989). The concept is that certain loci or chromosomal regions are physically "marked" in the germline cells due to passage through either oogenesis and/or spermatogenesis. As such, this imprinting can later cause these copies to act in a functionally distinct manner when compared to unmarked copies (Solter, 1988).

Other genetic factors which can influence the development of heterochromatin in Drosophila are euchromatic mutant modifiers, or single locus modifiers of PEV. These can be either dominant or recessive. Genetic screens indicate that there may be anywhere from 30-50 PEV modifying loci within the Drosophila genome (Reuter et al., 1982). Dominant suppressors of variegation, [Su(var)s], usually restore over 50%, and often nearly full, activity of the variegating gene. Although the white locus is the one in which the most experimentation has been conducted, other variegating strains have been shown to respond to the effects of most Su(var)s. The suppressors, however, may not act identically on all of them (Sinclair reviewed, 1991 and Bishop,

1992). Additionally, one copy of some Su(var)s suppresses variegation whereas three copies of the same gene enhance variegation. These genes were termed Class I modifiers by Tartof and colleagues (Tartof et al., 1984, Locke et al., 1988, and Tartof et al., 1989). This finding suggests that at least some Su(var) loci are dosage sensitive and most importantly it implies that their products are produced in limited amounts (Locke et al. reviewed, 1991). At

7 least some of the Class I modifiers are structural components of heterochromatin, and thus their influence is easy to understand. An example of a Class I modifier is heterochromatin protein-1 or HP1. HP1 encodes a non- histone chromosomal protein which has been found to be an integral part of heterochromatin. It has been localized to alpha, beta, and intercallary heterochromatin as well as chromosome 4 (James and Elgin, 1986). This brings us to the discussion of Class II modifiers of variegation. They were identified as regions which acted opposite to Class I modifiers. They suppress variegation when duplicated and enhance variegation when present in a single gene copy (Locke et al., 1988 and Tartof et al., 1989).

Since the discovery of the concept of PEV many models have attempted to explain the mechanism involved in the phenomenon. Within recent times two models have been proposed to explain this process. In 1974, Zuckerkandl proposed his locking molecule model to explain the processes behind PEV. His model centers around the concept that there exists within the nucleus certain macromolecules, which he terms locking molecules. These molecules are found in limited amounts in the nucleus and act to change euchromatin into heterochromatin via interaction with repetitive sequences. Instead of siding with the simplistic view that chromosomes exist in only two defined states, Zuckerkandl believed that there was a "continuous inter gradation" between the pure euchromatin and pure heterochromatin. The differences in conformation which are encountered were proposed to reflect the differences in the region's DNA sequences. This was attributable to the region's natural affinity for the locking molecules as much as its tendency to naturally adopt higher order structures. He proposed several molecules that could possibly act as locking molecules; histones, enzymes that modify histones, and non-chromosomal

8 histone proteins. The concentration of these molecules was considered by Zuckerkandl to be as important as the sequences of the DNA to the establishment of either euchromatin or heterochromatin. He also proposed that the conformational changes seen in chromatin responded to the laws of mass action due to the available concentrations of locking molecules that were present. He based this premise on the alterations which are observed in chromatin structure when Y chromosome dosage is modified.

This model also states that the "creeping" of locking molecules from the heterochromatin-euchromatin breakpoint is responsible for the euchromatin adopting a specific higher-order structure. The idea is that the presence of heterochromatin at the breakpoint reduces the threshold requirement for a change from euchromatin into heterochromatin. This occurs, according to the model, by providing a nucleation site rich in locking molecules. The degree to which spreading occurs would be the result of the interaction between the locking molecules' target sites, DNA sequences, and the locking molecules themselves within and around the heterochromatin and the euchromatin. (Zuckerkandl, 1974).

A second model has been proposed by Tartof et al. (1984) to explain the effects seen in PEV. It has been called the boundary model and is based upon the premise that a heterochromatin domain contains discrete sites which initiate and terminate its formation. Initiation sites were believed to be regions where specific proteins, which would interact with each other, were thought to bind and then compact regions into heterochromatin via a polarized spreading effect. PEV is seen when a heterochromatin-euchromatin breakpoint is encountered within this spreading domain. Thus, heterochromatin is formed past the

9 breakpoint into the euchromatic region until a site that mimics the terminator is encountered. Since its initial proposal the boundary model has been modified to include the effects of mass action due to the results of studies dealing with modifiers of PEV (Locke et al., 1988 and Tartof et al., 1989). These indicated that the relative extent of heterochromatin formed was related to the concentration of these modifier proteins present in the nucleus.

When the two models are compared one finds differences within them, in terms of initiation, propagation, and termination, that should be able to determine which of them is correct. The greatest difference concerns the way in which heterochromatin initially forms. The boundary model states that a specific initiator site is required. When this is addressed using the locking molecule model, however, one is led to believe that all that is needed is a sequence with some repetitive elements. Both models agree that the propagation of heterochromatic compaction progresses in a linear manner from the breakpoint. In the boundary model this propagation is believed to continue until the terminator site is encountered. For the locking model, termination is believed to occur when the nucleation of the heterochromatin is no longer favored by the structural changes and/or the euchromatin becomes relatively resistant to this nucleation. Presently, there exists research which supports both of the proposed models (Weiler and Wakimoto, 1995).

To add to a greater understanding of the processes at work in the formation of heterochromatin we proposed to utilize molecular biology, specifically the use of a genetically engineered construct which could be activated at any specific time during development. Up until the advent of molecular biology, scientists could only rely on classical genetic manipulations and/or

10 mutagenesis screening to attempt to dissect out the processes at work. Now that we can utilize the tools available via molecular biology, we are better able to pinpoint and manipulate exact processes to precisely further our understanding.

This experiment uses a heat shock gene to initiate the production of reporter gene products at the desired time so that their specific effects can be addressed. The heat shock response was first described by Ritossa (1962). He saw a change in the condensation properties of larval salivary glands upon exposure to an increased temperature. This was found to be due to specific genes (later called heat shock genes), which become activated via a variety of stressor conditions. Conditions which have been identified experimentally are; noxious stimuli (such as heat and chemicals), cell injury conditions (such as those associated with ischemia and infection), and in some instances cell proliferation and differentiation (reviewed in Linquist, 1986). Transcription of major heat shock loci can be increased greater than 100-fold upon the initiation of heat shock (Gilmour and Lis, 1985). This large and rapid response triggers a high level of induction in as little as three minutes (O'Brien and Lis, 1985) and is mediated by factors which are present in the cells prior to induction (Zimarino and Wu, 1987). Of interest is that the heat shock response appears to be a universal reaction of living organisms to these inducer conditions and not just of Drosophila. It has been identified in organisms as diverse as bacteria and humans and the sequences of the heat shock genes have been found to be one of the most evolutionarily conserved class of proteins (reviewed in Linquist and Craig, 1988).

11 In 1997, Jedlicka, Mortin, and Wu isolated several mutations of the heat shock factor (hsf) locus. The induction of the heat shock response is dependent upon a transcription factor which is encoded by this gene. The heteroallelic combination of hsf 1 (amorph) and hsf4 (temperature sensitive allele) is viable at 19 C throughout the entire developmental range. If this heteroallelic combination is shifted to 29-30 C (sufficient to elicit a modest heat shock response) prior to pupariation it is lethal, but it is viable if the shift occurs after the onset of pupation. Although these individuals survive the temperature shift, they remain incapable of producing a heat shock response. It was this fact which is central to the genetic manipulations which were undertaken in this experimentation.

The actual heat shock construct that was used involved a P-element cassette including a Hsp70 promoter-driven lacZ reporter (Lu et al., 1996). Bonner et al. (1984) report that the Hsp70-lacZ construct is inducible in most cells. Henikoff (1981) previously demonstrated that an endogenous Hsp 70 driven promoter, when placed near heterochromatin, may be inactivated by compaction into heterochromatin. For this experimentation a new construct was designed. It consisted of the following: the heat shock lacZ chimera, an eye specific enhancer (for mini-white expression), the mini-white reporter gene, and the entire cassette was flanked by specialized chromatin structures (scs and scs' elements) to establish a region of independent gene activity removed from euchromatic position effects but not from the actions of position-effect variegation (See figure 1) (Kellum and Schedl, 1991).

From this initial stock several transgenic lines were generated by X-ray mutagenesis. One of these lines displays a 'salt and pepper' variegation of

12 white, it is called In(3L)BL1. Another, (Tp(3;Y)BL2), displays Y-linked sectored or 'large patch' mosaicism Upon heat shocking both of the above constructs, strong variegation effects in observable structures, like the eye, are found (Lu et al., 1996).

Lu et al. (1996) reports that the In(3L)BL1 stock contains an inversion of the left arm of chromosome 3. One breakpoint was identified at the 65F site, near the transgene site of insertion at 65E. The other breakpoint was found to be within the pericentric heterochromatin of the left arm of the third chromosome.

The second construct (Tp(3;Y)BL2) resulted from a transposition of a small piece of chromosome 3, containing the transgene, into the heterochromatic Y chromosome.

The variegation encountered in In(3L)BL1 and Tp(3;Y)BL2 not only meets the structural criteria for silencing by heterochromatin but also is responsive to classic genetic modifiers of PEV. An example of this is the dosage sensitive effects that some loci, which encode structural components of heterochromatin or their modifiers, have on heterochromatin-mediated variegation. For example, both In(3L)BL1 and Tp(3;Y)BL2 have also been found to display increased silencing with the addition of a PEV enhancer such as Dp(2;2)P90 (Wustmann et al., 1989) or decreased silencing with the addition of a PEV suppressor such as Su(var)2-1 (Dorn et al., 1986). They also reported that the expected suppression of the PEV effects by increases in Y chromosome dosage was observed. This expected enhancement or suppression was found in In(3L)BL1 and Tp(3;Y)BL2 in concordance with anticipated results for both the white variegation in the eye and the Hsp 70-lac-Z variegation within the entire fly.

13 When these lines were examined for the effect of heat shock-induced lacZ expression at different developmental times, they demonstrated that heterochromatin-induced gene repression begins at embryogenesis and is then propagated continually in the imaginal discs until the late third instar stage. They found that the construct was compacted into heterochromatin early in development and that nearly no expression of the construct was seen through the third instar phase. An interesting result was observed during pupariation when a derepression of the inactivation was observed. At the end of pupation, when the adult emerges, the eye tissue had progressed from a nearly totally repressed state in the third instar to a mosaic of repressed and unrepressed cells. The expression of the lacZ gene displayed a mosaic pattern consistent with that observed in the adult eye for the mini-white gene.

Bishop (unpublished) initiated a series of temperature shift experiments with

In(3L)BL1 to determine the timing of the derepression, specifically to define the most temperature sensitive windows for derepression. In his experiment, embryos were collected at a low temperature, 19 C (which has been demonstrated to enhance the phenomenon of PEV), and a portion were shifted to the higher temperature of 29 C (which has been demonstrated to suppress PEV). This was done on each day of development. The samples were allowed to complete development at the temperature to which they had been shifted and then were subjected to a standard red eye pigment deposition assay at 470 nm. The results indicated that the curve for early shifts was consistent with what one would find for the classic variegating allele of In(1)wm4 (Jackson and Bishop, submitted). Once pupariation occurred, however, the direction of the curve was found to reverse. Instead of seeing the continual decline in pigment

14 levels that one would expect, as in In(1)wm4, there was a gradual increase in the amount of pigment deposited.

This finding lead to speculation that this might be attributable to the presence of the temperature inducible Hsp70 promoter placed immediately adjacent to the mini-white gene. Thus, It was hypothesized that upon heat shock initiated by the shift to 29 C the partial activation of this promoter was facilitating a derepression of the mini-white gene. If this hypothesis was correct, the increase in pigment deposition would not be seen in a genetic background that prevented the initiation of a heat shock response. To test this hypothesis the following experiment was designed which created a genetic line containing

In(3L)BL1 and a combination of hsf1/hsf4 which would render these progeny incapable of undergoing a heat shock response and a line containing

In(3L)BL1 and hsf4/+ which would be capable of initiating a heat shock response. The amount of red eye pigment deposited was assayed and compared to determine the extent of compaction of the P-element construct in both of the lines. The following report indicates that the ability to initiate a heat shock response does facilitate the derepression of the mini-white gene in the construct.

15 Material and Methods Description and Rearing of Fly Stocks All of the stocks used in these experiments are described in either Lindsley and Grell (1968) or Locke, Kotarski, and Tartof (1988). A brief description of mutations used in this study is presented below. Stocks were all grown on standard fly food and raised at 25o C unless otherwise noted.

Genetic Mutations Mutation Function/Purpose w Null allele of the white gene. In(1)wm4 Classical variegating allele of white; caused by an inversion of the X chromosome.

SM5 Second chromosome balancer; prevents crossing-over on the second chromosome. Carries the dominant mutation

Curly as a visible marker and a wild type allele of hsf. hsf heat shock factor gene; encodes for a transcription factor required to initiate a heat shock response. Superscript indicates different mutant alleles.

TM3, Sb Third chromosome balancer that prevents crossing-over on the third chromosome. Carries the dominant mutation

Stubble as a visible marker. In(3L)BL1 Variegating allele of the P element construct described in Figure 1. Variegates for expression of both a heat shock

promoter driven bacterial lacZ gene and a Drosophila mini- white allele. dp cl cn bw Visible recessive markers present on the hsf4 chromosome. + A wild type allele or chromosome.

16 Figure 1

P-element transgene construct

[P - scs - HS-lacZ - enh - mini-white - scs' - P]

Fig. 1. Schematic representation of the transgene construct used in this experimentation. 'mini-white' refers to the white gene coding sequences and minimal promoter sequences found in the pCaSpeR transformation vector (Pirrota, 1988). 'enh' refers to the eye specific enhancer element, 'HS-lacZ' refers to the minimal D. melanogaster Hsp70 promoter, 5' UTR and the first seven codons of the Hsp70 gene fused to the E.coli lacZ gene coding sequence and the D. melanogaster Hsp70 3'UTR, scs and scs' refer to the domain boundary elements flanking the heat shock locus. Scs elements block euchromatic but not heterochromatic position effects (Kellum and Schedl, 1991). 'P' refers to the P-element transposon terminal elements which support insertional transposition (Spradling, 1986). Transcription of both the lacZ and mini-white sequences occurs in a left to right direction.

17 Figure 2

Classical Genetics for the construction of the temperature shift stock

w; TM3, Sb/In(3L) BL1 x w;T(2;3)Xa/SM5; TM3, Sb / w; T(2;3)Xa/SM5; TM3, Sb x CyO/hsf1

/ / w; SM5/+; TM3, Sb/In(3L) BL1 x w; T(2;3)Xa/hsf1 Select: non-Xa, SM5, Sb Xa / w; SM5/hsf1; TM3, Sb/+ x w; SM5/hsf1; In(3L) BL1/+ Select : non-Xa, SM5, Sb non-Xa, SM5, PEV eyes / w; SM5/ hsf1; TM3, Sb/ In(3L)BL1 Select : Sb and PEV eyes

Figure 2: Classical genetic techniques were employed to create a stock for use in the temperature shift experiments. From four initial stocks the final balanced test stock of w; SM5/hsf1; TM3, Sb/In (3L) BL1 was constructed for use in the temperature shift experiments.

18 Classical Genetics- Classical genetic techniques were employed to create a stock for use in the temperature shift experiments (see Figure 2).

From four initial stocks the final balanced test stock of w; SM5/hsf1; TM3, Sb/ In (3L) BL1 was constructed for use in the temperature shift experiments. This stock was mated with a temperature-sensitive allele of the heat shock factor (hsf) gene as follows: w; SM5/hsf1; TM3, Sb/ In (3L)BL1 X dp cl cn bw hsf4 = cross 1

This cross generated progeny that will be addressed below.

For additional controls In(1)wm4 X w; SM5/hsf1; TM3, Sb/ In (3L)BL1 = cross 2.

Temperature shift experiments- Temperature shift experiments were conducted on the progeny from the above crosses. Three replicates of each cross were set at 19 C. Each group of flies was allowed to lay eggs for 24 hours before it was transferred to a new vial at 19 C. This step was performed so that for each vial the temperature shifts would occur at the same time and developmental window. This would insure that individuals within the same vial would have no more than one day difference in their age. Cultures were allowed to mature to pupation at 19 C. At pupation, vials to undergo the temperature shift were placed into a 29 C environment. The shifted vials were cleared of any non-pupated larvae several times over the next few days.

19 After eclosion the adults were moved to 25 C, where they were allowed to mature over the next four days. This was done so that eye pigment deposition could be accomplished. After this time they were sorted into male or female experimental or control groups (see below).

Two types of male and female progeny from cross 1 (females w; SM5/hsf1; TM3, Sb/ In (3L)BL1 X dp cl cn bw hsf4 males) were collected. male offspring: w; hsf4 /hsf1; In (3L) BL1/+: labeled experimental, (incapable of a heat shock), and w; SM5/ hsf4; In (3L) BL1/+: labeled controls, (capable of a heat shock).

For cross 2, or the second chromosome control cross, (females In(1)wm4 X w; SM5/hsf1; TM3,Sb/ In (3L)BL1 males) male progeny were collected. w;SM5 /+;TM3,Sb /+. labeled control and w; hsf1/+;TM3,Sb /+. labeled experimental This cross was conducted to ensure that the observed differences were the result of an inability to induce a heat shock response and not due to a second site mutation located elsewhere on the SM5 or the hsf1 second chromosomes, cross 2 was conducted and male progeny were collected. Then, the SM5 progeny were compared to the hsf1 progeny for the amount of red eye pigmentation.

Eye pigment determination- The amount of red eye pigment was assayed using standard techniques at an absorbence of 470 nm(see Ephrussi and Herold, 1944, as modified in Locke, Kotarski, and Tartof, 1988 and Bishop and

20 Jackson, 1996) and the results were tabulated and subjected to statistical analysis, (specifically, calculation of standard error and pair comparisons using Tukey-Kramer HSD).

Sample size Each data point is derived from the mean of a minimum of 3 samples of 10 heads each.

Double Blind Study- The above temperature shift experiment was conducted a second time in its entirety to insure that the initial results were both valid and reliable. The eye pigment assay portion of the experiment was subjected to a stringent double blind control procedure.

21 Results Male flies heteroallelic for two mutations at the heat shock locus (w; hsf4/ hsf1; In (3L) BL1/+ experimental flies) displayed a 41% decrease in the amount of eye pigment deposited when compared to flies that contained one functional copy of the heat shock gene (w; SM5/ hsf1; In (3L) BL1/+ , control flies) (Table 1). These results were also supported by the female progeny of the same cross (Table 2). These flies were the progeny of temperature shift cross 1.

To ensure that the observed differences were the result of an inability to induce a heat shock response and not due to a second site mutation located elsewhere on the SM5 or the hsf1 second chromosomes, the w; SM5/hsf1; TM3, Sb/In(3L)BL1 stock was crossed to In(1)wm4, cross 2 (Figure 2). Then, the In(1)wm4;SM5/+;TM3,Sb/+ progeny were compared to the In(1)wm4;hsf1/+;TM3,Sb/+ progeny for the amount of eye pigmentation present. This experimentation did not show any significant difference in the amount of pigment deposited in hsf1 versus SM5 progeny (Student's t= 0.12393; ts0.05=2.776, Table 3).

The double blind experimentation verified the results obtained from the original experiment (data not shown). In this experiment both male and female progeny of the test crosses were also assayed for pigment deposition. Their results supported the results produced by the first temperature shift experiment.

22 Table 1

Red Eye Pigmentation Assay for Cross 1 Male Progeny: w; SM5/hsf1; TM3, Sb/ In (3L)BL1 x +; hsf4

Absorbence at 470 nm Absorbence at 470 nm (Least Sqr. Means)+/-SE (Least Sqr. Means)+/-SE Experimental Control Replicate w; hsf 4 /hsf 1 ; In (3L) BL1/+ w; SM5/ hsf 4 ; In (3L) BL1/+ A 0.02250+/- 0.00366 0.04260+/-0.00253 B 0.02830+/-0.00301 0.04250+/-0.00223 C 0.02800+/-0.00108 0.04557+/-0.00227

Table 1: Red eye pigmentation assay for cross 1 male progeny. Displayed is the least square mean of the red eye pigmentation absorbence at 470 nm for at least 40 individuals for each replicate. Statistics: Comparison for experimental/control using Tukey-Kramer HSD= 0.013085 (positive values indicates that the pairs of means are significantly different).

23 Table 2

Red Eye Pigmentation Assay for Cross 1 Female Progeny: w; SM5/hsf1; TM3, Sb/ In (3L)BL1 x +; hsf4

Absorbence at 470 nm Absorbence at 470 nm Least Sqr. Means+/-SE Least Sqr. Means+/-SE Experimental Control Pair Comparison using Replicate w/+; hsf 4 /hsf 1 ; In (3L) BL1/+ w/+; SM5/ hsf 1 ; In (3L) BL1/+ Tukey-Kramer HSD* A 0.45800+/- 0.00532 0.68400+/-0.00582 0.170826 B 0.46400+/-0.00583 0.77475+/-0.00532 0.261576 C 0.47000+/-0.00583 0.91550+/-0.00517 0.390326

Table 2: Red eye pigmentation assay for cross 1 female progeny. Displayed is the least square mean of the red eye pigmentation absorbence at 470 nm for at least 40 individuals for each replicate.

*- positive values show pairs of means that are significantly different.

24 Table 3

Red Eye Pigmentation Assay for Control Cross 2:

In(1)wm4 X w; SM5/hsf1; TM3, Sb/ In (3L)BL1

Absorbence at 470 nm Absorbence at 470 nm (Least Sqr. Means)+/-SE (Least Sqr. Means)+/-SE Experimental Control Replicate In(1)w m4 ; hsf 1 /+;TM3,Sb/+ In(1)w m4 ; SM5/+;TM3,Sb/+ A 0.01230+/- 0.00132 0.00960+/-0.00246 B 0.0127+/-0.00120 0.01450+/-0.00236 C 0.01230+/-0.00116 0.01320+/-0.00146

Table 3: Red eye pigmentation assay for cross 2 male progeny. Displayed is the least square mean of the red eye pigmentation absorbence at 470 nm for at least 40 individuals for each replicate. No significant differences in the amount of pigment deposited in hsf1 versus SM5 progeny were detected (Student's t= 0.12393; ts0.05=2.776).

25 Discussion Cavalli and Paro (1998) have shown that, in euchromatin, the binding of proteins from the Polycomb group of genes (PcG) can be reversed by the expression of a nearby promoter, thus changing the epigenetic repression of gene expression. To examine this question, they constructed a transgenic fly containing a Fab-7 element adjacent to a GAL-4 driven lacZ reporter gene and a mini-white gene. In the absence of GAL 4, proteins from the PcG bind to the

Fab-7 element and repress expression of the mini-white gene. Early transient expression of the Gal 4 promoter is sufficient to permanently erase the repressed state of the mini-white gene. This occurs even though the mini-white gene is not expressed in the eyes until the late third instar period. In the experiment presented in this paper, a similar construct (Hsp70 driven lacZ and a mini-white gene) has been used to examine the influence of adjacent promoters on heterochromatin-induced gene inactivation.

By transforming a P element construct into the euchromatic 65E region on the third chromosome and by using x-ray mutagenesis, Lu, Bishop, and Eissenberg (1996) designed a variegating system that could be followed throughout development and expressed in nearly every cell. The P element construct inserted contained two novel reporter systems, the Hsp70-lacZ reporter plus a mini-white reporter system (Figure 1). Upon X-ray mutagenesis two subsequent variegating lines were isolated (In(3L)BL1 and Tp(3;Y) BL2) by juxtaposing the P-element construct next to a region of heterochromatin. This resulted in variegated expression of the mini-white gene and the adjacent lacZ gene. When the effect of heat shock-induced lacZ expression was examined throughout development, the results indicated that heterochromatin-induced

26 gene repression begins during embryogenesis. It is then propagated in the imaginal discs until the late third instar stage. They found that the construct was compacted into heterochromatin early in development and that virtually no expression of the construct was seen through the third instar phase. During pupariation an interesting result was observed, it was found that a derepression of the heterochromatic activation occurred at this time. At this stage the eye tissue progressed from a nearly totally compacted repressed state to a mosaic of repressed and unrepressed cells. At this developmental time, the expression of the lacZ gene displayed a mosaic pattern consistent with that observed in the adult eye for the mini-white gene.

Bishop (unpublished) examined In(3L)BL1 in more depth to determine the timing of the derepression. Specifically, he used a series of temperature shift experiments to define the most temperature sensitive windows for derepression. Embryos were collected at a low temperature, 19 C (which has been demonstrated to enhance the phenomenon of PEV), and a portion were shifted to the higher temperature of 29 C (which has been demonstrated to suppress PEV). This was done on each day of development. The samples completed development at their shifted temperature. The resulting individuals were then subjected to a standard red eye pigment deposition assay at 470 nm. The results indicated that the curve for early shifts was consistent with what one would find for the classic variegating allele of In(1)wm4 (Jackson and Bishop, submitted). After pupariation, however, the direction of the curve was found to reverse. After this time, there was a gradual increase in the amount of pigment deposited instead of seeing the continual decline in pigment levels that one would expect, as in In(1)wm4. It was speculated that this might be attributable to the presence of the temperature inducible Hsp70 promoter

27 placed immediately adjacent to the mini-white gene in In(3L)BL1. It was hypothesized that a heat shock response, initiated by the shift to 29 C, resulted in the partial activation of the Hsp70 promoter, thus facilitating a derepression of the mini-white gene. If this hypothesis was correct, the increase in pigment deposition would not be seen in a genetic background that prevented the initiation of a heat shock response.

Jedlicka, Mortin, and Wu (1997) isolated mutants for the heat shock factor (hsf) locus, which encodes a transcription factor that is required for the induction of a heat shock response. The heteroallelic combination of hsf1 (amorph) and hsf4 (temperature sensitive allele) is viable at a low temperature throughout all of development. Once this heteroallelic combination is shifted to 29 C, however, it is lethal if this shift occurs prior to pupariation. If the shift occurs after the onset of pupariation this genetic combination is viable but the individuals are found to be incapable of initiating a heat shock response. Because of this we were able to utilize these constructs to further test the hypothesis put forth in the previous paragraph. Since a shift from 19 C to 29 C will initiate a modest heat shock response (Ashburner, 1989) we can test the effects on derepression of In(3L)BL1 in a genetic background allowing for a heat shock response (SM5, hsf+/hsf4) and in one in which it is prevented (hsf1/hsf4).

The following cross produced both the control and the experimental genotypes, differing in their ability to induce a heat shock response:

w; SM5, hsf+/hsf1; TM3, Sb/In (3L)BL1 X dp cl cn bw hsf 4

Control: w;SM5, hsf+/hsf4; In(3L)BL1/+

28 The control group genotype has only one functional copy of the hsf gene. This is, however, enough to initiate a heat shock response. In males, the variegating mini-white gene derived from In(3L)BL1 is the only source of the white gene.

Experimental: w; hsf1/hsf4; In(3L)BL1/+ The experimental genotype has been genetically manipulated to be functionally hemizygous for the hsf4 temperature sensitive allele and as such is incapable of initiating a heat shock response at 29 C. In males, as was seen in the above control, the only source of the white gene is the mini-white gene contained within the In(3L)BL1.

The females of both genotypes will be heterozygous for the endogenous white+ locus (w/+) in addition to the variegating In(3L)BL1 allele. Since a wild type w allele is only incompletely dominant to a w mutation (Lindsley and Grell, 1972), it can be reasoned that these females may also be examined for derepression of In(3L)BL1.

The results of our experiments clearly demonstrate that both males and females undergo derepression to a much greater extent when a heat shock response is genetically and environmentally possible. Males in particular show a significantly greater amount of eye pigment deposited (41% difference) when they are capable of initiating a heat shock response. Females also show a significant difference in the amount of pigment deposited in the heat shocked experimental in relation to the control individuals.

The control cross was conducted to insure that the observed differences were due to the hsf mutations and not due to second site modifiers located

29 elsewhere on the second chromosome. When the results of the control cross were analyzed, it was found that there were no significant differences found in the amount of red eye pigment deposited in the experimental progeny

(In(1)wm4;hsf1/+; TM3,Sb/+) when compared to the control (In(1)wm4;SM5/+; TM3,Sb/+). Because of this, the possibility of the difference being due to the action of second-site modifiers of variegation elsewhere on the second chromosome can be eliminated as a source for the results obtained.

Through this research we have established that the expression of a nearby promoter can facilitate the developmentally programmed derepression of heterochromatin which was observed during pupation. Before this derepression occurs, the heat shock promoter is well compacted into the heterochromatin and as such is incapable of being expressed in the majority of cells (Lu et al., 1996). Since the gene is compacted into heterochromatin and repressed, it is not able to initiate or facilitate derepression. While heterochromatin-induced gene repression is present (during the period prior to pupariation), raising Drosophila at a low temperature favors the formation of even more heterochromatin. Thus, when conditions favor the formation of heterochromatin, the result is even greater inactivation of the compacted gene. This is thought to occur by further compacting the underlying DNA into an even more repressive form of heterochromatin (for example, more extensive post translational modification of the histones), or the physical effect of surrounding the DNA in an even greater quantity of heterochromatic proteins. Both of the following situations lessen the probability that derepression of the region will occur. When the programmed derepression occurs enough to allow for the expression of the Hsp 70 gene, its expression can facilitate the derepression of adjacent DNA (the mini-white construct in our experimentation). This might be

30 accomplished for instance, by moving the DNA or chromosomal region into an area of the nucleus that favors the breakdown of heterochromatin. Jackson and Bishop (submitted) have indicated that the formation of heterochromatin appears to be a progressive event. The temperature shift data for In(3L)BL1 argues that the derepression of the gene also occurs in a progressive manner (Bishop, unpublished). In other words, as development proceeds past pupariation, the later the heat shock promoter is induced the greater the likelihood that it has been derepressed and therefore can be more fully expressed. This stands to reason for the early compaction of the DNA into more dense regions of heterochromatin is followed by a developmentally pre- programmed derepression at pupation (Lu et al., 1996). It is possible that as this derepression proceeds the Hsp 70 promoter becomes continually less compacted in a greater majority of cells. Alternatively, the ability to induce a heat shock response in all cells could be increasing with time. Whichever the case, when a heat shock response is induced more cells can initiate a more forceful response as the derepression advances. This in turn can act to facilitate the derepression of the compacted nearby genes to an ever increasing greater degree.

This experimentation suggests that cooperative derepression occurs and may be playing a role in the expression of genes contained within the B- heterochromatic regions (at the heterochromatic/euchromatic junction and on the fourth chromosome). In these regions there appears to be a dispersal of euchromatic genes around heterochromatic genes. Perhaps the mechanisms proposed in this experimentation delineate the means of expression of genes contained within these areas.

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39 Curriculum Vita

Daniel R. McNeill was born on September 09, 1968 in West Virginia. He attended West Virginia University for his undergraduate schooling and obtained a B.A. in Biology in 1990. He then spent 3 years in the Medical School at West Virginia University. During this time he was honored as a Van Liere Medical Scholarship in 1991. In 1993, He returned to the Biology Department at West Virginia University for graduate studies. During this time he functioned as a researcher and teaching assistant in this department. In 1998, he obtained a Masters of Science in Molecular and Cellular Biology from this department.