François Karch

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

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 φC31-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 φC31 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 φC31-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.

The Abdominal Region of the Bithorax Complex

The homeotic mutations in the right half of the bithorax complex of Drosophila cause segmental transformations in the second through the eighth segments of the fly. A chromosomal walk in the bithorax complex has now been extended 215 kb through the right half of the complex, and lesions for over 40 mutations have been located on the DNA map. The mutations can be grouped in a series of phenotypic classes, one for each abdominal segment, although each mutation typically affects more than one segment. The mutant lesions of each class are clustered, and they are aligned on the chromosome in the order of the body segments that they affect. Complementation tests suggest interactions between widely spaced DNA regions; indeed, the right half cannot be split anywhere without some loss of function.

A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation

The bithorax complex specifies the identity of parasegments 5‐14 of Drosophila. Although nine parasegment‐specific functions, abx/bx, bxd/pbx and iab‐2 to iab‐8,9 have been identified, the whole bithorax complex appears to encode only three classes of proteins, Ubx, abd‐A and Abd‐B. Many observations suggest that the parasegment‐specific functions act as positive cis‐regulatory elements of Ubx, abd‐A and Abd‐B. We report the molecular genetics of a new gain‐of‐function mutation, Fab‐7, which transforms parasegment 11 into parasegment 12. Induction of Abd‐B mutations in cis (one of which removes the Abd‐B homeobox) causes reversion of the dominant phenotype, demonstrating that Fab‐7 misregulates Abd‐B. A 4 kb deletion, 30 kb downstream from the Abd‐B transcription unit, is solely responsible for the Fab‐7 phenotype. We consider that the parasegment‐specific functions lie in DNA domains that are sequentially and independently ‘opened’ along the chromosome. Once a domain is opened, the cis‐regulatory sequences within it can carry out their function. We propose that the Fab‐7 deletion removes a boundary separating the iab‐6 and iab‐7 cis‐regulatory regions (the functions specific for parasegments 11 and 12) allowing the open configuration of iab‐6 to invade iab‐7 in parasegment 11. This is strongly supported by our finding that Fab‐7 can be caused to revert by lesions not only in iab‐7 but also in iab‐6.

The iab-7 Polycomb Response Element Maps to a Nucleosome- Free Region of Chromatin and Requires Both GAGA and Pleiohomeotic for Silencing Activity

ABSTRACT In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogasterbithorax complex (BX-C). Previous studies mapped the iab-7PRE to an 860-bp fragment located just distal to the Fab-7boundary. Located within this fragment is an ∼230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of amini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of theiab-7 PRE in vivo.

The ABC of the BX-C: the bithorax complex explained

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.

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.

Homeo box genes of the Antennapedia and Bithorax Complexes of Drosophila

The Antennapedia, Ultrabithorax, and fushi tarazu genes of Drosophila melanogaster each contain a very similar protein coding sequence, the homeo box. Previously cloned homeo box sequences were used to isolate additional well conserved members of the homeo box gene family. The most strongly conserved members of the homeo box gene family map within either the Antennapedia or Bithorax gene complexes. The tissue distribution of transcripts encoded by the two rightmost homeo box genes of the Bithorax complex are compared with the iab-2 and iab-7 phenotypes.

Histone Chaperones ASF1 and NAP1 Differentially Modulate Removal of Active Histone Marks by LID-RPD3 Complexes during NOTCH Silencing

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.

The bluetail transposon: Evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex

An extremely large cis‐regulatory region generates the parasegment‐specific expression patterns of the homeotic genes in the bithorax complex. We present evidence supporting the idea that this cis‐regulatory region is subdivided into independent cis‐regulatory domains. We describe a Ubx‐lacZ transposon which is inserted into one of these domains, iab‐7. The PS12‐specific pattern of LacZ expression from this reporter indicates that it is subject to the control of the iab‐7 cis‐regulatory domain, but is protected from the effects of adjacent regulatory domains. Protection on the proximal side appears to be provided by the Fab‐7 boundary element. Deletion of this boundary results in the ectopic activation of iab‐7 in PS11 (where the iab‐6 cis‐regulatory domain normally functions). We show that the Fab‐7 boundary, like other boundaries, has an unusual chromatin structure.

Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification

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, ∼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.