SU(VAR)3-7, a Drosophila heterochromatinassociated protein and companion of HP1 in the genomic silencing of position-effect variegation of homeotic gene complexes (i.e. Paro and Hogness, 1991; Fabienne Clé ard, Marion Delattre and Dorn et al., 1993; Fauvarque and Dura, 1993; Farkas Pierre Spierer 1 Tschiersch et al., 1994).Department of Zoology and Animal Biology, University of Geneva,The genetic modifiers of PEV could identify components 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland of the mechanisms of genomic silencing (Eissenberg, 1989;Reuter and Spierer, 1992). In Drosophila melanogas-1 Corresponding author ter, haplo-suppressor loci with a triplo-enhancer effect on PEV are candidates for being structural components of An increase in the dose of the Su(var)3-7 locus of the heterochromatin conformation that spreads silencing Drosophila melanogaster enhances the genomic silencon euchromatic genes. Among these, we have cloned the ing of position-effect variegation caused by centromeric modifier locus Su(var)3-7, and shown that an increase in heterochromatin. Here we show that the product of its dose enhances PEV ; Cléard et al., Su(var)3-7 is a nuclear protein which associates 1995). The deduced protein sequence predicts that it with pericentromeric heterochromatin at interphase, encodes a large protein with seven widely separated zinc whether on diploid chromosomes from embryonic fingers. Another candidate, HP1, initially isolated as a nuclei or on polytene chromosomes from larval salivary heterochromatin-associated protein (James and Elgin, glands. The protein also associates with the partially 1986), later was found to be encoded by the haploheterochromatic chromosome 4. As these phenotypes suppressor triplo-enhancer of PEV Su(var)2-5 (Eissenberg and localizations resemble those described by others for et al., 1990, 1992). In polytene chromosomes, HP1 was the Su(var)2-5 locus and its heterochromatin-associated found to associate with centromeric heterochromatin and protein HP1, the presumed co-operation of the two chromosome 4, and to a lesser extent with a subset of proteins was tested further. The effect of the dose of telomeres and a few euchromatic bands (James et al., Su(var)3-7 on silencing of a number of variegating 1989). In embryonic nuclei, HP1 co-localizes with centrorearrangements and insertions is strikingly similar to meric heterochromatin in interphase chromosomes the effect of the dose of Su(var)2-5 reported by others. (Kellum et al., 1995). The involvement of HP1 in the In addition, the two loci interact genetically, and genomic silencing of PEV is also supported cytologically the two proteins co-immunoprecipitate from nuclear by its association with euchromatic loci inactivated by extracts. The results suggest that SU(VAR)3-7 and HP1PEV (Belyaeva et al., 1993). co-operate in building the genomic silencing associated It has been proposed that the heterochromatic conformwith heterochromatin.ation spreads silence by co-operative assembly of a variety Keywords: Drosophila/genom...

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.

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.

Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine-tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by these two signals and is sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing leads to a dramatic reduction in suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips) through a compensatory mechanism. Through the functional analysis of these four MYBs, we generated plants allowing unbiased investigation of endodermal suberin function, without accounting for confounding effects due to Casparian strip defects, and were able to unravel specific roles of suberin in nutrient homeostasis.

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.

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.

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.

An increase in the number of copies of the Drosophila locus Suvar(3)7 enhances position-effect variegation, i.e. the inactivation in some cells of genes brought close to heterochromatin by a chromosomal rearrangement. The locus produces two transcripts of 5047 and 4203 nt that differ solely by the length of their 3' untranslated region. That these transcripts encode the modifier of variegation Suvar(3)7 is demonstrated by genetic transformation with the corresponding cDNAs. The deduced protein is 1169 amino acids long and contains seven widely spaced zinc fingers. These fingers are each preceded at 11-16 amino acids before the N-terminal cysteine by a tryptophan-containing motif. The transcripts are maternally transmitted, but are also found throughout development. The ubiquitous distribution of transcripts in embryos and the different sequence motifs support our speculation that the locus encodes a chromosomal protein implicated in heterochromatin-mediated DNA silencing.