Gligorov D, Sitnik J L, Maeda R K, Wolfner M F, Karch F

Department of Genetics and Evolution and NCCR Frontiers in Genetics, University of Geneva, Geneva, Switzerland.

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.

Gummalla M, Maeda R K, Castro Alvarez J J, Gyurkovics H, Singari S, Edwards K A, Karch F, Bender W

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

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.

Gibert J M, Karch F

Department of Zoology and Animal Biology, University of Geneva, Science III, 30 Quai Ernest Ansermet, 1211, Geneva 4, Switzerland, Jean-Michel.Gibert@unige.ch.

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.

Maeda R K, Karch F

Department of Zoology and Animal Biology and NCCR Frontiers in Genetics, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland.

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.

Gibert J M, Karch F, Schlotterer C

Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland.

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.

Iampietro C, Gummalla M, Mutero A, Karch F, Maeda R K

National Centre of Competence in Research (NCCR) Frontiers in Genetics and Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland.

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.

Maeda R K, Karch F

NCCR Frontiers in Genetics, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland.

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).

Moshkin Y M, Kan T W, Goodfellow H, Bezstarosti K, Maeda R K, Pilyugin M, Karch F, Bray S J, Demmers J A, Verrijzer C P

Department of Biochemistry, Center for Biomedical Genetics, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing.

Pilyugin M, Demmers J, Verrijzer C P, Karch F, Moshkin Y M

Department of Zoology and National Research Center Frontiers in Genetics, University of Geneva, Geneva, Switzerland.

Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.

Maeda R K, Karch F

Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland.

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.

Iampietro C, Cleard F, Gyurkovics H, Maeda R K, Karch F

NCCR, Frontiers in Genetics, University of Geneva, Department of Zoology and Animal Biology, 30 Quai Ernest Ansermet, 1211 Geneva 11, Switzerland.

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.

Maeda R K, Karch F

Department of Zoology and Animal Biology and NCCR Frontiers in Genetics, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland. robert.maeda@zoo.unige.ch

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.