Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1

Abstract

The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a p160 coactivator-interacting protein that may inhibit transcriptional activity of nuclear receptors. Here, we have characterized the transcriptional regulatory domains of ANCO-1. Two intrinsic repression domains (RD) were identified: an N-terminal RD1 at residues 318-611 and a C-terminal RD2 at 2369-2663. ANCO-1 also contains an activation domain (AD) capable of stimulating transcription in both mammalian and yeast cells. The minimal AD was delimited to a 70-amino acid region at residues 2076-2145. Overall, full-length ANCO-1 exhibited transcriptional repressor activity, suggesting that RD domains may suppress the AD activity. We further demonstrated that ANCO-1 silencing by siRNA enhanced progesterone receptor-mediated transcription. Together, these results indicate that the transcriptional potential of ANCO-1 may be modulated by a combination of repression and activation signals.

Fig. 1. Delineation of Transcriptional Regulatory Domains of ANCO-1

(A) Schematic representation of G4-ANCO-1 constructs. G4 represents the DNA binding domain (aa 1-147) of the yeast (S. cerevisiae) GAL4 protein. The starting and ending amino acids of individual ANCO-1 fragments are shown next to each fragment. (B) Transcriptional activities of G4-ANCO-1 constructs on a G4-dependent luciferase reporter MH100-tk-Luc. COS-7 cells were transfected with indicated plasmids and relative luciferase/β-galactosidase activities were determined. Fragments C (RD1 or repression domain-1) and J (RD2 or repression domain-2) show 6- and 9-fold repression activities relative to G4 alone, respectively. Fragment H (AD or activation domain) shows 12-fold activation relative to G4 alone. (C) Transcriptional activities of G4-ANCO-1 K (G4-ANCO-1F or full-length), L (ΔRD2), and M (AD + RD2) constructs, in comparison to G4 and ANCO-1 H and J fragments. Fold-repression of G4 reporter activity in comparison to G4 alone is represented. (D) Dose-dependent repression of the G4 reporter by G4-ANCO-1 J (RD2) fusion. Fold-repression relative to G4 alone is shown. Expression of G4-ANCO-1 J (RD2) fusion (indicated by an arrow at right) was detected by Western blot with anti-G4 antibody (Santa Cruz Biotechnology, Inc.). Asterisk indicates a nonspecific signal in all samples detected by the G4 antibody. The amounts of plasmid DNA used in each transfection are shown at bottom.

Fig. 2. ANCO-1 AD Domain Activates Transcription in Yeast

Yeast (S. cerevisiae) Y190 cells were transformed with plasmids encoding the indicated G4 fusion proteins or the empty vector pGBT9. Colony X-Gal filter assay (A) and liquid ONPG assay (B) were conducted to measure β-galactosidase activity as described in Materials and Methods. The control pGBT9 vector, ANCO-1 Ank (aa 126-295) and C (aa 2369-2663) domains had no detectable activity, while the RAC3.1 [12] and ANCO-1 AD (aa 1851-2145) activated lacZ gene expression strongly. (C) Schematic representation of G4-ANCO-1 AD sub-fragments fusions. Five ANCO-1 AD sub-fragments (A through E) are shown with indicated starting and ending amino acids beside each fragment. G4, Gal4 DNA binding domain. (D) Transcriptional activities of G4-ANCO-1 AD sub-fragments in yeast cells. Y190 cells were transformed with indicated expression vectors and average β-galactosidase activities were determined from three colonies. The E fragment 9aa 2076-2145) possesses the most transcriptional activation function. (E) Transcriptional activation by the G4-ANCO-1 AD sub-fragments in mammalian cells. COS-7 cells were transiently transfected with the G4 empty vector or G4-ANCO-1 AD sub-fragments and relative luciferase activities were determined from three independent transfections.

Fig. 2. ANCO-1 AD Domain Activates Transcription in Yeast

Yeast (S. cerevisiae) Y190 cells were transformed with plasmids encoding the indicated G4 fusion proteins or the empty vector pGBT9. Colony X-Gal filter assay (A) and liquid ONPG assay (B) were conducted to measure β-galactosidase activity as described in Materials and Methods. The control pGBT9 vector, ANCO-1 Ank (aa 126-295) and C (aa 2369-2663) domains had no detectable activity, while the RAC3.1 [12] and ANCO-1 AD (aa 1851-2145) activated lacZ gene expression strongly. (C) Schematic representation of G4-ANCO-1 AD sub-fragments fusions. Five ANCO-1 AD sub-fragments (A through E) are shown with indicated starting and ending amino acids beside each fragment. G4, Gal4 DNA binding domain. (D) Transcriptional activities of G4-ANCO-1 AD sub-fragments in yeast cells. Y190 cells were transformed with indicated expression vectors and average β-galactosidase activities were determined from three colonies. The E fragment 9aa 2076-2145) possesses the most transcriptional activation function. (E) Transcriptional activation by the G4-ANCO-1 AD sub-fragments in mammalian cells. COS-7 cells were transiently transfected with the G4 empty vector or G4-ANCO-1 AD sub-fragments and relative luciferase activities were determined from three independent transfections.

Fig. 3. Multiple Regions Are Involved in RD2-meidated Repression

(A) Schematic representation of G4-ANCO-1 RD2 sub-fragments. The RD2 domain (aa 2369-2663) is subdivided into four smaller fragments (A through D). Each fragment is fused individually or in combination with Gal4 DNA binding domain (G4). The starting and ending amino acids are indicated beside each fragment. (B) Luciferase reporter assay showing repression activity of each RD2 sub-fragment. COS-7 cells were transfected with indicated G4-ANCO-1 RD2 sub-fragments. Normalized luciferase activities were determined from three independent transfections.

Fig. 4. Silencing of ANCO-1 by siRNA Enhances PR Transcriptional Activity

(A) ANCO-1 shRNA sequence and its predicted hairpin structure. The ANCO-1 shRNA targets nucleotide 458 to 478 of the ANCO-1 cDNA (corresponding to aa 57 to 63). The siRNA sequences that targets ANCO-1 mRNA are shown in red. (B) ANCO-1 shRNA inhibits expression of ANCO-1 in transfected cells. HeLa cells were transfected with indicated plasmids and analyzed by Western blot with anti-HA antibody. The ANCO-1 shRNA specifically inhibited the expression of HA-ANCO-1N but not HA-DaxxF. (C) Effects of ANCO-1 knock down on PR-mediated transcription. HeLa cells were transiently transfected with hPR-B, MMTV-luciferase reporter, and the ANCO-1 shRNA expressing vector or pSUPER vector as control. Progesterone (0.1 nM) was added at indicated hours before harvesting at 48 hours after transfection. (D) ANCO-1 shRNA enhances PR-mediated transcriptional activation in a concentration-dependent manner. Transfected cells were treated with progesterone (0.1 nM) for 6 hours before harvesting for luciferase assay.