Interbands behave as decompacted autonomous units in Drosophila melanogaster polytene chromosomes

Abstract

We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.

Dissection of open chromatin domain formation by site specific recombination inDrosophila

“…4C-C0. Because this 4-kb fragment is 8.3 kb downstream of the 4.7-kb fragment containing the distal boundary (Semeshin et al, 2008) the 61C7-8 interband includes at least 17 kb DNA (Fig. 5A), more than previously estimated (Demakov et al, 1993).…”

“…The extent of the decondensed 3C6-7 interband had previously been mapped by genetics and high-resolution in situ hybridization (Keppy and Welshons, 1977;Rykowski et al, 1988) and it has been suggested that it forms independently of transcription (Rykowski et al, 1988;Semeshin et al, 2008).…”

“…Data from the Zhimulev laboratory also suggest a transcriptionindependent mechanism for the formation of open chromatin by the 61C7-8 interband (Semeshin et al, 1989;Demakov et al, 1993;Demakov et al, 2004;Semeshin et al, 2008). DNA from the 61C7-8 interband was first cloned starting from a P-element insertion that formed a new band within this interband (Demakov et al, 1993).…”

“…Therefore, based on the knowledge on interbands available at that time, the size of the 61C7-8 interband DNA was estimated to extend to 2-5 kb surrounding the P-element insertion. Later, a corresponding 4.7-kb DNA fragment from 61C7-8 was demonstrated to form decondensed chromatin autonomously (Semeshin et al, 2008). However, walking across the 61C7-8 region by high resolution in situ hybridization (H. S. unpublished), we determined that the 61C7-8 interband is much larger and covers a ,17-kb DNA segment starting 3.5 kb distal to the P-element insertion that was used to initiate the cloning of this interband ( Fig.…”

“…Condensed chromatin can be established at ectopic sites by transposition of transcriptionally inactive DNA (Semeshin et al, 1986;Semeshin et al, 1989), and decondensed chromatin also autonomously maintains an open state in a new chromosomal environment (Semeshin et al, 2008). These observations offer the possibility to assay for DNA sequences required for formation of decondensed chromatin.…”