Paucity and preferential suppression of transgenes in late replication domains of the D. melanogaster genome

Abstract

Background: Eukaryotic genomes are organized in extended domains with distinct features intimately linking genome structure, replication pattern and chromatin state. Recently we identified a set of long late replicating euchromatic regions that are underreplicated in salivary gland polytene chromosomes of D. melanogaster.

Results: Here we demonstrate that these underreplicated regions (URs) have a low density of P-element and piggyBac insertions compared to the genome average or neighboring regions. In contrast, Minos-based transposons show no paucity in URs but have a strong bias to testis-specific genes. We estimated the suppression level in 2,852 stocks carrying a single P-element by analysis of eye color determined by the mini-white marker gene and demonstrate that the proportion of suppressed transgenes in URs is more than three times higher than in the flanking regions or the genomic average. The suppressed transgenes reside in intergenic, genic or promoter regions of the annotated genes. We speculate that the low insertion frequency of P-elements and piggyBacs in URs partially results from suppression of transgenes that potentially could prevent identification of transgenes due to complete suppression of the marker gene. In a similar manner, the proportion of suppressed transgenes is higher in loci replicating late or very late in Kc cells and these loci have a lower density of P-elements and piggyBac insertions. In transgenes with two marker genes suppression of mini-white gene in eye coincides with suppression of yellow gene in bristles.

Conclusions: Our results suggest that the late replication domains have a high inactivation potential apparently linked to the silenced or closed chromatin state in these regions, and that such inactivation potential is largely maintained in different tissues.

Figure 1

The underreplicated regions are enriched in suppressed transgenes. Examples of eye with strong (A), moderate (B) and weak (C) suppression of mini-white. (D) Eye color in wild type fly. (E) Distribution of transgenes from Selected set with different suppression of mini-white marker gene in URs, the flank regions and the genome. Color coding is explained at the bottom of the graph. The proportion of suppressed transgenes is 3.6 times higher in URs than in flanks (P < 7.6E-10). (F) Distribution of transgenes with different level of mini-white suppression indicates that URs contain higher proportion of inserts with strong suppression. Probability to have such trend by chance is 3.7E-15.

Figure 2

Example of distribution of the suppressed and active transgenes within and around the underreplicated region in the 11A region of the X-chromosome. The modified screenshot of USCS genome browser [41] encompassed 1 Mb of genomic DNA (chrX:11,650,001-12,650,000; dm3 genome assembly) is shown. Numbers on top mark the position on the chromosome (in kbs). Red and black bars indicate non-suppressed and suppressed insertions, respectively. Black rectangle corresponds to the underreplicated region and yellow rectangles correspond to the flank regions. Only one annotated isoform of protein-coding FlyBase Genes 5.12 for each gene is shown at the bottom.

Figure 3

Suppression of yellow gene in bristles. (A) Wild type wing. Examples of wings with weak (B) and moderate (C) suppression of yellow resulting in appearance of both dark and yellow bristles. Some dark bristles are indicated by arrows. (D) Strong suppression of transgene results in very weak staining of nearly all bristles. (E) Wing in yellow mutant.

Figure 4

Preferential suppression of transgenes in testis-specific loci and loci replicating late in Kc cells. (A) Distribution of the transgens from Selected set in testis-specific loci and all other loci. (B) Distribution of the transgenes in loci replicating late (LR) and early (ER) in Kc cells. Color coding is on the right.