The C-module-binding factor supports amplification of TRE5-A retrotransposons in the Dictyostelium discoideum genome

Abstract

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery.

Fig. 1.

CbfA is involved in regulation of TRE5-A steady-state RNA levels. AX2 and JH.D cells were grown to late logarithmic phase in the absence or presence of 80 μM antimycin A as described previously (22). Total RNA was prepared, separated by agarose gel electrophoresis, and blotted. The filter was first hybridized with a (−) RNA strand-specific probe, then stripped and hybridized with a (+) RNA strand-specific TRE5-A DNA probe, and finally stripped again and hybridized to histone H3 (hstC) RNA as a loading control. Size markers to the left refer to the migration positions of 26S and 17S rRNAs (ca. 4.1 and 1.9 kb, respectively).

Fig. 2.

Ectopic expression of CbfA in a CbfA-depleted mutant rescues TRE5-A expression. A Northern blot was prepared with total RNAs from the indicated strains: lane 1, AX2 cells; lane 2, untransformed JH.D cells; lane 3, JH.D cells transformed with empty expression vector; lane 4, JH.D cells expressing CbfA-CTD; and lane 5, JH.D cells expressing full-length CbfA. The filter was hybridized successively with (−) RNA strand-specific and (+) RNA strand-specific TRE5-A DNA probes, followed by a probe detecting histone H3 (hstC) RNA as a loading control. Size markers to the left refer to the migration positions of 26S and 17S rRNAs (ca. 4.1 and 1.9 kb, respectively).

Fig. 3.

Activities of the A module and the C module in the CbfA mutant strain JH.D. AX2 and JH.D cells were transformed with plasmid pGEM-A-luc (gray bars) or pGEM-C-luc (black bars) and with pISAR. After G418 selection, clones were pooled, and the average copy numbers of the expression plasmids were determined by quantitative PCR. Only pools with average plasmid copy numbers between 25 and 80 were selected for luciferase reporter assays. The values resulted from 4 to 6 pools per transformation and were normalized for 100 plasmid copies. Background activities of AX2 cells and JH.D cells transformed only with pISAR were 9 ± 10 and 17 ± 17 arbitrary units, respectively.

Fig. 4.

Outline of the TRE trap assay.

Fig. 5.

TRE5-A retrotransposition activity in JH.D cells. TRE^trap plasmids without a tRNA gene as an integration target (gray bars) or with a D. discoideum Val^UAC tRNA gene as bait to attract integration of mobile TREs (black bars) were transformed into DH1[ura⁻] or JH.D[ura⁻] cells. Mean clone numbers (± SD) from 5 petri dishes are shown.

Fig. 6.

Complementation of TRE5-A retrotransposition in the CbfA mutant. (A) TRE5-A.1 expression was quantified in JH.D[TRE^trap/ura⁺] transformants by real-time RT-PCR. Expression of TRE5-A in AX2 cells versus untransformed JH.D[ura⁻] cells served as a control (white bar, column 1). JH.D[ura⁻] cells were transformed with TRE^trap plasmids without a tRNA gene as an integration target (gray bars, columns 2 to 4) or with a D. discoideum Val^UAC tRNA gene as bait to attract mobile TREs (black bars, columns 5 to 7). Resulting JH.D[TRE^trap/ura⁺] cells were then supertransformed with empty expression vector (columns 2 and 5) or with plasmids that allowed for the expression of either CbfA-CTD (columns 3 and 6) or full-length CbfA (columns 4 and 7) in the JH.D background. All expression data were obtained from 4 to 8 independent transformants and are given relative to untransformed JH.D[ura⁻] cells, meaning that values of >1 indicate more TRE5-A expression in the transformants or AX2 cells than in untransformed JH.D[ura⁻] cells. (B) JH.D[TRE^trap/ura⁺] cells were subjected to selection in 5-FOA and uracil. Numbers of 5-FOA-resistant clones were calculated for 5 petri dishes and are given as means ± SD. Columns are the same as in panel A. The experiment was repeated twice with similar results.