Sequence elements in cis influence heterochromatic silencing in trans

Abstract

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin, which causes the locus to aberrantly associate with heterochromatin in interphase nuclei and silences the wild-type allele in heterozygotes. Transgenes placed near the bw(+) locus, in trans to bw(D), can also be silenced. The strength of silencing (called trans inactivation) varies with the regulatory sequences of the transgene and its distance away from the bw(D) insertion site in trans. In this study, we examine endogenous sequences in cis that influence susceptibility of a reporter gene to trans inactivation. Flanking deletions were induced in two parental lines containing P-element transgenes showing trans inactivation of the mini-white reporter. These new lines, which have mini-white under the influence of different endogenous sequence elements, now show varied ability to be silenced by bw(D). Determination of the deleted regions and the levels of mini-white expression and trans inactivation has allowed us to explore the correlation between cis sequence elements and susceptibility to trans inactivation and to identify a 301-bp sequence that acts as an enhancer of trans inactivation. Intriguingly, this region encompasses the upstream regions of two divergently transcribed genes and contains a sequence motif that may bind BEAF, a protein involved in delimiting chromatin boundaries.

FIG. 1.

Moving a P-element closer to the bw^D insertion site trans inactivates a P-element reporter gene. (Left) Schematic representations of the genomic region around the brown locus showing the various deletion and insertion strains over the bw^D insertion chromosome. Each box represents a gene (determined from published sequence accession no. AE003461.2). The dotted line represents a deletion. The figure is to scale with the exception of the bw^D insert, which is very large, containing approximately 1.6 Mbp of the simple satellite sequence AAGAG. mini-white is denoted as mw in the figures. Distal and proximal refer to the absolute position along the chromosome: proximal toward the centromere and distal toward the telomere. (Right) Fly eye photographs. Within each photograph, the left eyes (bw⁺) show the corresponding fly line with the transgene heterozygous to wild-type brown. The right eyes (bw^D) are from the corresponding fly line with the transgene heterozygous to the bw^D allele and P{cos bw⁺} on the third chromosome. To the right of the fly eyes are the spot count, standard deviation (in parentheses), and number of eyes examined. The notation n.s. indicates no spots. An approximately 29-kbp distal flanking deletion from Dcp-1 generated the ahf6 line. The ahf6 deletion endpoint is at the same site as the k11531 insertion site. The k11531 insertion is not trans inactivated (n t-x), while the k15608 insertion is trans inactivated (t-x). trans inactivation of the k15608 insertion is difficult to see in photographs but is apparent when examined under the microscope.

FIG. 2.

A distal flanking sequence influences mini-white expression without influencing trans inactivation. The notation is the same as in Fig. 1. An approximately 20-kbp proximal flanking deletion from Dcp-1 generated the csa7 line. The csa7 deletion endpoint is 3 bp from the chrw insertion site. To the right of the fly eyes are the spot count, standard deviation (in parentheses), and number of eyes examined.

FIG.3.

A 301-bp region acts as a cis enhancer of trans inactivation. Proximal is to the left, and distal is to the right. (A) A distal flanking deletion from the chrw insertion reveals a 301-bp region acting as an enhancer element. (B) A proximal flanking deletion from the Dcp-1 insertion reveals a 301-bp region acting as an enhancer element. (C) Enhanced silencing of insertions of P{EPgy2}. To the right of the fly eyes are the spot count, standard deviation (in parentheses), and number of eyes examined.

FIG. 4.

Sequence analysis of the 301-bp region. (A) The 5′ ends of the flanking genes are depicted, with translation start sites depicted by broken arrows. Putative transcription start sites are at the end of the box under the gene name. The sequences deleted by flanking deletions csc1, csc2, and ahd2, as well as the insertion sites of lines EY03568 and EY01834, are also indicated. Additionally, predicted BEAF, Mad, and ftz binding sites are indicated. (B) The sequence conserved between D. melanogaster (m), D. yakuba (y), and D. pseudoobscura (p) is shaded. The 301-bp region is in bold. Sequences of interest are highlighted in the color designated in the key. (C) Location of the possible BEAF binding sites. All sites include one BEAF palindrome and at least one additional site within 250 bp. The BEAF site in the 301-bp region is boxed. The entire sequence shown in Fig. 1 and 5 was searched, but only the region where sites were found is shown.

FIG. 5.

An approximately 108-kbp proximal flanking deletion from Dcp-1 generated the csa3 line. The csa3 deletion endpoint is located 25 bp proximal of the s4830 insertion site. Of interest, the csa3 line is missing a gene-poor and possibly heterochromatin-like region, while the s4830 line contains the gene-poor region. The region immediately proximal (not shown) is gene rich. Spots were exceedingly rare and faint in s4830/bw^D flies, making quantitation unreliable.