Molecular genetics of the bithorax complex in Drosophila

François Karch

Full Professor

  • T: +41 22 379 63 31
  • office 3004a (Sciences III)

Outline

Although it is now possible to engineer living organisms to express almost any gene in a controlled fashion, the mechanisms governing this control still remain elusive. In eukaryotes, genes are large, with regulatory elements often tens, or even hundreds of kilobases away from their target promoter. Yet, even given this distance, these distal elements can still faithfully find and control their target promoter in a precise fashion. The experiments performed in our laboratory aim at understanding on how these distal regulatory elements can control gene expression. As our model system, we study the regulation of the homeotic genes (Hox gene) of the Drosophila bithorax complex (BX-C) Regulatory elements, spanning a 300 kilobases-long region of the BX-C DNA are required for proper expression of three hox genes, Ubx, abd-A and Abd-B. Homeotic genes are strikingly conserved between invertebrates and vertebrates. The genes are found in clusters or 'complexes' that are arranged on the chromosome in the order of their function along the anteroposterior body axis. This striking correspondence between genomic organization and antero-posterior axis gives us a unique opportunity to study gene regulation in its chromosomal context. The amazing evolutionary conservation in the genomic organization of hox complexes is further highlighted by the recent finding of 2 conserved micro-RNAs at similar location within the complexes of arthropods and vertebrates.

Synopsis of the BX-C

Synopsis of the BX-C. The Drosophila BX-C is one of two homeotic gene clusters in the fly and is responsible for determining the segmental identity of the posterior thoracic segment and each of the fly abdominal segments (Lewis, 1978; Sanchez-Herrero et al., 1985). It does this by using a >300 kb cis-regulatory region (shown by the horizontal line) to control the parasegement-specific expression of the three BX-C homeotic genes: Ubx, abd-A and Abd-B (for review, see (Maeda and Karch, 2006)). From the early genetic analysis of the BX-C to the most recent data involving enhancer trap lines, it was shown that the cis-regulatory sequences of the BX-C can be divided into nine parasegment-specific chromosomal domains (abx/bx, bxd/pbx, and iab-2 through iab-8), where each domain controls the activation of one of the three BX-C homeotic genes in a pattern appropriate for that parasegment (Bender et al., 1983; Bender and Hudson, 2000; Celniker et al., 1990; Karch et al., 1985; Peifer et al., 1987; Sanchez-Herrero, 1991). The parasegment-specific regulatory domains and the parasegments in which they act on the body axis of the fly are depicted in colors. While the orange/red regulatory domains direct Ubx expression, the bluish regulatory domains control abd-A, and the greenish domains control Abd-B. Remarkably enough the parasegment-specific cis regulatory domains are aligned on the chromosome in the same order as the parasegments they specify along the antero-posterior avis of the fly.

Ongoing projects

In situ dissection of regulatory domains and boundaries

For much of the last few years, we have been developing tools to precisely dissect this complex locus in situ (see ref (Bischof et al., 2007)). These tools now provide us with a unique opportunity to delve into the intricacies governing the regulation of this complex locus. Our research is presently centered on 3 main questions. The 1st project focuses on how different regulatory elements, such as enhancers, repressors, silencers and insulators interact with one another to generate the final regulatory output on their target gene. Using the advanced genetic methods developed in our lab, we have identified and characterized particular regulatory elements, called initiators. We have found that in vivo, these enhancer-like elements function as switches to coordinate the activity of the neighboring regulatory elements. As such, initiators do not provide cell-type specificity or patterning information, but instead limit the function of other regulatory elements. To our knowledge this is the 1st example of a hierarchical organization between regulatory enhancers (Iampietro et al., submitted). A second type of element that we are currently investigating, is called a chromatin boundary (or insulators). Within the BX-C, boundary elements seem to flank each of the cis-regulatory domains, allowing the domains to function autonomously across the A-P axis (for review see ref (Maeda and Karch, 2007). An important part of our in situ dissection focuses on one particular boundary that we identified, named Fab-7. Using our genetic tools, we can now efficiently and rapidly target specific point mutations to the Fab-7 boundary, and are thus, presently addressing the role of specific binding sites in boundary activity.

The Fab-7boundary region

The Fab-7 boundary region is characterized by a set of 3 major nuclease hypersensitive sites (HS1, HS2 and HS3. While the major sites, HS1 and HS2 (along with a minor site marked as a star) correspond to the boundary, a 3rd prominent site, HS3 corresponds to the iab-7PRE (Aoki et al., 2008; Karch et al., 1994). Using gene conversion, we have replaced the whole region by an attp sites, that allows us to re-integrate modified boundary via the φC31 integration system (Bischof et al., 2007). Using this platform, we have quickly established that the region required for the boundary activity spans the HS1 region. In order to gain further information about the sequence requirements for the Fab-7 boundary function we replaced HS1 by the corresponding region from D. yacuba and D. erecta (belonging to the melanogaster subgroup) as well as by the Fab-7 sequences from D.pseudoobscura a further distant specie, from the obscura subgroup. As all 3 elements were able to substitute for the melanogaster boundary, sequence comparisons led us to consider that the presence of multiple binding sequences for the GAGA factor may be one element required for boundary function. We thus mutated 5 GAGAG motifs spanning the HS. The abdominal cuticles shown below indicate that the mutation is associated with a weak and low penetrance homeotic phenotype in which PS11/A6 is slightly transformed into PS12/A7. While this result clearly establishes a role for the GAGA factor into boundary function, it also indicates the presence of additional elements involved into Fab-7 activity.

Non-coding RNAs of the BX-C

As with many loci across eukaryotic genomes, the BX-C is the site of intense intergenic transcription (Bae et al., 2002; Drewell et al., 2002; Lipshitz et al., 1987; Sanchez-Herrero and Akam, 1989). In the 2nd main topic of research, we are currently investigating the biological role of these ncRNAs in cis or trans regulation of the BX-C homeotic genes. Our working hypothesis is that initiator elements coordinate the activity of the nearby regulatory elements within a domain by activating intergenic transcription. We are presently testing this hypothesis in the BX-C by inserting transcriptional terminators to block intergenic transcription. Another line of research in the lab revolving around ncRNAs, focuses a peculiar ncRNA from the BX-C that is over 120 kilobases long and is known to serve as the template for a Ubx-specific microRNA. Remarkably, this microRNA is conserved at a similar position in the hox clusters of other arthropods and vertebrates (Bender, 2008; Ronshaugen et al., 2005; Stark et al., 2008; Tyler et al., 2008). Given the incredible length of this ncRNA, we have been investigating other roles that this ncRNA may playing in BX-C regulation.

Double in situ hybridization

Double in situ hybridization with a probe spanning the iab-6 initiator (blue) and with a probe detecting the pair-rule gene even-skipped (red). The eve pattern is detected in red in odd numbered parasegemnts (even relates to segments boundaries that are shifted by one parasegment). While the staining is weak, careful examination reveals the presence of blue signal in PS12 and PS11.

Studying Abd-B regulation with BACs

The readouts of Abd-B activity in assigning segment identities are visible in the cuticle of the adult abdominal segments and in the embryonic expression expression pattern. Surprisingly little is known about Abd-B expression during the 3 larval stages and metamorphosis. The problem stems from the fact that larvae and pupae are difficult to analyze with antibody or in situ hybridization based methods. In the 3rd line of experiments, we have been using BACs spanning the Abd-B gene and its large 3' cis-regulatory region to study Abd-B regulation and to identify new tissues in which Abd-B is expressed. By crossing BAC transgenic inserts that faithfully express the yeast Gal4 activator in the pattern of Abd-B to UAS-GFP reporters, we can now monitor Abd-B expression in vivo at all developmental stages and in all tissues. We are presently focusing on Abd-B expression and activity in the male accessory glands. The male accessory gland is the synthesis site of numerous Accessory-Gland-Specific-Proteins (Acps; more than 100 are identified) that elicit a number of post-mating behaviors in females after copulation. These behaviors include: increasing egg production, ovulation, and oviposition; regulating sperm utilization and storage; and decreasing receptivity to remating (for review, see ref. (Wolfner, 2009). Thus far, we have identified the cis-acting elements responsible for Abd-B expression in the accessory gland and recovered mutants specifically deleting these enhancers. Phenotypic analysis of these mutants is currently underway.

Abd-B expression in the accessory glands

Abd-B expression in the accessory glands. The picture on the left shows strong GFP expression (under Abd-B-BAC-Gal4 regulation) that leaks out from the nucleus and label the cytoplasm, where one observes the numerous vacuoles that are characteristic for the secondary cells. On the right, staining of accessory glands with antibodies directed against Abd-B. Note the binucleated secondary cells.

References

  • Aoki, T., Schweinsberg, S., Manasson, J. and Schedl, P. (2008). A stage-specific factor confers Fab-7 boundary activity during early embryogenesis in Drosophila. Mol Cell Biol 28, 1047-60.
  • Bae, E., Calhoun, V. C., Levine, M., Lewis, E. B. and Drewell, R. A. (2002). Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila bithorax complex. Proc Natl Acad Sci U S A 99, 16847-52.
  • Bender, W. (2008). MicroRNAs in the Drosophila bithorax complex. Genes Dev 22, 14-9.
  • Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S. (1983). Molecular genetics of the bithorax complex in Drosophila melanogaster. Science 221, 23-9.
  • Bender, W. and Hudson, A. (2000). P element homing to the Drosophila bithorax complex. Development 127, 3981-3992.
  • Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104, 3312-7.
  • Celniker, S. E., Sharma, S., Keelan, D. J. and Lewis, E. B. (1990). The molecular genetics of the bithorax complex of Drosophila: cis- regulation in the Abdominal-B domain. Embo J 9, 4277-86.
  • Drewell, R. A., Bae, E., Burr, J. and Lewis, E. B. (2002). Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex. Proc Natl Acad Sci U S A 99, 16853-8.
  • Iampietro, C. Gummalla, M., Mutéro, A., Karch, F. and Maeda, R. Initiators elemements function to determine the activity states of BX-C enhancers. Submitted to PLoS Genetics
  • Karch, F., Galloni, M., Sipos, L., Gausz, J., Gyurkovics, H. and Schedl, P. (1994). Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster. Nucleic Acids Res 22, 3138-46 Issn: 0305-1048.
  • Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I., Celniker, S., Crosby, M. and Lewis, E. B. (1985). The abdominal region of the bithorax complex. Cell 43, 81-96.
  • Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276, 565-70.
  • Lipshitz, H. D., Peattie, D. A. and Hogness, D. S. (1987). Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev 1, 307-22.
  • Maeda, R. K. and Karch, F. (2006). The ABC of the BX-C: the bithorax complex explained. Development 133, 1413-1422.
  • Maeda, R. K. and Karch, F. (2007). Making connections: boundaries and insulators in Drosophila. Curr Opin Genet Dev 17, 394-9.
  • Peifer, M., Karch, F. and Bender, W. (1987). The bithorax complex: control of segmental identity. Genes Dev 1, 891-98.
  • Ronshaugen, M., Biemar, F., Piel, J., Levine, M. and Lai, E. C. (2005). The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings. Genes Dev 19, 2947-52.
  • Sanchez-Herrero, E. (1991). Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos. Development 111, 437-49.
  • Sanchez-Herrero, E. and Akam, M. (1989). Spatially ordered transcription of regulatory DNA in the bithorax complex of Drosophila. Development 107, 321-9.
  • Sanchez-Herrero, E., Vernos, I., Marco, R. and Morata, G. (1985). Genetic organization of Drosophila bithorax complex. Nature 313, 108-13.
  • Stark, A., Bushati, N., Jan, C. H., Kheradpour, P., Hodges, E., Brennecke, J., Bartel, D. P., Cohen, S. M. and Kellis, M. (2008). A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands. Genes Dev 22, 8-13.
  • Tyler, D. M., Okamura, K., Chung, W. J., Hagen, J. W., Berezikov, E., Hannon, G. J. and Lai, E. C. (2008). Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci. Genes Dev 22, 26-36.
  • Wolfner, M. F. (2009). Battle and ballet: molecular interactions between the sexes in Drosophila. J Hered 100, 399-410.

  • A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands. J Vis Exp 2019 Sep;(151):. 10.3791/60218.

    abstract

    To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

    view more details on Pubmed

  • Vps11 and Vps18 of Vps-C membrane traffic complexes are E3 ubiquitin ligases and fine-tune signalling. Nat Commun 2019 04;10(1):1833. 10.1038/s41467-019-09800-y. 10.1038/s41467-019-09800-y. PMC6478910.

    abstract

    In response to extracellular signals, many signalling proteins associated with the plasma membrane are sorted into endosomes. This involves endosomal fusion, which depends on the complexes HOPS and CORVET. Whether and how their subunits themselves modulate signal transduction is unknown. We show that Vps11 and Vps18 (Vps11/18), two common subunits of the HOPS/CORVET complexes, are E3 ubiquitin ligases. Upon overexpression of Vps11/Vps18, we find perturbations of ubiquitination in signal transduction pathways. We specifically demonstrate that Vps11/18 regulate several signalling factors and pathways, including Wnt, estrogen receptor α (ERα), and NFκB. For ERα, we demonstrate that the Vps11/18-mediated ubiquitination of the scaffold protein PELP1 impairs the activation of ERα by c-Src. Thus, proteins involved in membrane traffic, in addition to performing their well-described role in endosomal fusion, fine-tune signalling in several different ways, including through ubiquitination.

    view more details on Pubmed

  • Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity. Traffic 2018 Nov;():. 10.1111/tra.12622.

    abstract

    The male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long-term post-mating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open-access imaging resource to display our complete findings regarding Rab localization in the accessory gland. This article is protected by copyright. All rights reserved.

    view more details on Pubmed

  • Using a phiC31 "Disintegrase" to make new attP sites in the Drosophila genome at locations showing chromosomal position effects. PLoS ONE 2018 ;13(10):e0205538. 10.1371/journal.pone.0205538. PONE-D-18-24553.

    abstract

    An engineered phiC31 "Disintegrase" able to make an attP site in Drosophila out of an attR-attL pair is described. This was used to generate attP sites at genomic locations where a mini-white (mini-w) transgene was subject to chromosomal position effects (CPE). The first step was random genomic integration of a P-element-based transposon with an insulated mini-w transgene. We then removed the upstream insulator using FLP recombinase to detect CPE. Next mini-w and the downstream insulator were "dis-integrated" leaving behind an attP site. The location is marked by a yellow+ transgene that is flanked by loxP sites, so it can also be removed. Using this system, we generated 10 new attP landing platforms. Three of these showing strong activating CPE were selected for further analysis. We show that the attP sites are functional by integrating in plasmids with attB sites. The CPE is recapitulated and can be blocked by insulators. We show that a dimerized 215 bp fragment of the 500 bp BEAF-dependent scs' insulator containing a high affinity BEAF binding site blocks the CPE, while a monomer of the sequence is less effective. This indicates that two BEAF binding sites make a stronger insulator than a single site. This system could be useful for generating attP sites at prescreened sites for other purposes, such as studying CPE in embryos or other tissues or for use with "trapped" enhancers of interest.

    view more details on Pubmed

  • The BEN Domain Protein Insensitive Binds to the Fab-7 Chromatin Boundary To Establish Proper Segmental Identity in Drosophila Genetics 2018 Aug;():. genetics.118.301259. 10.1534/genetics.118.301259.

    abstract

    Boundaries (insulators) in the bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The boundary separates the and regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks crosstalk between and and facilitates boundary bypass. Here, we show that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ~100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (~420 kD and ~265-290 kD, respectively) in nuclear extracts. Using a sensitized genetic background we show that the Insv protein is required for boundary function, and that PS11 identity is not properly established in mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.

    view more details on Pubmed

  • The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility. PLoS Genet. 2018 Jul;14(7):e1007519. 10.1371/journal.pgen.1007519. PGENETICS-D-17-01962.

    abstract

    Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. Here, we show that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues.

    view more details on Pubmed

  • Different Evolutionary Strategies To Conserve Chromatin Boundary Function in the Bithorax Complex. Genetics 2017 Feb;205(2):589-603. genetics.116.195586. 10.1534/genetics.116.195586.

    abstract

    Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7, which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a "gene" replacement strategy, we show that Fab-7 boundaries from two closely related species, D. erecta and D. yakuba, and a more distant species, D. pseudoobscura, are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

    view more details on Pubmed

  • Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex. PLoS Genet. 2016 Jul;12(7):e1006188. 10.1371/journal.pgen.1006188. PGENETICS-D-15-02937. PMC4948906.

    abstract

    Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

    view more details on Pubmed

  • Stuxnet Recruits the Proteasome to Take Down Polycomb. Dev. Cell 2016 Jun;37(6):485-6. S1534-5807(16)30375-6. 10.1016/j.devcel.2016.06.006.

    abstract

    In this issue of Developmental Cell, Du et al. (2016) describe a gene named stuxnet that regulates Polycomb protein stability, thereby influencing the activity of the Polycomb-group repressive chromatin complexes.

    view more details on Pubmed

  • The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster. Genetics 2016 Mar;202(3):1029-41. genetics.115.181644. 10.1534/genetics.115.181644. PMC4788108.

    abstract

    Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP) and are maintained by SP's binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6(cocu) males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6(cocu) males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR.

    view more details on Pubmed

  • MoD Special issue on "Upstream and Downstream of Hox Genes. Mech. Dev. 2015 Nov;138 Pt 2():63. S0925-4773(15)00079-9. 10.1016/j.mod.2015.11.003.

    abstract

    view more details on Pubmed

  • In vivo studies of the Drosophila insulator factor CTCF reach a Catch 22. BMC Biol. 2015 ;13():71. 10.1186/s12915-015-0182-9. 10.1186/s12915-015-0182-9. PMC4557222.

    abstract

    Mutations in the proteins that bind insulator DNA elements that define the boundaries of chromatin domains can give morphogenetic readouts in Drosophila, as recently reported in BMC Biology by Bonchuk et al. in the Georgiev laboratory. But disentangling the effects on the phenotype may not be simple.See research article: http://www.biomedcentral.com/1741-7007/13/63.

    view more details on Pubmed

  • Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex. Mol. Cell. Biol. 2015 Nov;35(21):3739-52. MCB.00456-15. 10.1128/MCB.00456-15. PMC4589599.

    abstract

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

    view more details on Pubmed

  • Following the intracellular localization of the iab-8ncRNA of the bithorax complex using the MS2-MCP-GFP system. Mech. Dev. 2015 Nov;138 Pt 2():133-40. S0925-4773(15)30021-6. 10.1016/j.mod.2015.08.004.

    abstract

    Homeotic genes are aligned on the chromosome in the order of the segments that they specify along the antero-posterior axis of the fly. In general the genes affecting the more posterior segments repress the more anterior genes, a phenomenon known as "posterior dominance". There is however a noticeable exception to this rule in the central nervous system of Drosophila melanogaster where the posterior Abd-B gene does not repress the immediately more anterior abd-A gene. Instead, abd-A repression is accomplished by a 92 kb-long ncRNA (the iab-8ncRNA) that is transcribed from the large inter-genic region between abd-A and Abd-B. This iab-8ncRNA encodes a microRNA to repress abd-A and also a second redundant repression mechanism acting in cis and thought to be transcriptional interference with the abd-A promoter. Using in situ hybridization, a previous work suggested that the iab8ncRNA transcript forms discrete foci restricted to the nuclear periphery and that this localization may be important for its function. In order to better characterize the intra-cellular localization of the iab-8ncRNA we used the MS2-MCP system, which allows fluorescent labeling of RNA in cells and relies on the interaction between GFP-tagged MS2 coat protein (MCP-GFP) and MS2 RNA stem loops. Our results indicate that the large foci seen in previous studies correspond to the site of iab8ncRNA transcription and that the foci seen may simply be an indication of the level of transcription at the locus. We find no evidence to suggest that this localization is important for its function on abd-A repression. We discuss the idea that the iab-8ncRNA may be a relic of a more general ancient mechanism of posterior dominance during the emergence of the hox clusters that was mediated by transcriptional interference.

    view more details on Pubmed

  • The open for business model of the bithorax complex in Drosophila. Chromosoma 2015 Sep;124(3):293-307. 10.1007/s00412-015-0522-0. PMC4548009.

    abstract

    After nearly 30 years of effort, Ed Lewis published his 1978 landmark paper in which he described the analysis of a series of mutations that affect the identity of the segments that form along the anterior-posterior (AP) axis of the fly (Lewis 1978). The mutations behaved in a non-canonical fashion in complementation tests, forming what Ed Lewis called a "pseudo-allelic" series. Because of this, he never thought that the mutations represented segment-specific genes. As all of these mutations were grouped to a particular area of the Drosophila third chromosome, the locus became known of as the bithorax complex (BX-C). One of the key findings of Lewis' article was that it revealed for the first time, to a wide scientific audience, that there was a remarkable correlation between the order of the segment-specific mutations along the chromosome and the order of the segments they affected along the AP axis. In Ed Lewis' eyes, the mutants he discovered affected "segment-specific functions" that were sequentially activated along the chromosome as one moves from anterior to posterior along the body axis (the colinearity concept now cited in elementary biology textbooks). The nature of the "segment-specific functions" started to become clear when the BX-C was cloned through the pioneering chromosomal walk initiated in the mid 1980s by the Hogness and Bender laboratories (Bender et al. 1983a; Karch et al. 1985). Through this molecular biology effort, and along with genetic characterizations performed by Gines Morata's group in Madrid (Sanchez-Herrero et al. 1985) and Robert Whittle's in Sussex (Tiong et al. 1985), it soon became clear that the whole BX-C encoded only three protein-coding genes (Ubx, abd-A, and Abd-B). Later, immunostaining against the Ubx protein hinted that the segment-specific functions could, in fact, be cis-regulatory elements regulating the expression of the three protein-coding genes. In 1987, Peifer, Karch, and Bender proposed a comprehensive model of the functioning of the BX-C, in which the "segment-specific functions" appear as segment-specific enhancers regulating, Ubx, abd-A, or Abd-B (Peifer et al. 1987). Key to their model was that the segmental address of these enhancers was not an inherent ability of the enhancers themselves, but was determined by the chromosomal location in which they lay. In their view, the sequential activation of the segment-specific functions resulted from the sequential opening of chromatin domains along the chromosome as one moves from anterior to posterior. This model soon became known of as the open for business model. While the open for business model is quite easy to visualize at a conceptual level, molecular evidence to validate this model has been missing for almost 30 years. The recent publication describing the outstanding, joint effort from the Bender and Kingston laboratories now provides the missing proof to support this model (Bowman et al. 2014). The purpose of this article is to review the open for business model and take the reader through the genetic arguments that led to its elaboration.

    view more details on Pubmed

  • In vivo analysis of a fluorescent SUMO fusion in transgenic Drosophila. Fly (Austin) 2014 ;8(2):108-12. 10.4161/fly.28312. PMC4197013.

    abstract

    Sumoylation, the covalent attachment of SUMO, a 90 amino acid peptide related to ubiquitin, is a major modulator of protein functions. Fluorescent SUMO protein fusions have been used in cell cultures to visualize SUMO in vivo but not in multicellular organisms. We generated a transgenic line of Drosophila expressing an mCherry-SUMO fusion. We analyzed its pattern in vivo in salivary gland nuclei expressing Venus-HP1 to recognize the different chromatin components (Chromocenter, chromosome IV). We compared it to SUMO immunostaining on squashed polytene chromosomes and observed similar patterns. In addition to the previously reported SUMO localizations (chromosome arms and chromocenter), we identify 2 intense binding sites: the fourth chromosome telomere and the DAPI-bright band in the region 81F.

    view more details on Pubmed

  • DamID as an approach to studying long-distance chromatin interactions. Methods Mol. Biol. 2014 ;1196():279-89. 10.1007/978-1-4939-1242-1_17.

    abstract

    How transcription is controlled by distally located cis-regulatory elements is an active area of research in biology. As such, there have been many techniques developed to probe these long-distance chromatin interactions. Here, we focus on one such method, called DamID (van Steensel and Henikoff, Nat Biotechnol 18(4):424-428, 2000). While other methods like 3C (Dekker et al., Science 295(5558):1306-1311, 2002), 4C (Simonis et al., Nat Genet 38(11):1348-1354, 2006; Zhao et al., Nat Genet 38(11):1341-1347, 2006), and 5C (Dostie et al., Genome Res 16(10):1299-1309, 2006) are undoubtedly powerful, the DamID method can offer some advantages over these methods if the genetic locus can be easily modified. The lack of tissue fixation, the low amounts of starting material required to perform the experiment, and the relatively modest hardware requirements make DamID experiments an interesting alternative to consider when examining long-distance chromatin interactions.

    view more details on Pubmed

  • Hox gene regulation in the central nervous system of Drosophila. Front Cell Neurosci 2014 ;8():96. 10.3389/fncel.2014.00096. PMC4005941.

    abstract

    Hox genes specify the structures that form along the anteroposterior (AP) axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called "posterior dominance," states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. Here we review a noticeable exception to this rule where the more-posteriorly expressed Abd-B Hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS). While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92 kb long non-coding RNA (lncRNA) encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA). Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The first mechanism is mediated by a microRNA (mir-iab-8) encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila Hox complexes.

    view more details on Pubmed

  • A novel function for the Hox gene Abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila. PLoS Genet. 2013 Mar;9(3):e1003395. 10.1371/journal.pgen.1003395. PGENETICS-D-12-00167. PMC3610936.

    abstract

    In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), we discovered that Abd-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 from the iab-6 regulatory domain that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. We determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of sex peptide in female mates of iab-6 mutant males. We also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. Our results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR.

    view more details on Pubmed

  • abd-A regulation by the iab-8 noncoding RNA. PLoS Genet. 2012 ;8(5):e1002720. 10.1371/journal.pgen.1002720. PGENETICS-D-11-02462. PMC3359974.

    abstract

    The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This "iab-8 RNA" produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only "in cis." Transcriptional interference with the abd-A promoter is the most likely mechanism.

    view more details on Pubmed

  • Gene expression in time and space: additive vs hierarchical organization of cis-regulatory regions. Curr. Opin. Genet. Dev. 2011 Apr;21(2):187-93. S0959-437X(11)00033-5. 10.1016/j.gde.2011.01.021.

    abstract

    In higher eukaryotes, individual genes are often intermingled with other genes and spread out across tens to hundreds of kilobases, even though only small portions of their sequence are devoted to protein coding. Yet, in this seemingly extended and tangled mess, the cell is able to precisely regulate gene expression in both time and space. Over the past few decades, numerous elements, like enhancers, silencers and insulators have been found that shed some light on how the precise control of gene expression is achieved. Through these discoveries, an additive model of gene expression was envisioned, where the addition of the patterning details imparted by regulatory elements would create the final pattern of gene expression. Although many genes can be described using this model, recent work in the Drosophila bithorax complex suggests that this model may be somewhat simplistic and, in fact, regulatory elements sometimes seem to communicate with each other to form a functional hierarchy that is far from additive.

    view more details on Pubmed

  • The Polycomb group protein CRAMPED is involved with TRF2 in the activation of the histone H1 gene. Chromosoma 2011 Jun;120(3):297-307. 10.1007/s00412-011-0312-2.

    abstract

    CRAMPED (CRM), conserved from plants to animals, was previously characterized genetically as a repressive factor involved in the formation of facultative and constitutive heterochromatin (Polycomb silencing, position effect variegation). We show that crm is dynamically regulated during replication and identify the Histone gene cluster (His-C) as a major CRM target. Surprisingly, CRM is specifically required for the expression of the Histone H1 gene, like the promoter-bound transcription factor TRF2. Consistently with this, CRM genetically interacts and co-immunoprecipitates with TRF2. However, the Polycomb phenotypes observed in crm mutants are not observed in TRF2 hypomorphic mutants, suggesting that they correspond to independent roles of CRM. CRM is thus a highly pleiotropic factor involved in both activation and repression.

    view more details on Pubmed

  • Segregating variation in the polycomb group gene cramped alters the effect of temperature on multiple traits. PLoS Genet. 2011 ;7(1):e1001280. 10.1371/journal.pgen.1001280. PMC3024266.

    abstract

    The phenotype produced by a given genotype can be strongly modulated by environmental conditions. Therefore, natural populations continuously adapt to environment heterogeneity to maintain optimal phenotypes. It generates a high genetic variation in environment-sensitive gene networks, which is thought to facilitate evolution. Here we analyze the chromatin regulator crm, identified as a candidate for adaptation of Drosophila melanogaster to northern latitudes. We show that crm contributes to environmental canalization. In particular, crm modulates the effect of temperature on a genomic region encoding Hedgehog and Wingless signaling effectors. crm affects this region through both constitutive heterochromatin and Polycomb silencing. Furthermore, we show that crm European and African natural variants shift the reaction norms of plastic traits. Interestingly, traits modulated by crm natural variants can differ markedly between Drosophila species, suggesting that temperature adaptation facilitates their evolution.

    view more details on Pubmed

  • Initiator elements function to determine the activity state of BX-C enhancers. PLoS Genet. 2010 ;6(12):e1001260. 10.1371/journal.pgen.1001260. PMC3009686.

    abstract

    A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain.

    view more details on Pubmed

  • Cis-regulation in the Drosophila Bithorax Complex. Adv. Exp. Med. Biol. 2010 ;689():17-40.

    abstract

    The discovery of the first homeotic mutation by Calvin Bridges in 1915 profoundly influenced the way we think about developmental processes. Although many mutations modify or deform morphological structures, homeotic mutations cause a spectacular phenotype in which a morphological structure develops like a copy of a structure that is normally found elsewhere on an organism's body plan. This is best illustrated in Drosophila where homeotic mutations were first discovered. For example, Antennapedia mutants have legs developing on their head instead of antennae. Because a mutation in a single gene creates such complete structures, homeotic genes were proposed to be key "selector genes" regulating the initiation of a developmental program. According to this model, once a specific developmental program is initiated (i.e., antenna or leg), it can be executed by downstream "realizator genes" independent of its location along the body axis. Consistent with this idea, homeotic genes have been shown to encode transcription factor proteins that control the activity of the many downstream targets to "realize" a developmental program. Here, we will review the first and perhaps, best characterized homeotic complex, the Bithorax Complex (BX-C).

    view more details on Pubmed

  • The bithorax complex of Drosophila an exceptional Hox cluster. Curr. Top. Dev. Biol. 2009 ;88():1-33. S0070-2153(09)88001-0. 10.1016/S0070-2153(09)88001-0.

    abstract

    In his 1978 seminal paper, Ed Lewis described a series of mutations that affect the segmental identities of the segments forming the posterior two-thirds of the Drosophila body plan. In each class of mutations, particular segments developed like copies of a more-anterior segment. Genetic mapping of the different classes of mutations led to the discovery that their arrangement along the chromosome paralleled the body segments they affect along the anteroposterior axis of the fly. As all these mutations mapped to the same cytological location, he named this chromosomal locus after its founding mutation. Thus the first homeotic gene (Hox) cluster became known as the bithorax complex (BX-C). Even before the sequencing of the BX-C, the fact that these similar mutations grouped together in a cluster, lead Ed Lewis to propose that the homeotic genes arose through a gene duplication mechanism and that these clusters would be conserved through evolution. With the identification of the homeobox in the early 1980s, Lewis' first prediction was confirmed. The two cloned Drosophila homeotic genes, Antennapedia and Ultrabithorax, were indeed related genes. Using the homeobox as an entry point, homologous genes have since been cloned in many other species. Today, Hox clusters have been discovered in almost all metazoan phyla, confirming Lewis' second prediction. Remarkably, these homologous Hox genes are also arranged in clusters with their order within each cluster reflecting the anterior boundary of their domain of expression along the anterior-posterior axis of the animal. This correlation between the genomic organization and the activity along the anteroposterior body axis is known as the principle of "colinearity." The description of the BX-C inspired decades of developmental and evolutionary biology. And although this first Hox cluster led to the identification of many important features common to all Hox gene clusters, it now turns out that the fly Hox clusters are rather exceptional when compared with the Hox clusters of other animals. In this chapter, we will review the history and salient features of bithorax molecular genetics, in part, emphasizing its unique features relative to the other Hox clusters.

    view more details on Pubmed

  • Boundary swapping in the Drosophila Bithorax complex. Development 2008 Dec;135(24):3983-7. dev.025700. 10.1242/dev.025700.

    abstract

    Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, we tested whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, we exchanged the Fab-7 and Fab-8 boundaries within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, we find that the Fab-7 boundary can almost completely replace the Fab-8 boundary. Our results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries.

    view more details on Pubmed

  • Making connections: boundaries and insulators in Drosophila. Curr. Opin. Genet. Dev. 2007 Oct;17(5):394-9. S0959-437X(07)00153-0. 10.1016/j.gde.2007.08.002.

    abstract

    In eukaryotes, enhancers must often exert their effect over many tens of kilobases of DNA with a choice between many different promoters. Given this situation, elements known as chromatin boundaries have evolved to prevent adventitious interactions between enhancers and promoters. The amenability of Drosophila to molecular genetics has been crucial to the discovery and analysis of these elements. Since these elements are involved in such diverse processes and show little or no sequence similarity between them, no single molecular mechanism has been identified that accounts for their activity. However, over the past approximately 5 years, evidence has accumulated suggesting that boundaries probably function through the formation of long-distance chromatin loops. These loops have been proposed to play a crucial role in both controlling enhancer-promoter interactions and packing DNA.

    view more details on Pubmed

  • An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. U.S.A. 2007 Feb;104(9):3312-7. 0611511104. 10.1073/pnas.0611511104. PMC1805588.

    abstract

    Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.

    view more details on Pubmed

  • Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification. Nat. Genet. 2006 Aug;38(8):931-5. ng1833. 10.1038/ng1833.

    abstract

    A cis-regulatory region of nearly 300 kb controls the expression of the three bithorax complex (BX-C) homeotic genes: Ubx, abd-A and Abd-B. Interspersed between the numerous enhancers and silencers within the complex are elements called domain boundaries. Recently, many pieces of evidence have suggested that boundaries function to create autonomous domains by interacting among themselves and forming chromatin loops. In order to test this hypothesis, we used Dam identification to probe for interactions between the Fab-7 boundary and other regions in the BX-C. We were surprised to find that the targeting of Dam methyltransferase (Dam) to the Fab-7 boundary results in a strong methylation signal at the Abd-Bm promoter, approximately 35 kb away. Moreover, this methylation pattern is found primarily in the tissues where Abd-B is not expressed and requires an intact Fab-7 boundary. Overall, our work provides the first documented example of a dynamic, long-distance physical interaction between distal regulatory elements within a living, multicellular organism.

    view more details on Pubmed

  • Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex. Development 2006 Aug;133(15):2983-93. dev.02451. 10.1242/dev.02451.

    abstract

    The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.

    view more details on Pubmed

  • The ABC of the BX-C: the bithorax complex explained. Development 2006 Apr;133(8):1413-22. 133/8/1413. 10.1242/dev.02323.

    abstract

    As one of two Drosophila Hox clusters, the bithorax complex (BX-C) is responsible for determining the posterior thorax and each abdominal segment of the fly. Through the dissection of its large cis-regulatory region, biologists have obtained a wealth of knowledge that has informed our understanding of gene expression, chromatin dynamics and gene evolution. This primer attempts to distill and explain our current knowledge about this classic, complex locus.

    view more details on Pubmed

  • Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex. Genes Dev. 2005 Aug;19(15):1755-60. 19/15/1755. 10.1101/gad.347005. PMC1182336.

    abstract

    Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.

    view more details on Pubmed

  • GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 2005 Mar;17(5):695-707. S1097-2765(05)01091-9. 10.1016/j.molcel.2005.02.013.

    abstract

    The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. Here, we show that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). We purified USP7 from embryo nuclear extracts as a stable heteromeric complex with guanosine 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation.

    view more details on Pubmed

  • Ensuring enhancer fidelity. Nat. Genet. 2003 Aug;34(4):360-1. 10.1038/ng0803-360. ng0803-360.

    abstract

    view more details on Pubmed

  • Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing. Development 2002 Nov;129(21):4915-22.

    abstract

    The Fab-7 chromatin domain boundary insures functional autonomy of the iab-6 and iab-7 cis-regulatory domains in the bithorax complex (BX-C). We have previously shown that chromatin insulators such as gypsy or scs(min) are potent insulators that cannot substitute for Fab-7 function within the BX-C. During the early stages of these swapping experiments, we initially used a fragment of scs that was slightly larger than a minimal scs element (scs(min)). We report that this scs fragment, unlike scs(min), interferes in an orientation-dependent manner with the output of a regulatory region covering 80 kb of DNA (from iab-4 to iab-8). At the core of this orientation-dependent phenotype is a promoter located immediately adjacent to the scs insulator. In one orientation, the promoter traps the activity of the iab-3 through iab-5 cis-regulatory domains, diverting them from the abd-A gene. In the opposite orientation, the promoter is transcribing the iab-7 cis-regulatory domain, resulting in ectopic activation of the latter. Our data suggest that transcription through a Polycomb-Response Element (PRE) interferes with the maintenance of a Polycomb repression complex.

    view more details on Pubmed

  • Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster. Nucleic Acids Res. 1994 Aug;22(15):3138-46. PMC310287.

    abstract

    A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.

    view more details on Pubmed

  • abdA expression in Drosophila embryos. Genes Dev. 1990 Sep;4(9):1573-87.

    abstract

    The abdominal A (abdA) gene is one of three transcription units in the Bithorax Complex of Drosophila encoding a homeo box protein; it is flanked by Ultrabithorax (Ubx) and Abdominal B (AbdB). The abdA gene is required for segmental identity of the second through eighth abdominal segments. The transcription unit of abdA is approximately 20 kb long and encodes a protein of 330 amino acids. The abdA homeo box is almost identical to the homeo box of Ubx but is quite different from the AbdB homeo box. A polyclonal antibody to abdA protein stains embryonic nuclei in segments A1-A7 (parasegments 7-13). The iab-2, 3, and 4 mutant classes define positive cis-regulatory elements that induce expression of abdA in segments A2-A4 (parasegments 7-9), respectively. Once a pattern of abdA expression is turned on in a given parasegment, it remains on in the more posterior parasegments, so that the complex pattern of expression is built up in the successive parasegments. The abdA product appears to repress expression of Ubx whenever they appear in the same cell, but abdA is repressed by AbdB only in the eighth and ninth abdominal segments.

    view more details on Pubmed

  • The bithorax complex: control of segmental identity. Genes Dev. 1987 11;1(9):891-898. 1/9/891. 10.1101/gad.1.9.891.

    abstract

    view more details on Pubmed

see all publications

alumni

Nothing to show yet