Evidence for a regulatory protein complex on RNA polymerase III promoter of human retroposons of Alu family

Abstract

Abundant human retroposons of the Alu family produce few RNA polymerase III (RPIII)-dependent transcripts in vivo. This suggests that either the bulk of the repeats has no proper promoter elements or that transcription of Alu by RPIII is repressed. In this study, we analyzed complexes formed by human nuclear proteins with the Alu B-box and with an adjacent downstream sequence (DB-sequence). Four complexes (C1-C4) were detected and two of them (C2 and C3) were found to be induced by different proteins. C3 formation was found to be sensitive to minor sequence variation within the Alu DB-sequence. The C2 complex is specifically repressed by the competing VA1 B-box oligonucleotide and was found to be very stable. In addition, it is downregulated in human cells transformed by adenovirus 5. This is consistent with a view that the C2 complex is formed by a protein (designated as ACR1) that is different from TFIIIC2. The ACR1 protein may be involved in the modulation of Alu transcription in vivo by interfering or cooperating with TFIIIC2. A similar complex is detected with the efficiently transcribed adenovirus VA1 RNA gene B-box. We compared the affinity of complexes formed by ACR1 with Alu and VA1 B-boxes. It was found that both B-boxes bind ACR1 with equal affinity with a dissociation constant of about 2 nM. However, DB-sequences in Alu and VA1 promoters are non-homologous, and C3/C4 complexes are found to be formed with Alu DB, but not formed with VA1 DB sequences. The Alu-specific protein forming C3 (named as ACR2) may cooperate with ACR1 in selective repression of RPIII-dependent Alu transcription in vivo.

A novel human DNA‐binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA‐polymerase‐III‐driven transcription of the Alu‐family retroposons in vitro

“…In our previous work, evidence was presented for the assembly of specific protein complexes on an Alu sub‐sequence located within and just downstream of its promoter B‐box [34–36]. In this study we show that one of the complexes is down‐regulated in adenovirus‐5‐infected cells, which is consistent with the view that it may be a Pol‐III‐repressor complex.…”

“…Deletion mapping of the internal promoter elements essential for the Pol‐III‐dependent Alu transcription in vitro [13,14] indicated that one of the elements (directing element) is located in the Alu subregion between nt 63 and 140 covering the promoter B‐box. This subregion was also found to interact in vitro with HeLa nuclear proteins [34,35,41]; one of which may be basal transcription factor TFIIIC which preferentially binds to the B‐box region [3,8]. Figure 1 shows part of the corresponding Alu subregion and oligonucleotide probes used in this study.…”

“…F, free oligonucleotide. Labeling of oligonucleotides, preparation of nuclear extracts and EMSA were performed as described earlier [35,37]. All EMSA incubation mixes contained 5 µg of total protein and DBc oligonucleotide was used.…”

“…Electrophoretic mobility shift assay (EMSA) was performed essentially as described earlier [34–37]. Oligonucleotides were synthesized either at Gibco‐BRL or BioTeZ (Berlin‐Buch, Germany).…”

The function of the epigenome in cell reprogramming

“…The latter finding is reminiscent of the localization of other insulator proteins that were proposed to establish chromatin loops to define independent domains of transcriptional activity [160]. In mammals, about 500 000 copies of Alu repeats, which account for 5% of the genome content, contain B-box sequences [165]. This large fraction of potential TFIIIC-binding sites indicates a potential role for at least a fraction of Alu elements in the spatial organization of the mammalian genome.…”