The structural organization of the cell nucleus and chromosomes is a great challenge of biological research. Various genetic phenomena have underscored the importance of long-range chromatin architecture in gene expression and chromosomestability. Chromosome organization must be dynamic, allowing for motions of the chromatin fiber in processes such as chromosome condensation, homologous recombination, formation of replication centers and the regulated, long-range order interactions between enhancers and promoters (enhancer looping) in gene expression. The long-term goal is to identify and dissect the higher-order subunits of chromosomes and to characterize its cis-acting elements and structural components.
Chromatin domains and boundary elements
Chromatin domains are thought to be the ‘functional’ subunits of the nucleus and delimited by flanking cis-acting DNA sequences called boundary elements (BEs). These elements prevent promiscuous, unwanted interactions between regulatory enhancer elements and promoters (Figure 1, arrow 1), but allow or facilitate desired gene activities within a domain (arrow 2). BEs (not all) also serves as a barrier to block the spreading of genetically silenced heterochromatin (H) into the protected domain (arrow 3).
Although the experimental concepts of BEs are quite simple, there is currently no dominant molecular model. Different models have been discussed, those that include local interactions and others that are based on nuclear compartmentalization and/or tethering. Our fascination for these elements stem from the premise, that understanding the molecular mechanism of BEs will establish a functional link between gene expression and nuclear/chromosomal order.
Figure 1. Some properties of boundary elements (BEs) are depicted.
The Drosophila boundary protein called BEAF
Only a handful of BE-binding proteins have been characterized so far. This laboratory isolated a boundary activity termed BEAF which binds the scs’ element of the Drosophila heat shock domain ( Zhao et al., 1995 ). We immunolocalized BEAF to numerous interbands and puff boundaries on polytene chromosomes (Figure 2). This observation suggested the existence of a common class of boundary elements in Drosophila and that the band-interband structure of polytene chromosomes might be related to chromatin domains. Consequently, we isolated several genomic BEAF binding sites as candidate boundary elements and then demonstrated that these elements reduce PEV in transgenic flies ( Cuvier et al., 1998 ) Sequence comparison revealed that the BEAF family of BEs are defined at the sequence level by clustered, variably spaced and oriented CGATA motifs. Interestingly, BEAF was found to interacted well with these different sites despite the varied arrangement of CGATA motifs: this protein may assemble into different sized complexes to adapt to these binding sites. Importantly, we established the functionality importance of the CGATA motifs in transgenic flies by mutagenesis ( Cuvier et al., 1998 ).
BEAF consists of two related proteins 32A and 32B derived from the same gene. They form in solution predominantly heteromeric trimers but also larger complexes. We characterized three protein domains in BEAF. Heterocomplex formation is mediated by their identical C-terminal domain (C) and DNA-binding by their unique N-terminal fragment (N). The middle domain (M) is dispensable in vitro, but harbors part of boundary function; the main activity is in domain C.
Figure 2. Visualization of chromosomal domains defined by BEAF.
Drosophila BEAF acts as a boundary activity in S. cerevisiae
One of the experimental difficulty to dissect the molecular mechanism of BEs is the cumbersome assay, that is, the generation of transgenic flies or stably transformed cell lines. We are therefore very exited by our observation that Drosophila BEAF functions as a boundary activity in the powerful model system, yeast S. cerevisiae. Examples of chromosome domains in the yeast include the silent mating type loci, (HML & HMR) and proximity to telomeres. Genes inserted at the silent mating type loci HML or HMR are subject to a gene-nonspecific repression via heterochromatinization.
To assay BEAF activity in yeast, we inserted into the HML locus two distinguishable marker genes and flanked gene A with BEs (Figure 3). Both of these genes are epigentically silenced (off) in the absence of BEAF as mediated by the heterochromatic state of HML. In contrast, if BEAF is targeted to the indicated BEs, then expression of gene A but not gene B occurs (Figure 3). Importantly, this reporter procedure was found to distinguish between boundary function and transcription activation. We will exploit this assay in a major way to dissect BEAF function further and to identify its molecular partners. In particular, we are engaged in a genetic screen to identify new evolutionarily conserved boundary proteins in yeast and other biological systems. We believe this approach will go along way toward a molecular dissection of the boundary activity.
DNA sequence-specific drugs: amazing chromosome and gene tools
Targeting DNA satellites Genome projects not only discover a daunting number of new genes, they also yield an enormous amount of "non-genic" sequence data which must include "architectural" DNA elements. Architectural DNA is proposed to harbor sequences that mediate nuclear order, chromosome stability and dynamics, sister chromatid cohesion, centromere and telomere formation.
Biological assays to study architectural DNA are extremely limited. For example, although the phenomena of position effect variation (PEV) is attributed to the positioning of genes near centric heterochromatin, genetic tools to dissect the functions (if any) of centric satellite DNA are lacking. Is PEV mediated by the sequence satellite repeats, by base composition, by their epigenetic state or simply by the repetitive nature of its chromatin? In view of the difficulties one encounters of assigning functions to large fractions of the genome, we consider the development of new approaches and tools of major importance.
The approach we successfully developed in the last 3 years is based on the synthesis of DNA sequence-specific pseudopeptides (polyamides). Considerable progress was recently made in the synthesis of polyamides, composed of the aromatic amino acids N-methylpyrrole (Py) and N-methylimidazole (Im) that can bind specific DNA sequences with remarkable affinities (Dervan's laboratory). We made an number of chemical improvements and synthesized compounds that target different Drosophila melanogaster satellites ( Janssen et al., 2000a ). Compound P31 specifically binds the GAGAA satellite V and P9 targets the AT-rich satellites I and III. This was demonstrated by various techniques including epifuorescence microscopy of stained nuclei and chro-mosomes (Figure 4). Remarkably, these drugs, when fed to developing Drosophila flies, caused gain or loss of function phenotypes. While polyamide P9 (not P31) suppressed PEV of white-mottled flies (increased gene expression), P31 (not P9) mediated homeotic trans-formations (loss of function) exclusively in brown-dominant flies. Both phenomena are explained at the molecular level by chromatin opening (increased accessibility) of the targeted DNA satellites. Chromatin opening of satellite III by P9 is proposed to suppress PEV of white-mottled flies whereas chromatin opening of satellite V by P31 is proposed to create an inopportune ‘sink’ for the GAGA factor (GAF).
In the future, we will continue to use polyamides as novel tools for structural studies of chromosomes and to further refine cis-acting elements of PEV and chromosome architecture.