Henrik Gyurkovics

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 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.

Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex

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.

Efficient and specific targeting of polycomb group proteins requires cooperative interaction between grainyhead and pleiohomeotic

ABSTRACT Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. Here we show that the grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, we propose that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity.

The transcriptional coactivator SAYP is a trithorax group signature subunit of the PBAP chromatin remodeling complex

ABSTRACT SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome

SummaryFour dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3) 14 maps in 86CD, Su-var(3) 13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.

abd-A Regulation by the iab-8 Noncoding RNA

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.

Long-distance interactions between enhancers and promoters

Abdominal‐B (Abd‐B) is a complex homeotic gene with a difficult task: one transcript determines the identity of four different abdominal segments throughout development in Drosophila. Although an increasing amount of information is available about the structure and the functioning of the regulatory regions that determine the expression pattern of Abd‐B, it is still not clear how these regulatory regions can contact the distantly located (several tens of kilobases away) promoter in the nucleus, what mechanism restricts promiscuous enhancers to this specific interaction, and how different regulatory regions replace one another at the same promoter in subsequent abdominal segments. Moreover, several of these regulatory regions have to act over chromatin domain boundaries and extensive inactive chromatin domains, similarly to the situation found in the chicken beta‐globin cluster. In this minireview we survey mechanisms and factors that may be involved in mediating specific interactions between the Abd‐B promoter and its regulatory regions.

Boundary swapping in the Drosophila Bithorax complex

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.

Genomic organization and functional analysis of a deletion variant of the 87A7 heat shock locus of Drosophila melanogaster.

The major 70,000 Mr heat-induced protein (hsp 70) of Drosophila melanogaster is encoded at two loci, 87A7 and 87C1. The 87A7 locus has two hsp 70-coding sequences (zc) about 2·8 × 103 bases apart in a divergent orientation. We have identified a variant 87A7 locus, the Sze-13 chromosome, which has a reduced heat shock puff. Characterization of this altered 87A7 locus reveals that it has a small deletion in the spacer between the two genes that inactivates the proximal gene. The mutant locus has been cloned and the breakpoints of the deletion determined by sequence analysis. We find that the deletion removes the entire 5′ half of the proximal hsp 70 gene and extends to position −479 base-pairs upstream from the start of transcription of the distal gene. Using flies that retain only the mutant 87A7 hsp 70 locus of Sze-13 we have shown that the one remaining “intact” hsp 70 gene is still active. Since a partially deleted hsp 70 gene lacking the 3′ half of the transcription unit is also active, it is likely that the initial portion of the transcription unit (zc) plus the conserved znc sequence that is immediately adjacent to the 5′ end of the gene contain the sequences necessary for hsp 70 expression.