Exploring regulation and expression of the Abd-B Homeotic Gene using BAC technology in Drosophila: a new role in reproduction
We discovered that the hox gene Abdominal B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer in the iab-6 regulatory domain that is responsible for Abd-B expression in these cells removal of which results in visible morphological defects in the secondary cells. Mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity phenotypes. These data for the first time uncovered part of the function of the secondary cells. Transcriptome analysis from WT and mutant accessory glands led us to consider a list of 73 target genes that are down regulated in the mutant gland. The results RNAi experiments on these genes are presented in detail within the thesis. Interesting case of transvection as well as Gal4 toxicity are also included in these thesis.
GLIGOROV, Dragan. Exploring regulation and expression of the Abd-B Homeotic Gene using BAC technology in Drosophila: a new role in reproduction. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4562
URN : urn:nbn:ch:unige-290620
DOI : 10.13097/archive-ouverte/unige:29062
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITE de GENEVE FACULTE DES SCIENCES Département de génétique et evolution Professeur François Karch
Exploring Regulation and Expression of the Abd-B Homeotic Gene Using BAC Technology in Drosophila: A New Role in Reproduction
par Dragan Gligorov
Thèse No – 4562 -
Atelier d’impression ReproMail Mai 2013
Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
Table of content
Résumé français 3
Abd-B regulation 11
The domain model 14
Long-distance interaction in the BX-C 18
Boundary Elements in Long-Distance Chromatin Interactions 20
Part I. Ectopic trans-activation of Abd-B in the salivary gland by the Abd-B Gal4 Bac
Adult salivary gland shows ectopic Abd-B expression 26
DNA FISH 28
Salivary gland morphological phenotype 30
Secondary cell enhancer studies suggest Gal-4 toxicity 35
Part II. A novel function for Abd-B in the male accessory gland 43
Creation of BAC reporters/fusions for Abdominal-B 47
Previously unknown location of Abd-B expression in adult flies 51
Secondary cell enhancer 55
Abd-B expression in secondary cells is independent of the initiator 61 Part III. Dissection of the secondary cell transcriptome 62
Preliminary in situ hybridization results 69
Abd-Bm ISH 70
Antibody development 71
Vacuolar markers 71
New insights into Abd-B regulation 74
Initial observation from the mRNA-seq data 77
Possible crosstalk between the different cell types in the male reproductive system
Cluster or group of genes are simultaneously regulated 80
RNAi of candidate genes 81
The vacuoles 83
Abd-Bm in situ hybridization 84
My Summary 86
Materials and Methods 89
Table 1. The 73 candidate genes, predicted molecular function, maximal tissue expression
Table 2. RNAi experiment egg laying and secondary cell morphological data 116
My First Paper (added as a pdf print out from the journal PLOS Genetic with supplementary figures)
A novel function for the hox gene abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila.
Les gènes homéotiques (également connus sous le nom de gènes architectes, ou gènes Hox) sont connus pour le rôle qu’ils jouent dans la détermination des structures qui se forment le long de l’axe antéropostérieur des organismes à symétrie bilatérale. C’est l’analyse génétique chez la drosophile (Drosophila melanogaster) qui a conduit à l’identification des gènes homéotiques, grâce à la découverte de mutations qui transforment les antennes des drosophiles en pattes, ou des mutations qui conduisent à l’apparition de mouches avec 2 paires d’ailes au lieu d’une seule.
De façon très intrigante, les gènes architectes sont arrangés le long du chromosome dans le même ordre que les segments qu’ils spécifient le long de l’axe antéropostérieur. De façon encore plus remarquable cette correspondance entre organisation génomique et axe antéro-postérieur a été conservée au cours de l’évolution, et des complexes similaires de gènes architectes (ou complexes Hox) se retrouvent chez les vertébrés, les mammifères et chez l’homme. Au cours de mon travail de thèse j’ai étudié la régulation du gène homéotique Abdominal-B (Abd-B) qui appartient au complexe bithorax et qui détermine l’identité des segments abdominaux postérieur de la drosophile (A5 à A8). Le gène Abd-B est très complexe et occupe avec ses séquences régulatrices beaucoup d’espace sur le chromosome. Afin de mieux comprendre son mode de régulation, nous avons créer un chromosome artificiel d’origine bactérienne qui exprime une protéine fluorescente de méduse (GFP) dans le contexte génomique normal d’Abd-B.
L’expression de la GFP mimant celle du gène Abd-B, peut alors être suivie in vivo, en observant des embryons, larves ou mouches adultes à la lumière ultra-violette. J’ai ainsi découvert, que le
4 gène Abd-B est exprimé dans les « glandes accessoires » de l’appareil reproductif mâle. Ces
« glandes accessoires », (qui pourraient s’apparenter à la prostate chez l’homme), synthétisent un cocktail de peptides qui composent le liquide séminal et qui modifient le comportement des femelles une fois l’accouplement terminé. Lors de l’accouplement, la drosophile femelle reçoit le sperme qu’elle stocke dans des structures spécialisées, les spermathèques et les glandes séminales, pour féconder de manière autonome les nombreux ovoyctes qu’elle libère durant la dizaine de jours suivants. Ce sperme contient en plus des spermatozoïdes, des composés, parmi lesquels le « sex peptide » qui induit chez la femelle, des transformations physiologiques telles que la stimulation de l’ovogenèse ou du système immunitaire, ainsi qu’un changement de comportement. Les femelles fraichement fécondées repoussent les sollicitations d’autres mâles.
En d’autres termes, en transférant son « sex petide », le male s’assure de l’exclusivité de la transmission de ses propres gènes. Les « glandes accessoires » sont des organes tubulaires composés d’un millier de cellules d’origine mésodermale (les cellules principales). A l’extrémité distale de la glande se trouvent une soixantaine de cellules (dites secondaires) à la morphologie clairement distincte du reste des cellules de la glande, par la présence de grandes vacuoles dans leur cytoplasme. Le gène Abd-B est exprimé spécifiquement dans ces cellules secondaires. En puisant dans la collection d’allèles du laboratoire , j’ai identifié une mutation qui élimine l’expression d’Abd-B dans les cellules secondaires des glandes accessoires. La perte de l’expression d’Abd-B dans ces cellules ne cause pas leur transformation en cellules principales. Le gène Abd-B n’agit donc pas comme déterminant de l’identité cellulaire dans les glandes accessoires. Les cellules secondaires restent secondaires, mais les vacuoles présentes dans leur cytoplasme disparaissent. Cette mutation n’a aucune autre conséquence sur la spécification des segments abdominaux et les mouches éclosent avec une morphologie externe
5 parfaitement normale.. En collaboration avec le laboratoire de Mariana Wolfner de l’Université de Cornell dans l’état de New York, nous avons démontré que les femelles fécondées par les males mutants ne repoussent plus les nouveaux courtisans après un accouplement. En référence à ce phénotype cet allèle a été baptisé iab-6cocu. Le « sex peptide » qui est synthétisé par les cellules principales est bien transmis aux femelles. Mais, s’il reste détectable pendant une dizaine de jours chez les femelles fécondées par des males normaux, le « sex peptide » disparaît beaucoup plus rapidement des femelles fécondées par des males iab-6cocu. Cette découverte a une certaine résonnance dans le petit monde intéressé par la question du changement de comportement des femelles suite à l’accouplement, car elle démontre l’importance des cellules secondaires des glandes accessoires. L’existence de deux types cellulaires distincts dans les glandes accessoires, cellules primaires et secondaires et l’expression du gène Abd-B uniquement dans ces dernières, suggère un mécanisme de compartimentation. On peut imaginer que le liquide séminal doit être activé lors de l’éjaculation, pour que le « sex petide » soit stabilisé une fois transféré à la femelle.. Des expériences dites de « proteomic » ont permis l’identification d’environ 180 protéines spécifiques des glandes annexes (appelées Acps pour « Accessory gland-specific proteins). Avec notre nouvelle lignée exprimant la GFP spécifiquement dans les cellules secondaires et l’identification de la mutation qui élimine l’expression d’Abd-B dans ces mêmes cellules, le laboratoire dispose de nouveaux outils pour identifier les Acps spécifiques des cellules secondaires et ainsi mieux comprendre la complexité de la biologie de la reproduction chez la drosophile.
Ces observations pourraient également apporter un éclairage sur des questions évolutives.
L’arrangement des gènes homéotiques au sein des complexes Hox reflète l’ordre des segments ou métamères dans lesquels ils sont actifs. Le gène Abd-B et ses homologues chez les vertébrés
6 (hox10-13) sont positionnés à l’extrémité des complexes Hox active dans les segments ou métamères postérieurs de l’embryon. Chez la drosophile, le gène Abd-B est actif dans les segments abdominaux 5 à 8 et dans la « primordia » qui va donner naissance aux structures génitales externes. Cependant, chez des insectes plus primitifs tels que la sauterelle, ou chez les araignées, le gène Abd-B n’est exprimé que dans les structures génitales externes. Cette observation a incité plusieurs chercheurs à proposer que la fonction ancestrale du gène Abd-B était de spécifier les structures génitales externe et qu’il avait été recruté plus tard au cours de l’évolution, comme gène de spécification des segments abdominaux postérieurs. Chez les mammifères les gènes Hox10-13 (homologues du gène Abd-B) sont également actifs dans le bouton génital, la prostate et la glande séminale. Ainsi, cette conservation de l’expression dans la prostate chez les mammifères et dans les glandes accessoires chez les drosophiles pourrait renforcer l’hypothèse que la fonction primordiale du gène Abd-B chez les arthropodes et de ses homologues hox10-13 chez les mammifères serait de spécifier les organes de reproductions. La position d’Abd-B et de ses homologues hox10-13 au sein des complexes Hox expliquerait la raison pour laquelle les organes génitaux se développent toujours à l’extrémité postérieure des organismes à symétrie bilatérale.
Homeotic genes (also known as “architect genes”, or Hox genes) are known for their role in the specification of the structures that form along the anteroposterior axis of bilateria (organisms from the animal kingdom with bilateral symmetry at the same stage of development). Homeotic genes were discovered almost a century ago in Drosophila through the identification of mutations that transform antennas to legs, or mutations that lead to the emergence of flies with 4 wings instead of 2. Strikingly, these architect genes are arranged along the chromosome in the same order as the body segments they specify along the anteroposterior axis of the fly.
Furthermore this remarkable correspondence between genomic organization and anteroposterior axis has been conserved through evolution, and similar complexes of architect genes (Hox complex) are found in other invertebrates, in vertebrates, mammals and human. During my thesis work, I have analyzed the regulation of the Abd-B homeotic gene from the bithorax complex (BX-C) in Drosophila. The Abd-B gene species the identities of the segments that form the posterior abdomen of the fly (A5 to A8). The Abd-B gene is quite complex with regulatory regions that are spread over large regions of DNA. In order to better understand how it is regulated, I have reconstructed the entire Abd-B locus on a bacterial artificial chromosome. In this reconstituted locus however, the Abd-B coding sequences were replaced by sequences coding for a green fluorescent protein from jellyfish (GFP). After introducing this large construct into the fly genome, one can follow Abd-B expression in living embryos, larvae or adults flies by simple illumination with ultraviolet light under the microscope. This procedure
8 enabled me to discover that Abd-B is expressed in the "accessory glands" of the male reproductive tract. The "accessory glands" (which correspond to the male prostate and seminal vesicles in mammals) is responsible for the synthesis of a cocktail of proteins that constitute the seminal fluid. Some of these proteins have the power to change the female’s behavior after mating (the so-called post-mating response or PMR). It should be noticed that in Drosophila, females store the sperm in specialized structures (called spermathecae and seminal receptacles).
This storage enables the female to fertilize autonomously, the many oocytes that she will release during ~10 days that follow mating. In addition to transmitting his sperm, the male adds a cocktail of proteins, including the “sex peptide”, which will elicit different physiological and behavior changes in the female, ensuring thereby optimal use of his sperm. Among these changes (eg, stimulation of oogenesis or of the immune system), freshly fertilized females reject other courting males for a period of about 10 days after mating. In other words, by transferring his "sex petide", the male ensures the fidelity of the female for a period of ten days. The induced physiological changes in the female result in a genetic advantage for the genes of the copulating male in populating the next generation. Accessory glands are tubular organs composed of a thousand cells of mesodermal origin named the “main cells”. At the distal end of the gland, there are about 40 cells per lobe (the secondary cells) that are clearly distinguishable from the main cells by the presence of large vacuoles in their cytoplasm. Abd-B is specifically expressed in these secondary cells. Looking in the large collections of Abd-B alleles available in the laboratory, I have identified a small deletion that eliminates Abd-B expression in these secondary cells. The loss of expression of Abd-B does not cause the transformation of secondary cells into main cells. Thus, Abd-B does not act as a determinant of cell identity in the accessory glands.
While the secondary cell fate is not affected in the mutant, the cells appearance changes as
9 revealed by the loss of their characteristic large vacuoles. The small deletion that eliminates expression of Abd-B in the secondary cells has no other effect on the specification of the abdominal segments and flies hatch with a completely normal external morphology. However, male fertility is affected. In collaboration with the laboratory of Mariana Wolfner from Cornell University in upstate New York, we have discovered that females fertilized by the mutant males do not exhibit the long-term behavioral change post mating and do not reject subsequent courtship by males. In reference to this phenotype the allele was named iab-6cocu. The "sex peptide" which is synthesized by the main cells is transmitted to females. But although the "sex petide" remains detectable in females for ten days after mating by normal males, it disappears much faster in females fertilized by iab-6cocu males. This finding has some importance in the small community interested in the post mating response, because it demonstrates the importance of the secondary cells in this process. The existence of two distinct cell types in the accessory glands suggests a mechanism of compartmentalization. One can imagine that the seminal fluid must be enabled upon ejaculation, so that the "sex petide" is stabilized once transferred to the female. In general, the vacuoles are formed by the fusion of small vesicles involved in intra- cellular trafficking and in secretion. One can imagine that the large vacuoles of the secondary cells are the storage site of compounds that must be strictly separated from other compounds synthesized by the main cells. Proteomic technology led to the identification of 208 Accessory gland-specific proteins (Acps). With the Drosophila line expressing GFP specifically in secondary cells and with the identification of the mutation that eliminates expression of Abd-B specifically in these cells, the laboratory has now powerful tools to identify Acp specific for the secondary cells and to better understand the complexity of the biology of reproduction in Drosophila.
10 These findings may also have evolutionary implications. During embryogenesis, the establishment of the anteroposterior axis in organisms with bilateral symmetry such as worms, arthropods or vertebrates, proceeds by segmentation and/or metamerisation into repetitive structures. Each segment, or metamer, subsequently acquires an identity through the activity of the homeotic genes. As mentioned above, the arrangement of homeotic genes in the Hox complex reflects the order of the metamers or segments in which they are active. The Abd-B gene, and its orthologous counterparts in vertebrates (hox10-13), are positioned at the end of the Hox complexes and are active in the posterior segment or metamers. In Drosophila, Abd-B is active in abdominal segments 5 to 8/9, as well as, in the "primordia" of the external genital structures (the so-called genital disc). However, in more primitive insects such as grasshoppers and in spiders, the Abd-B gene is solely expressed in the external genital structures. This observation has led several researchers to propose that the ancestral function of the Abd-B gene was the specification of the external genital structures, and that Abd-B was later recruited during evolution as a gene to specify posterior abdominal segments. Interestingly, the function of the Abd-B class genes in seminal protein producing tissues seems to be an ancient and conserved function, as the orthologs of Abd-B in mammals, the hox10-13 class of genes, are expressed in the mammalian prostate and seminal vesicle. This conservation of function supports the hypothesis that the primary function of the Abd-B gene in arthropods (and its hox10-13 counterparts in mammals) is the specification of the genital structures. The position of Abd-B and its counterparts within the Hox clusters may explain why the reproductive organs always develop at the posterior end of bilateria.
The homeobox-containing transcription factor, Abdominal B (Abd-B), determines the segmental identity of the last five segments of the fly (the 5th through 9th abdominal segments) (Figure 1). Together with the abdominal-A (abd-A) and Ultrabithorax (Ubx) genes, Abd-B makes up one of the two Drosophila homeotic gene clusters, known of as the Bithorax complex (BX-C) (Lewis 1954), (Maeda and Karch 2006). These three genes are responsible for patterning the posterior 2/3 of the fly, from the posterior thorax to the posterior tip of the abdomen.
Figure 1. Diagram of the BX-C. The multicolored bar represents the DNA of the BX-C. Map coordinate numbering follows the numbering established by the original Drosophila Genome Project sequencing of the BX-C (Martin et al.,1995). The three BX-C homeotic genes,Ubx, abd-A and Abd-B are indicated below this bar (with exons indicated by the black horizontal bars and introns indicated by the diagonal lines connecting the bars). The individual cis- regulatory domains are indicated by the different colored regions on this bar. The orange and red regions (abx/bx and bxd/pbx) control Ubx expression. The regions shaded in blue (iab-2, 3 and 4) control abd-A expression. And theregions shaded in green (iab-5 through iab-8) control Abd-B expression. The corresponding adult segments affected by mutations in each cis-regulatory region are indicated on the diagram of the adult fly using the same color code. (Figure adopted from Maeda and Karch 2006 - The ABC of the BX-C: the bithorax complex explained)
12 As alluded to above, the Abd-B transcription factor is expressed in the 5th through 9th abdominal segments of the fly. In the embryo, however, where segmental borders are not easily seen until later stages, metameric units known as parasegments (PSs) are generally used to describe expression patterns. Early in embryonic development the Drosophila embryo is divided into 14 PSs by the products of the gap and pair-rule genes. Each embryonic PS roughly corresponds to the posterior half of one future adult segment and the anterior half of the next adult segment. Abd-B is thus expressed in embryonic PS 10-14 of the Drosophila embryo (Figure 2). In this thesis, I will use both segment and/or parasegment nomenclature depending upon the stage of development referred to.
Figure 2. The Drosophila embryo is metamerized into 14 parasegments. The segments and parasegments are slightly shifted relative to one another. In the thorax and the abdomen, this shift is approximately half a segment, meaning that a parasegment comprises the posterior half of one segment and the anterior half of the next. For example, PS6 comprises the posterior of segment T3 and the anterior segment A1. (Figure adopted from Maeda and Karch 2006 - The ABC of the BX-C: the bithorax complex explained)
The expression of Abd-B is set very early in development and can be detected at the protein level by about 3-4 hours of embryonic development (Celniker, Keelan et al. 1989). There are two isoforms of Abdominal B, the “morphogenic” (m) form, which is expressed in parasegments 10-13, and the “regulatory” (r) form, which is expressed only in parasegment 14.
Expression of both Abd-B isoforms is controlled by a large cis-regulatory region that spans about
13 100kb. Mutational analysis has subdivided this region into four cis-regulatory domains, called infrabdominal (iab) domains, each seemingly controlling the expression of the Abd-Bm isoform in a specific parasegment of the embryo (Lewis 1978; Zavortink and Sakonju 1989). For example, the iab-5 domain controls the expression of Abd-Bm in PS10 (making up the visible portion of the Abdominal segment 5 (A5) of the adult fly). Meanwhile, the iab-6 domain controls the expression Abd-Bm in PS11 (A6 of the adult fly) (Figure 1). Interestingly, these iab domains are aligned along the chromosome in an order colinear to the segments in which they function along the A-P axis.
Genetic data suggests that only one iab domain controls Abd-B expression in a given parasegment of the embryo (Mihaly, Barges et al. 2006). This is supported by the fact that deletion of a single domain generally affects the development of only one parasegment. In parasegments anterior to where a domain is required to activate transcription, Abd-B is thought to be kept in a repressed state by the Polycomb repression system (Zink and Paro 1989; Zink, Engstrom et al. 1991). Given the complex regulatory interactions in the BX-C, it has become a model system to study gene expression and the interplay between chromatin structure and gene expression.
Since the identification of the cis-regulatory domains of the BX-C, these domains have been dissected using transgenic reporter assays to identify individual regulatory elements capable of modifying reporter gene expression. This analysis has revealed that each cis-regulatory domain of the BX-C seems to be composed of a similar set of cis-regulatory elements (for a review see (Maeda and Karch 2006)). Among the elements identified were early embryonic enhancers (also called initiators), cell-type-specific enhancers, silencers and insulators.
Interestingly, although homeotic gene expression is restricted along the A-P axis, many of the
14 elements identified by transgenic analysis only control reporter gene expression in a tissue- specific manner, but not in an A-P position-restricted manner. Thus, within their normal genomic context, the cis-regulatory elements must be modulated by other cis-regulatory elements to gain A-P restriction. These data have led to the driving hypothesis in BX-C research where the cis- regulatory elements of the BX-C are controlled as a group through the activation or repression of parasegment-specific (PS-specific) chromatin domains (Peifer, Karch et al. 1987; Bender and Hudson 2000).
The domain model
According to this model, the BX-C functions through multiple layers of control. First, there are the enhancers that directly activate homeotic gene expression in a pattern appropriate for a specific segment. These are the cell-/tissue-specific enhancers. Numerous elements of this type have been found in the BX-C including enhancers driving gene expression in the CNS, epidermis and gut mesoderm (Mihaly, Barges et al. 2006). Genetic deletion analysis has shown that these enhancers are grouped so that all the enhancers required to produce a pattern appropriate for a given segment/parasegment are clustered into a single region of the BX-C cis- regulatory sequence. Once again, as the transgenic data reveals, these enhancers, on their own, drive gene expression in specific tissues, but are not restricted along the A-P axis.
The second layer of control comes from Polycomb-response element silencers (PREs).
Within each cis-regulatory domain there seems to be at least one PRE (and probably multiple PREs). These silencers are thought to turn off the clusters of enhancers in parasegments where they are not needed, via modification of the local chromatin structure around the enhancers (Simon, Chiang et al. 1992; Orlando and Paro 1993; Chiang, O'Connor et al. 1995; Fitzgerald
15 and Bender 2001; Akbari, Bousum et al. 2006; Maeda and Karch 2006; Müller and Kassis 2006).
The stable gene silencing resulting from this modification of chromatin structure has led to these elements often being called maintenance elements. The maintenance activity of PREs can be observed in transgenic assays when combined with enhancers. For example, the iab-6 initiator fragment (a specific type of early embryonic enhancer, see below) drives reporter gene expression in PS11 and more- posterior parasegments early in development. Later, this pattern becomes disrupted and, depending upon transgene insertion site, reporter gene expression becomes chaotic (Figure 3 A and B). Panels C and D show the case of the iab-5 initiator that contains also a PRE, the PRE element placed next to the iab-5 fragment preserves the reporter gene pattern of expression in the late embryo stages (Figure 3 C and D). This PRE can come from any domain, once again indicating that PREs do not, by themselves, sense postional information.
Figure 3. Reporter constructs identify initiator and maintenance elements. Drosophila embryos immunostained for the B-galactosidase protein. (A,C) Early embryos at germband extension, where the posterior parasegments have curved around towards the dorsal side; (B,D) later stage embryos. (A,B) Embryos in which lacZ expression is driven by an element from the iab-6 region. (A) In early embryos, lacZ expression is restricted to the posterior of the embryo, with its anterior border positioned at PS11. (B) At later stages of development, the repression of lacZ anterior to PS11 is lost, as the iab-6 element becomes active throughout the embryo. (C,D) Embryos in which lacZ expression is driven by a DNA fragment derived from iab-5. (C) In early embryos, the anterior border of lacZ expression is positioned at PS10. (D) Later in development, the anterior border of lacZ expression is maintained, indicating the presence of both an initiator and a maintenance element on this fragment. ant., anterior; post., posterior. (Figure adopted from Maeda and Karch 2006 - The ABC of the BX-C: the bithorax complex explained)
16 Domain boundary elements form a third layer of control. Each of the PS-specific enhancer clusters seems to be flanked by boundary elements. In situ, we have shown that loss of a domain boundary causes the fusion of PS-specific domains, resulting in mutant phenotypes, where the affected segments displays phenotypes characteristic of the more-posterior segment (for review see (Gurudatta and Corces 2009; Maeda and Karch 2011)). For example, the deletion of the Fab-7 boundary, which lies between the iab-6 (controlling Abd-B expression in PS11/A6) and iab-7 domains (controlling Abd-B expression in PS12/A7), results in a homeotic transformation of A6 towards A7 (Gyurkovics, Gausz et al. 1990). In transgenic assays, these elements have been shown to behave as insulators, blocking both positive and negative effects of cis-regulatory elements on reporter gene activity (Hagstrom, Muller et al. 1996; Zhou, Barolo et al. 1996). These results have led to the hypothesis that domain boundary elements are required to maintain the autonomous function of each enhancer cluster from the influences of neighboring cis-regulatory domains. However, the presence of boundary elements cannot explain the A-P restriction of the BX-C regulatory elements. As with the enhancers and silencers, when taken out of the BX-C, boundary elements do not seem to have an A-P restricted activity.
The last layer of control is thought to be the initiator elements (Simon, Mark et al. 1990;
Qian, Capovilla et al. 1991; Mullerl and Bienz 1992; Zhou, Ashe et al. 1999; Shimell, Peterson et al. 2000). These elements, when taken out of the BX-C can sense an A-P positional address.
Indeed sequence analysis of known insulator elements shows that they contain binding sites for numerous maternal, gap and pair-rule genes. As each domain seems to have at least one initiator element, it was hypothesized that they might be hubs, controlling domain activation/repression.
This idea is supported by recent work from our lab. In this work, our lab showed that deletion of the initiator from iab-6 seemed to completely inactivate the iab-6 domain and that exchanging
17 the iab-6 initiator with that of iab-5 caused a homeotic transformation in which A5 took the identity of A6 (Iampietro, Cléard et al. 2008). These experiments showed, not only, that the iab- 6 initiator was necessary for domain function (other experiments showed that it was not sufficient), but that placing an initiator for iab-5 in the domain could activate the A6 enhancers one segment too anterior (in A5, where iab-5 is normally active).
Figure 4. Organization of a Abd-B regulatory domain. The upper portion of the figure represents the Abdominal B cis-regulatory region. The name of each domain is written bellow the line representing the region. On the lower portion of the figure is the zoomed in iab-6 domain. Within are marked the separate components of the the domain Init- initiator, PRE – polycomb response elements. The cylinder and the bar are tissue specific enhancers while the red circles are the boundaries of the domain. Within the simple model the relationship between the initiator the enhancers and the PREs is also represented. When the domain is active the initiator represses the PREs and allows the enhancers to activate Abd-B expression. (Figure adopted from Maeda and Karch 2011 - Gene expression in time and space: additive vs hierarchical organization of cis-regulatory regions)
Taken together, we, and others, have proposed a domain model for BX-C gene regulation.
According to the domain model, initiator elements act early in development to set up the state of the domain, active/inactive. The maintenance elements conserve this state throughout development by compacting non-initiated domains into silenced, heterochromatic-like chromatin. In active domains, tissue-specific enhancers are free to interact with the promoter, leading to Abd-B expression in an A-P-restricted, tissue-specific manner. Meanwhile, the
18 boundary elements keep the each domain autonomous from the regulatory influences of the neighboring domains (Figure 4) (Mihaly, Barges et al. 2006).
Long-distance interaction in the BX-C
The domain model for BX-C gene regulation is now fairly well accepted. Yet, even if we accept the domain model as mostly true (an assumption that we now know has many exceptions), it remains only a schematic model for how the BX-C works. There remain a number of important details to be answered before a mechanistic understanding of the BX-C can be claimed.
One important question still remaining is how elements in such a large cis-regulatory region can find and control gene expression at a promoter located tens of kbs away? Furthermore, how can these elements find this promoter when competing with so many other elements capable of controlling gene expression at the same promoter?
The question of how enhancers in the BX-C find their target promoter is one that has troubled BX-C researchers for some time. Even before the stucture of the genes was known, studies of the phenomenon of transvection in the BX-C hinted that elements must exist in the BX- C that aid gene expression across chromosomal distances.
In the 1950s, Ed Lewis uncovered different mutant combinations whose phenotypes varied depending upon chromosomal pairing. In general, these phenotypes increased in severity when one of the mutations was carried on a rearranged chromosome. Based on these results, he hypothesized that when chromosomes are paired, there must be a type of complementation that
19 occurs between them that helps the fly to compensate for specific mutations. He called this phenomenon, “transvection” (Lewis 1954).
In Drosophila, homologous chromosomes are paired in somatic cells. This pairing sometimes enables an enhancer from one chromosome to direct the expression of its gene target on a homologous chromosome (Lee and Wu 2006). Although the phenomenon of transvection has been observed at a number of fly loci, the BX-C remains a “hotspot” for examples of transvection. Because of this, a number of labs have tried to identify elements important for mediating transvection in the BX-C.
Both (Hopmann, Duncan et al. 1995) and (Sipos, Mihály et al. 1998) describe transvection events in the Abd-B cis-regulatory region. In both cases, they showed that deletions removing sequences around Abd-B could be complemented by deletions or rearrangements affecting in the cis-regulatory region controlling Abd-B expression. For example, a deletion of iab-7 (controlling Abd-B in PS12/A7) placed in trans over an Abd-B promoter deletion results in a phenotype that looks more or less wild type. Classical genetics would suggest that these flies should display an iab-7 phenotype (and A7 to A6 homeotic transformation), as the iab-7 chromosome should not be able to make Abd-B in PS12/A7 and the Abd-B mutant chromosome should not be able to make Abd-B at all. Using these types of phenotypes and different deletion mutations, Hopmann et al. identified a region downstream of the Abd-B transcription unit that is required to mediate this transvection event. Further dissection of this transvection mediating region (TMR) by the group of Mike Levine has suggested that the Fab-8 boundary or the nearby iab-8 PRE (both contained within the TMR fragment) may be the important elements mediating transvection (Zhou, Ashe et al. 1999). Sipos et al., on the other hand, showed the importance of the 5’Abd-B promoter region, which they called the tethering region.
20 In terms of trans-acting elements mediating transvection, not much is known. Outside of the zeste (z) gene, which has been shown to be responsible for some transvection events outside of the BX-C, little has been discovered. It has been suggested that Polycomb group genes may play a role in transvection, as the finding of the iab-8 PRE in the TMR may suggest. Consistent with this idea is the fact that PREs display a transvection-like phenomenon called pairing- sensitive repression, where paired PREs silence more than non-paired PREs (Kassis, VanSickle et al. 1991). However, more recent microscopy studies, looking at long distance chromatin interaction between BX-C elements in cells, seem hint more to a role for the boundary elements in transvection than the PREs (Bantignies, Grimaud et al. 2003; Vazquez, Muller et al. 2006; Li, Muller et al. 2011).
Boundary Elements in Long-Distance Chromatin Interactions
As mentioned above, each domain in the BX-C is thought to be bordered by elements called domain boundaries. In transgenic constructs, these domain boundary elements have the ability to act as insulators, blocking enhancer-promoter interactions when placed between the two elements. In the BX-C, however, we know that insulator activity is probably not the activity that domain boundaries perform, as many are located between enhancers and promoters that are known to interact. Since transgenic insulator activity is an artificial activity that could be explained through numerous mechanisms, we, and others, thought of possible mechanisms of domain boundary activity that might lead to insulator function on transgenes. One idea that arrose from these thoughts was that domain boundaries might be involved in mediating long distance chromatin interactions to help divide the BX-C into individual domains and potentially
21 aid in targeting domains to a promoter. This hypothesis was supported by a number of findings including those from the transvection studies mentioned above.
Studies on the Su(Hw) insulator from the gypsy retrotransposon also suggested that elements with insulator activity may mediate long-distance chromatin interactions. According to this work, enhancer controlled gene expression was shown to be blocked by one copy of a Su(Hw) insulator when place between the enhancer and the promoter. However, when two copies of the insulator were placed between the enhancer and the reporter gene promoter, gene expression was restored. This blocking could not be explained by simply stating that two, nearby insulators cancel each other out, as a second reporter, placed in between the two Su(Hw) insulators was still blocked from responding to the enhancer (Cai and Shen 2001; Muravyova, Golovnin et al. 2001). This led to the idea that the two insulators might be interacting to create chromatin domains (or loops), where adjacent domains were kept independent of neighboring cis-regulatory effects, but that domains further away could bypass these effects (Cai and Shen 2001; Muravyova, Golovnin et al. 2001).
Work from our lab showed that BX-C boundaries also mediate long distance interactions.
For this, we placed Gal-4 binging sites near the Fab-7 boundary element and used a Gal4-DNA binding domain-Dam methyltransferase fusion to perform Dam-ID. This work showed that targeting of the Dam methyl-transferase to the Fab-7 boundary led to methylation, not only at the Fab-7 boundary, but also at the Abd-B promoter and, to a lesser extent, at the Fab-8 boundary (Cléard, Moshkin et al. 2006). This “trans-methylation” meant that these three elements must be in close proximity within the nucleus. The finding that the Abd-B promoter was in close proximity to the Fab-7 boundary was consistent with a previous finding by Sipos et al., that suggested that an area near the Abd-B promoter, called the Promoter Proximal Tethering
22 Element, was required for long distance, enhancer-promoter interactions in the BX-C (Sipos, Mihály et al. 1998). Interestingly, the Dam-ID methylation pattern changed depending on the A- P position in the fly. Methylation at the promoter only occurred in the anterior areas of the fly.
In other words, Fab-7 seemed to be in close proximity to the Abd-B promoter in tissues where Abd-B was silenced. Combining this finding with the phenotype of Fab-7 deletion mutants led to a model in which boundaries are attached to a tethering region near the promoter and be released sequentially as domains become active. This sequential releasing would allow distal enhancers to be sequestered near the promoter, and if released from the tethering region, able to interact with the Abd-B promoter to activate transcription (Figure 5).
Figure 5. Boundary tethering model. In this model, boundaries are represented by red circles, the inactive regulatory regions are covered by green circles (representing Polycomb silencing), and active regulatory regions are depicted by black lines. Based on the DamID results (Cleard et al., 2006), we believe that the boundaries tether the inactive cis- regulatory domains to a region near the Abd-B promoter. In doing so, boundaries form chromatin domains, keeping each domain autonomous and preventing the imbedded enhancers from interacting with the Abd-B promoter. Once a domain is activated, the boundary element would release from the tethering region and allow the formerly enclosed enhancers to interact with Abd-B promoter. For example, in A5, Mcp is released allowing the enhancers contained in iab-5 to activate Abd-B. Since the next downstream regulatory domain (iab-6) is still tethered by the next boundary (Fab- 6), only the appropriate regulatory iab-5 domain is able to regulate Abd-B in A5.
The elements are not drawn to scale.
(Figure adopted from Maeda and Karch 2007 – Making connections: boundaries and insulators in Drosophila)
23 Most of the cis-regulatory elements described above were identified and studied using P element transgenes, where fragments of interest were placed into a reporter and inserted randomly into the fly genome. Although this approach has proven to be quite useful, it has some drawbacks when studying complex regulatory regions, where chromatin structure impacts regulatory activities. Increasing the size of P-element transgenes to try to incorporate more complexity has proven problematic, as increasing transposon size rapidly decreases transformation efficiency. Thus, far, our lab has tried to bypass these problems by performing most of our experiments on mutations affecting the actual BX-C. Although we have developed tools to help streamline this process, outside of a few regions where we have good tools, this approach is slow and labor intensive. During my thesis, I have tried to use BAC-based transgenes to model the complexities of the BX-C, while keeping some of the experimental flexibility of transgenic reporter analysis.
Part I. Ectopic trans-activation of Abdominal B in the salivary glands by the Abd-B-Gal4 BAC
Transvection is a phenomenon in which the regulatory sequences of a gene on one chromosome modify the expression of the gene allele on its homologous chromosome. A number of reports from both Drosophila (Lewis 1954; Henikoff and Dreesen 1989; Geyer, Green et al.
1990; Dreesen, Henikoff et al. 1991; Hagstrom, Muller et al. 1997; Li, Muller et al. 2011) and mammalian model systems (Spilianakis and Flavell 2004; Spilianakis, Lalioti et al. 2005; Ling, Li et al. 2006) have been published describing this phenomenon. In flies, where chromosomes are paired throughout the cell cycle, most studies are based on findings of pairing-dependent allelic complementation or silencing. Often, this takes the form of an enhancer of a gene with a mutated promoter driving expression from a second mutant allele on the homologous chromosome that contains a wild-type promoter but lacks a functional enhancer. While both alleles, by themselves are null, together they are able to reconstitute gene function. In general, this phenomenon is pairing-dependent. Chromosomal rearrangements that prevent pairing generally disrupt these transvection interactions.
Although classical transvection is defined as a pairing-dependent phenomenon, numerous transvection-like phenomena have been reported that are independent of chromosome pairing.
Perhaps not surprisingly, a number of these examples come from studies of the BX-C, the place
25 where transvection was first discovered. Within the Abd-B region of the BX-C there are at least five examples of transvection-like phenomena that are pairing independent (Hendrickson and Sakonju 1995; Hopmann, Duncan et al. 1995; Sipos, Mihály et al. 1998; Muller, Hagstrom et al.
1999; Bantignies, Grimaud et al. 2003). In each of these cases, elements located at disparate locations in the genome (even on other chromosomes) seemed to interact to regulate gene expression. In most of these examples, domain boundary elements were shown to play a key role in the long-range regulatory interactions (Sipos, Mihály et al. 1998; Muller, Hagstrom et al.
1999; Bantignies, Grimaud et al. 2003; Gohl, Muller et al. 2008; Li, Muller et al. 2011). Given the findings mentioned in the introduction, that boundaries seem to promote physical, long-range interactions, this is perhaps not surprising (Muravyova, Golovnin et al. 2001; Cléard, Moshkin et al. 2006). Thus, although chromosome pairing is not required for these regulatory interactions, the localized physical interactions between the two gene copies is reminiscent of classical transvection.
During the course of my thesis work, I observed many phenomena that seem to be transvection-related. In this chapter, I will describe these findings and my attempts to further investigate these phenomena. Unfortunately, these results do not provide any firm conclusions.
They do, however, clearly demonstrate how little we know about the complex mechanisms of gene regulation.
26 Adult salivary gland shows ectopic Abd-B expression.
Using our Abd-B-Gal::UAS-GFP reporter to discover new locations of Abd-B expression in the adult fly, we surprisingly detected a strong GFP signal in the adult salivary glands (Figure 1A). Expression of Abd-B in this tissue was never reported, and examination of the origins of the salivary gland made this expression pattern seem to be an artifact. The adult salivary glands are thin, transparent, single-cell-layered tubes that extend from the head until the first abdominal segment of the fly. They develop from a set of imaginal cells located at the border between the larval salivary duct and the larval salivary glands, called the ring cells. The posterior-most ring cells gives rise to the thin tube structures that will extend to the first abdominal segment, forming the secretory part of the adult salivary glands. Meanwhile, the anterior part of the ring cells produces the salivary duct, which fuses with its partner at the neck level and join to the pharynx (Demerec 1950).
Figure 1. Adult salivary glands. A) Adult salivary glands expressing the Abd-B-Gal4 UAS-GFP reporter. B) Abd-B antibody staining in flies carrying the Abd-B-Gal4 UAS- GFP reporter. Clear signal localized in the nucleus of the salivary gland cells can be detected, C) Abd-B antibody staining in adult salivary glands of wild type flies. No signal in the nucleus of the salivary gland cell is detected.
The green staining visible is overexposure of the background so the tissue would be visible.
In the larval gland, Abd-B is known to inhibit salivary gland formation in PS14, while the teashirt gene inhibits salivary gland formation in PS3-13 (Andrew 1998). Thus, it seemed likely that the GFP expression in the adult salivary gland was simply a result of position effect on the BAC reporter. Antibody staining confirmed the notion that no Abd-B protein is present in wild
27 type adult salivary glands. However, when performing this staining on Abd-B-Gal4::UAS-GFP reporter flies, we found distinct Abd-B staining in the adult salivary glands (Figure 1B).
Examining younger Abd-B-Gal4::UAS-GFP individuals showed that both GFP reporter expression and Abd-B expression could be detected in the ring and salivary duct cells of the larval salivary glands, as well as, in the precursors of the salivary gland in the embryo (Figure 2).
Figure 2. Ectopic activation of Abd-B expression in embryonic and larval salivary glands. Top three panels are embryos (A, B, C), three bottom panels are anterior part of the third instar larva salivary glands (D, E, F). A and D are Abd-B staining of wild type embryo and larval salivary gland. No signal in the anterior part of the embryo is detectable. There is also no detectable signal in the salivary glands, the red is the overexposure of the tissue in order to become visible. B and C are embryonic and larval salivary gland expression of the Abd-B-Gal4 UAS-GFP reporter. In the embryo ectopic expression can be detected in the anterior part of the CNS as well as in the salivary gland precursors (white arrow). In the larval salivary glands signal can be seen in the ring cells and the salivary gland duct cells (white arrows – ring cells, yellow arrow – duct cells). C and F are Abd-B staining of embryo and larval salivary glands in the background of the Abd-B-Gal4 UAS-GFP reporter. As with the signal coming from the reporter Abd-B staining can be detected in the precursor of the salivary glands in the embryo (white arrow) as well in the ring cells (white arrow) and larval salivary gland duct cells (yellow arrow) in the larval salivary glands.
This early expression was likewise dependent upon the presence of the Abd-B- Gal4::UAS-GFP reporter. As we knew from previous experiments that the Abd-B-Gal4 BAC does not express any Abd-B protein by itself, we concluded that the ectopic expression of Abd-B in the adult salivary glands must come from the endogenous locus in the BX-C. Since this Abd-B staining was also dependent upon the Abd-B-Gal4 BAC, we believe that the Abd-B expression in
28 the salivary glands is caused by transvection between the Abd-B-Gal4 BAC on the second chromosome and the endogenous Abd-B locus on the third (Figure 3).
Figure 3. Model of transvection between Abd-B and 51C locus. Model of what might be happening between the endogenous Abd-B locus on the third chromosome (green lines) and the Abd-B-Gal4 BAC integrated in the second chromosome (yellow lines, green part represents the BAC integrated at position 51C). The red stars represent possible enhancer that when brought in close proximity to the endogenous Abd-B promoter could activate Abd-B expression ectopically in the salivary glands.
In order to verify a physical interaction between the Abd-B-Gal4 BAC and the endogenous Abd-B locus, we performed two-color DNA FISH analysis. 15 kb probes were designed to the 51C region (the site of integrations of the Abd-B BAC) and a region in the Abd-B locus not included on the Abd-B-Gal4 BAC. We used these probes to stain adult, larval (ring cells) and embryonic salivary glands. While embryonic salivary glands could not be imaged, we were able to detect signals in the two remaining tissues. Unfortunately, in these cells we always detected two distinct signals in the nuclei. No dramatic increase in colocalization was ever detected between lines carrying the BAC and lines without the BAC. (Figure 4).
This lack of positive data, while disappointing, does not mean that no transvection exists.
Indeed, the genetic data strongly suggests that an interaction must exist between the Abd-B-Gal4 BAC transgene on the second chromosome and the endogenous Abd-B region on the third
29 chromosome. We simply could not visualize an interaction. There are a number of possible explanations for this problem.
Figure 4. Double labeled DNA fluorescent in situ hybridization. Abd-B locus in green, 51C locus in red. Images represent screen shots of 3D rendered confocal stacks by the software Imaris. A) is a control, a larval salivary gland nuclei where Abd-B expression is not detected with the reporter BAC in the background, no overlap between the two signal is expected and we didn’t detect any overlap. B) Ring cells of larval salivary glands, no overlap between the signals was detected. C) larval salivary gland duct cells, no overlap of the two signals detected. D) Adult salivary gland, no overlap of the two signals was detected. (videos are available on demand).
First and foremost among the possibilities stems from the fact that we do not know when the interaction will take place. We know from studies mentioned in the introduction that the transcriptional availability of the BX-C is generally set early in development and then maintained throughout later stages of development. Because of this maintenance of transcriptionally permissive chromatin, the transvection interaction might have occurred earlier in development in just a few cells to set up an open chromatin state that is then maintained until later stages. This change of chromatin state would lead to expression of Abd-B at earlier stages, as additional positive factors may be required to activate transcription. These factors might bind to elements within the BX-C that, under normal circumstances, would be packed into Polycomb silenced chromatin. The transient nature of such interactions has been suggested by other studies
30 examining chromatin interactions between BX-C elements. In these studies, the authors generally found that the two interacting loci did not show colocalization in the vast majority of cells (Bantignies, Grimaud et al. 2003).
Salivary gland morphological phenotype
While establishing stocks for an unrelated set of experiments, we noticed that flies carrying the Abd-B-Gal4::UAS-GFP reporter and different deletions in the Abd-B cis-regulatory region had markedly reduced adult salivary glands (Figure 5). This phenomenon was observed only in the newly established lines and was not seen in the stocks carrying the Abd-B-Gal::UAS- GFP reporter alone. Interestingly, after several generations, we noticed that this salivary gland morphological phenotype gradually weakened.
Figure 5. Collection of salivary gland phenotypes. The mutation in question is homozygous with the Abd-B-Gal4 UAS-GFP reporter in the background. The names of the mutants are written on the picture. WT is a wild type salivary gland. As can be seen WT is a long straight tube like organ with a squiggly end. In comparison most of the mutant salivary glands presented here are smaller with undetermined shape.
Based on these initial observations, we performed preliminary experiments to determine if different mutations in the Abd-B gene could disturb the morphology of the adult salivary gland.
These experiments made particular sense, given that we already observed ectopic Abd-B expression in the salivary glands and wondered if this ectopic expression led to the
31 morphological phenotypes. The experiment was designed as a simple F1 screen to test if heterozygosity for various mutations could modify the morphology of the adult salivary gland in the presence of a copy of the Abd-B-Gal4::UAS-GFP BAC. A numerical scale was established for scoring the phenotype based on the length of the gland and its morphology. This scale is presented in Table 1. From these crosses, it soon became evident that the BAC itself was also capable of modifying the morphology of the adult salivary glands, if outcrossed. This finding was consistent with the previous findings that the phenotype is suppressed over generations. As this suppression occurs over only a couple of generations, we hypothesize that this suppression is probably occurring at the level of chromatin structure and not an accumulation of suppressor mutations. The fact that flies carrying the BAC alone also show morphological defects in the salivary gland is consistent with the hypothesis that ectopic Abd-B may be the cause of the salivary gland phenotype.
Table 1. Scoring of adult salivary glands phenotype based on their length. The table explains the scoring system for the salivary glands based on their length, location and brief explanation of the phenotype. Column one names the three body parts of the fly and subdivides them. Depending on the mutant the salivary gland length can reach all the way to the abdomen or be just the head. Column two shows the numbers 1 to 6 which are used to score the phenotype, 1 - severe reduction in length, 6- normal looking salivary glands. Column three gives a short description of the phenotype for each number.
The results from crossing in Abd-B mutations into the BAC background yielded somewhat puzzling results. For these experiments, we used three Abd-B mutations: Df P9, which removes the entire BX-C, Df D18, which removes the entire Abd-Bm coding region plus a little of the neighboring sequences, and Abd-BD16 , which is an Abd-B point mutation that is a protein null. While outcrossed Abd-B-Gal4::UAS-GFP flies show mild defects in salivary gland
32 formation (scores between 4 and 5), crossing in either Df P9 or Df D18 mutations was able to suppress this mild effect (Figure 6). This effect was particularly noticeable if the Abd-B- Gal4::UAS-GFP came from male (see below). Thus, these results are consistent with the idea that reducing the level of Abd-B in the BX-C or changing the pairing interactions (as these deletions remove substantial portions of the Abd-B region), is able to decrease the salivary gland phenotype. The confusing result comes from the Abd-BD16 allele. When Abd-BD16 is crossed into the BAC background, an enhancement of the phenotype is seen. Like the other Abd-B mutations, the Abd-BD16 allele should show reduced Abd-B levels. The difference between Abd-BD16 and the other Abd-B alleles tested lies in the possibility that the other Abd-B alleles remove interaction motifs important for transvection. Relative to a wild type copy, however, D16 should differ only by the loss of one copy of Abd-B. Thus, if we believe that ectopic Abd-B expression causes the salivary gland phenotype, we must conclude that Abd-BD16 has some peculiar characteristics (like a mutation in a regulatory element binding site) that we still do not understand.
Continuing this mutational analysis, we also examined the effects that other elements or proteins known to be involved in transvection could have on the salivary gland phenotype. For these experiments, we crossed in mutations removing BX-C domain boundaries (Mcp1; Fab71), and mutations in known transvection-mediating molecules (zeste, Polycomb proteins (Pc, pcl and Asx) and boundary proteins (CTCF)). As before, the progeny of the cross were dissected and their salivary glands scored based on their length (Figure 6).
Interestingly, as implied above, the phenotypes change depending upon the parental origin of the BAC, with females often showing a weaker phenotype (more-wild-type) than males (Figure 7). One possible explanation for this is based on the different epigenetic inheritance from male and from female. As mentioned earlier, the quick suppression of the salivary gland
33 phenotype suggests that epigenetic/chromatin regulation may be involved in the manifestation of the salivary gland phenotype. In males, it is known that during spermatogenesis all the histones get replaced by protamines, thereby, removing all histone marks that regulate gene expression ((Daxinger and Whitelaw 2010) – review on transgenerational epigenetic inheritance). In
Figure 6. Chart showing salivary gland phenotype variation in different mutant backgrounds. The flies scored for their salivary gland phenotype were produced by crossing the stock Abd-B-Gal4 UAS-GFP reporter flies (striped bar marked with AGFP/Cy) with ten different mutants (Pcl,Asx; Pc3; Mcp; Zop6; Abd-BD16; CTCF0463; Fab-71; CTCFp366; Abd-BD18 and Abd-BP9) and Oregon R as control (Green bars marked with AGFP/+). The left side of the chart, left of the stripped bar, are results where the origin of the BAC reporter is from the male while the right side are results where the origin of the BAC reporter is from the female. The scored mutations were heterozygous. About 30 flies (15 male and 15 females) were scored for their phenotype based on the salivary gland length per mutation per origin of the BAC reporter. Score of one was given for a severe phenotype while score of six is for normal salivary gland phenotype (see table 1 for more detailed explanation of the scoring of salivary glands).
females, however, histone marks have the possibility to be transmitted to the next generation.
Potentially, this is what we observe in our experiments. When the Abd-B-Gal4 BAC comes from