The Drosophila Dot Chromosome: Where Genes Flourish Amidst Repeats

Abstract

The F element of the Drosophila karyotype (the fourth chromosome in Drosophila melanogaster) is often referred to as the "dot chromosome" because of its appearance in a metaphase chromosome spread. This chromosome is distinct from other Drosophila autosomes in possessing both a high level of repetitious sequences (in particular, remnants of transposable elements) and a gene density similar to that found in the other chromosome arms, ∼80 genes distributed throughout its 1.3-Mb "long arm." The dot chromosome is notorious for its lack of recombination and is often neglected as a consequence. This and other features suggest that the F element is packaged as heterochromatin throughout. F element genes have distinct characteristics (e.g, low codon bias, and larger size due both to larger introns and an increased number of exons), but exhibit expression levels comparable to genes found in euchromatin. Mapping experiments show the presence of appropriate chromatin modifications for the formation of DNaseI hypersensitive sites and transcript initiation at the 5' ends of active genes, but, in most cases, high levels of heterochromatin proteins are observed over the body of these genes. These various features raise many interesting questions about the relationships of chromatin structures with gene and chromosome function. The apparent evolution of the F element as an autosome from an ancestral sex chromosome also raises intriguing questions. The findings argue that the F element is a unique chromosome that occupies its own space in the nucleus. Further study of the F element should provide new insights into chromosome structure and function.

Keywords: F element; FlyBook; HP1a; chromosome evolution; fourth chromosome; heterochromatin.

Figure 1

D. melanogaster chromosome 4 is enriched for HP1a and POF. Immunohistochemical analysis of polytene chromosomes from the third instar larval salivary gland shows the genomic distribution of HP1a and POF. (A) D. melanogaster karyotype indicating the chromosome configuration and the six Muller elements A–F. Regions of constitutive heterochromatin are shown in dark grey. (B) Phase contrast image of a polytene chromosome spread from the salivary glands of a third instar larva (D. melanogaster). The chromosome arms have gone through ∼10 rounds of endoreduplication, while the pericentric heterochromatin is underreplicated and fuses in a common chromocenter. The rectangle marks the region containing chromosome 4 enlarged in (C–F). (C) Close-up of the phase contrast image. (D) Close-up showing staining with the monoclonal antibody C1A9 against HP1a (secondary goat anti-mouse-Alexa488 antibody); the pericentric heterochromatin and chromosome 4 are stained. (D) Close-up showing staining with the antibody MO459 against POF (secondary goat anti-rabbit-Alexa594 antibody); only chromosome 4 is stained. (F) Close-up merged image showing HP1a and POF signals superimposed. HP1a=green; POF=red. The chromosome squashing and staining protocol used is that described by Stephens and colleagues (Stephens et al. 2004).

Figure 2

Map illustrating the interspersed position-effect variegation-inducing (heterochromatic) and permissive domains on D. melanogaster chromosome 4. Hsp70-white reporter insertion sites are shown above the chromosome 4 map [diagram based on Riddle et al. (2008) with modifications]. Insertion sites that confer a red eye phenotype are indicated by red triangles, while insertion sites that confer a variegating eye phenotype are marked by dotted triangles. Above the map, two examples each of variegating and red eye phenotypes are shown. Beneath the chromosome map, the locations of transposable elements (TEs) and of genes are shown [FlyBase FB2018_02 (Gramates et al. 2017)]. The bottom track shows the distribution of the nine chromatin states (as defined by Kharchenko and colleagues) across chromosome 4 from BG3 cells (Kharchenko et al. 2011). State 1, enriched in H3K4me3 and other marks of active transcription, is in red; state 6, enriched in H3K27me3 and associated with Polycomb, is in dark gray; and states 7 and 8, enriched in H3K9me2/3 and associated with HP1a (heterochromatin), are in dark blue and light blue, respectively. ID, identifier.

Figure 3

Genes on chromosome 4 are enriched for chromatin marks typical of heterochromatin irrespective of their expression status. (A) Metagene profiles of expressed genes residing in the pericentric heterochromatin (left) or on chromosome 4 of D. melanogaster (right), illustrating their enrichment for HP1a, H3K9me2, H3K9me3, and H3K36me3 in BG3 cells. In contrast to pericentric genes, chromosome 4 genes have higher levels of the heterochromatic marks HP1a, H3K9me2, and H3K9me3 across the body of the genes compared to intergenic regions. The metagene profile averages the 2-kb regions upstream and downstream of the gene, along with gene spans that have been scaled to 3 kb in size. x-axis: position along the metagene with 0 indicating the transcription start site; y-axis: enrichment relative to input [figure panels modified from Riddle et al. (2011)]. (B) Browser view illustrating that both an expressed gene (Ephrin) and a silent gene (CG1909) on chromosome 4 show similar levels of enrichment for heterochromatic marks, although there is a shift in the ratio of H3K9me2 to H3K9me3, with higher levels of H3K9me2 associated with silencing. x-axis: position along the chromosome; y-axis for chromatin immunoprecipitation panels: enrichment relative to input (University of California Santa Cruz Genome Browser; Kent et al. 2002). RefGenes, reference genes.

Figure 4

Dot chromosome evolution within the Drosophilids illustrates the changeable nature of the chromosome, which nonetheless maintains common characteristics. Phylogenetic tree and chromosome diagram modified from Schaeffer and colleagues (Schaeffer et al. 2008) using additional information from Song et al. (2011) and Zhou and Bachtrog (2015). Chromosome size estimates of the F element arms (from the most proximal to most distal gene, not including the pericentric heterochromatin or telomeres) are from Schaeffer et al. (2008) and Zhou and Bachtrog (2015). Repeat content estimates for the F element gene-containing regions are derived from Leung et al. (2010, , . In the partial karyotype, fusions of the F element (orange) to other chromosomes are marked; in D. busckii, the F element is fused to the sex chromosomes (red), i.e., one copy of the F element fused to the Y chromosome (F/Y fusion), the other copy fused to the X chromosome, which is the A element in this species (F/A fusion). In D. willistoni, the F element is fused with the E element (purple).