Genome mapping and characterization of the Anopheles gambiae heterochromatin

Abstract

Background: Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized.

Results: To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1) and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin.

Conclusions: Our results demonstrate that Anopheles polytene chromosomes and whole-genome shotgun assembly render the mapping and characterization of a significant part of heterochromatic scaffolds a possibility. These results reveal the strong association between characteristics of the genome features and morphological types of chromatin. Initial analysis of the An. gambiae heterochromatin provides a framework for its functional characterization and comparative genomic analyses with other organisms.

Figure 1

The pericentric and intercalary heterochromatin of polytene chromosomes shown on a standard cytogenetic map of An. gambiae [91]. PH--pericentric heterochromatin, IHc--compact intercalary heterochromatin, IHd--diffuse intercalary heterochromatin.

Figure 2

Localization of HP1 and lamin Dm₀ Drosophila antibodies on An. gambiae chromosomes. Small numbers and letters indicate subdivisions of the chromosome map. The diffuse type of heterochromatin is shown by black arrowheads (a, b, c). The white arrowheads show compact heterochromatin (c) and sites of HP1 and lamin localization (e, f). Asterisks (d) show attachments of diffuse heterochromatin to the NE. X, 2R, 2L, 3R, 3L - chromosomal arms, C - centromeric areas.

Figure 3

Schematic representation of the heterochromatin amount in the An. gambiae genome. (a) Relative proportions of mapped chromatin types and unmapped sequences in the assembly. PH--pericentric heterochromatin, IHc--compact intercalary heterochromatin, IHd--diffuse intercalary heterochromatin, PEU--proximal euchromatin, EU--euchromatin, UNK--"unknown chromosome." (b) Comparison of sizes and positions of islands of genomic divergence (IGD) and regions of pericentric heterochromatin (HET) in the X chromosome, the 2L arm, and 3L arm. Position of a putative centromere corresponds to 0 bp.

Figure 4

Overrepresented GO terms in genes within the cytologically confirmed heterochromatin (a) and within "unknown chromosome" (b) of An. gambiae. The percentages of heterochromatic (red) and euchromatic (blue) genes containing the listed GO biological process (pink shading), cellular location (blue shading), and molecular function (green shading) terms are indicated. Numbers in parentheses refer to the actual number of heterochromatin or unmapped genes annotated with the listed GO domain. GO-Term-Finder, Bonferroni corrected p-value scores are shown to the right (grey shading).

Figure 5

Median values of gene density and repetitive element coverage in chromatin types of An. gambiae. Percentage of region length occupied per 1 Mb are indicated for all repetitive elements. PH--pericentric heterochromatin, IHc--compact intercalary heterochromatin, IHd--diffuse intercalary heterochromatin, PEU--proximal euchromatin, EU--euchromatin.

Figure 6

Genome landscapes of the An. gambiae heterochromatin and euchromatin. Median values of coverage of molecular features are displayed as 5-Mb intervals in euchromatin (open circles) and < 1-Mb intervals in heterochromatin. Red squares--pericentric heterochromatin, open diamonds--proximal euchromatin, blue stars--diffuse intercalary heterochromatin, blue triangles--compact intercalary heterochromatin.

Figure 7

Median values of gene density and repetitive element coverage in "unknown chromosome" of An. gambiae. EU--total euchromatin, H--total heterochromatin, U--"unknown chromosome."