Protein interactions on telomeric retrotransposons in Drosophila

Abstract

Telomere length in Drosophila is maintained by targeted transposition of three non-LTR retrotransposons: HeT-A, TART and TAHRE (HTT), but understanding the regulation of this process is hindered by our poor knowledge of HTT associated proteins. We have identified new protein components of the HTT array: Chromator (Chro), the TRF2/DREF complex and the sumoylation machinery. Chro was localized on telomeric HTT arrays by immunostaining, where it may interact with Prod directly, as indicated by yeast two-hybrid interaction, co-IP, and colocalization on polytene chromosomes. The TRF2/DREF complex may promote the open structure of HTT chromatin. The protein interactions controlling HTT chromatin structure and telomere length may be modulated by sumoylation.

Keywords: Chromator; Drosophila; Z4; chromatin; prod; putzig; retrotransposon; sumoylation; telomere.

Figure 1

Coimmunoprecipitation of Chro and Prod from S2 cells. (A) Arrowhead indicates that Prod was immunoprecipitated with anti-Prod antibody from the cell lysate (Lys) with minor left-over (Rest). The secondary anti-rabbit antibody used in the Western blot also detects the rabbit-anti-Prod antibodies in lane Prod-IP as major bands above and below Prod, as indicated in lane AB, where anti-Prod AB was run alone. The lower filter, stained for Chro, indicates that Chromator is also present in the Prod-IP lane, although most of it remained in the supernatant (Rest). The Prod-IP lane stained for Prod contains 1/20th of total IP, while Prod-IP lane stained for Chro contains all the rest (19/20th). As both lanes Lys and Rest contain 1/50th of total extract, and the Prod-IP lane is roughly estimated to contain half Chro compared to Rest, it follows that less than 1% of Chro is associated with Prod in vivo. (B) In the reciprocal experiment Chro was immunoprecipitated from an S2 extract and a small fraction of total Prod was co-immunoprecipitated with it (arrowhead). Control lanes (C) show that the same immunoprecipitates made in the absence of Prod or Chro antibodies contain neither Prod nor Chro.

Figure 2

Prod, Z4, Chro and DREF bind to the HTT array. (A) Double-staining of Tel/Oregon R polytene chromosomes with anti-Prod (red) and anti-Z4 (green) antibodies. 2L and 2R telomeres are shown, but other telomeres stain similarly. Prod and Z4 staining patterns of chromosome arms are different, but both proteins localize to the elongated HTT array (arrowhead), as indicated by the strong yellow color in the merged images. (B) Staining of Tel/Oregon R polytene chromosomes with anti-Chro antibodies show localization at the extended HTT array of the Tel homologue (arrowhead), similar to Prod and Z4. (C) The localization of Z4 (red) and Chro (green) completely overlap not only in interbands but also on telomeres (arrows). The left panel shows the y¹ w X chromosome, which lacks TAS , nevertheless Z4 and Chro telomeric localization remains the same as in wild type (not shown). The right panel shows an X chromosome with terminal deficiency, DfRT858 , which removes both TAS and HTT (white line connects identical bands in 1C) but retains a functional capping complex. Strong telomeric Z4 and Chro staining disappears from this telomere indicating that they are associated with HTT and not with the cap. (D) Double-staining of Tel/Oregon R polytene chromosomes with anti-Z4 (red) and anti-DREF (green) antibodies. The extended HTT homologues (arrowheads) stain with both antibodies.

Figure 3

SUMO staining of Tel/OreR telomeres indicates that proteins on HTT are intensely sumoylated, while the sumoylation level on the cap and TAS is not prominent. Only chromosome tips are shown, and arrows point at the elongated HTT array on 3R (A), 2R and 2L (B). The terminal 2L deficiency Df(2L)GB26/Cy (C) shows significant sumoylation only on the balancer chromosome (arrow), not at the terminal deficiency chromosome (arrowhead), although it also has a capping complex. The sumoylation level of the X chromosome telomere in wild type (E) is comparable to that of the y¹ w chromosome (D), which lacks X TAS, indicating that TAS associated proteins may not be sumoylated significantly.

Figure 4

Telomere length comparison of heterozygous mutants. Graph represents relative copy number of HeT-A (average 2^∆ct values ), normalized to the copy number of ribosomal protein S17 (RpS17). Error bars represent the standard deviation of threshold values from the average of triplicate reactions run on different days, each in triplicate. Blue columns represent negative controls; the green column shows the positive Tel¹control . All strains except Tel carry y w^67c23.