Dispersion Profiles and Gene Associations of Repetitive DNAs in the Euchromatin of the Beetle Tribolium castaneum

Abstract

Satellite DNAs are tandemly repeated sequences clustered within heterochromatin. However, in some cases, such as the major TCAST1 satellite DNA from the beetle Tribolium castaneum, they are found partially dispersed within euchromatin. Such organization together with transcriptional activity enables TCAST1 to modulate the activity of neighboring genes. In order to explore if other T. castaneum repetitive families have features that could provide them with a possible gene-modulatory role, we compare here the structure, organization, dispersion profiles, and transcription activity of 10 distinct TCAST repetitive families including TCAST1. The genome organization of TCAST families exhibit either satellite-like or transposon-like characteristics. In addition to heterochromatin localization, bioinformatic searches of the assembled genome have revealed dispersion of all families within euchromatin, preferentially in the form of single repeats. Dispersed TCAST repeats are mutually correlated in distribution and are grouped in distinct regions of euchromatin. The repeats are associated with genes, are enriched in introns relative to intergenic regions, and very rarely overlap exons. In spite of the different mechanisms of repeat proliferation, such as transposition and homologous recombination, all TCAST families share a similar frequency of spreading as well as dispersion and gene association profiles. Additionally, TCAST families are transcribed and their transcription is significantly activated by heat stress. A possibility that such common features of TCAST families might be related to their potential gene-modulatory role is discussed.

Keywords: Tribolium castaneum; gene regulation; repetitive DNA; satellite DNA; transcription.

Figure 1

Schematic representation of the Dna2-3_Tca transposon (A), which has 91% similarity to the full-size TCAST4 repeat of 744 bp. The transposon also contains the partial 576 bp-long reverse (rev) complement of TCAST4 (146–740 bp). TCAST4-like terminal inverted repeats of a transposon have similarity of 92% and are separated by 41 bp TCAST4-unrelated sequence (blue). (B) The Dna5-3_Tca transposon, which has 99% similarity to the full-size TCAST8 repeat of 294 bp. The transposon also contains the partial 86 bp long reverse complement of TCAST4 (208–294 bp). TCAST8-like terminal inverted repeats of transposon have similarity of 86% and are separated by 31 bp TCAST8-unrelated sequence (blue). (C) The TCAST10 repeat, which shares similarity with DNA transposons hAT-N16_Lmi [1–235 nucleotides (nt)] and IS3EU-1_TC [251–486 nucleotides (nt)]. (D) TCAST7 repeat, composed of two 735 bp-long, inverted repeats, which share mutual similarity of 90% and are separated by 300 bp of unrelated sequence (light blue). Short segments of TCAST7 sequence (indicated by red rectangles) share similarity with transposons Mariner-69_HSal (120–238 nt), Mariner-76_HSal (428–468), Dna-3-1_DPur (750–804 nt), and retrotransposon nonLTR/Tad1 (1002–1085 nt).

Figure 2

Polymerase chain reaction amplification using specific primers that amplify tandemly arranged dimers and larger multimers, and subsequent separation of products by agarose gel electrophoresis. Sequencing of products reveals satellite DNA-like tandem organization of TCAST3, TCAST5, TCAST6, and TCAST9 repeats within the T. castaneum genome.

Figure 3

Genomic content indicated as a percentage of the total genome and copy number (N) of repetitive families TCAST3–TCAST10 in the T. castaneum GA2 strain, estimated by dot blot hybridization.

Figure 4

The histograms show the percentage of all dispersed TCAST elements (red), as well as the percentage of genes (blue) along the length of 10 T. castaneum chromosomes. The percentage is calculated for the intervals corresponding to 1/10 of length of each chromosome. LG, linkage group.

Figure 5

Distances of intergenic TCAST elements to the nearest gene. Probability densities are plotted (y-axis) against log10-transformed distances (x-axis). Similarity of probability distributions between real data (blue curve) and simulated data (red) are calculated by Kolmogorov–Smirnov test and P-values are indicated for each TCAST element.

Figure 6

Dynamics of expression of repetitive TCAST families measured under standard conditions [no HS (heat shock)], immediately after long-term heat stress of 24 hr at 40° (0 min), at 30 min, or 1 or 2 hr of recovery at 25°. Relative No values are shown, which are obtained by dividing each No value by No value of control (no HS) for each repetitive DNA. Columns show averages of two different qRT-PCR experiments performed in triplicate and error bars represent SD.