Ataxin-1 and Brother of ataxin-1 are components of the Notch signalling pathway

Abstract

Ataxin-1 (ATXN1), a causative factor for spinocerebellar ataxia type 1 (SCA1), and the related Brother of ATXN1 (BOAT1) are human proteins involved in transcriptional repression. So far, little is known about which transcriptional pathways mediate the effects of ATXN1 and BOAT1. From our analyses of the properties of BOAT1 in Drosophila and of both proteins in mammalian cells, we report here that BOAT1 and ATXN1 are components of the Notch signalling pathway. In Drosophila, BOAT1 compromises the activities of Notch. In mammalian cells, both ATXN1 and BOAT1 bind to the promoter region of Hey1 and inhibit the transcriptional output of Notch through direct interactions with CBF1, a transcription factor that is crucial for the Notch pathway. Our results suggest that, in addition to their involvement in SCA1, ATXN1 and BOAT1 might participate in several Notch-controlled developmental and pathological processes.

Figure 1

BOAT1 inhibits Notch activity in the Drosophila wings. (A) The BOAT1-mediated wing-vein phenotype is enhanced by Notch mutation. Wings were prepared from adult female flies with the indicated genotypes; hh-Gal4 line directs protein expression in the posterior compartment; GFP was used as a negative control; N¹ is a loss-of-function allele and Df(1)N-8 is a deficiency line; the positions of the LV5 and the PCV are marked with arrowheads and asterisks, respectively. (B) BOAT1 inhibits E(spl)mβ expression. Wing discs isolated from late third instar larvae with the indicated genotypes were immunostained with antibodies against β-galactosidase and BOAT1, and then DAPI stained; E(spl)mβ-lacZ is a reporter line; ap-Gal4 line directs protein expression in the dorsal compartment. The anterior (A), posterior (P), dorsal (D) and ventral (V) compartments are marked. (C) The BOAT1-mediated LV5 phenotype is suppressed by Su(H) mutation. Su(H)¹ is a loss-of-function allele; the positions of LV5 and PCV are marked with arrowheads and asterisks, respectively. ap, apterous; BOAT1, Brother of ataxin-1; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; hh, hedgehog; LV5, longitudinal wing vein 5; PCV, posterior crossvein; Su(H), Suppressor of Hairless; wt, wild type.

Figure 2

BOAT1 and ATXN1 are direct CBF1-interacting factors. (A) BOAT1 and ATXN1 associate with CBF1 in mammalian cells. HEK293 cells expressing the indicated FLAG-tagged proteins were subject to coimmunoprecipitation experiments using FLAG antibody. The precipitants were analysed with western blot using the indicated antibodies; HES1 is a human homologue of E(spl); SMRT and Sin3A were used as controls. (B) Yeast two-hybrid assays show that BOAT1 and ATXN1 interact with CBF1. AH109 yeast cells were transformed with the indicated pGBT9- and pGAD424-based plasmids; SD-Leu/-Trp are growth plates and SD-Leu/-Trp/-His/-Ade are selection plates. ATXN1(1–703) has basal transcriptional activity owing to its lack of the (704–816) region of ATXN1; SMRT construct was used as a positive control. ATXN1, ataxin-1; BOAT1, Brother of ataxin-1; IP, immunoprecipitation; HEK, human embryonic kidney; Su(H), Suppressor of Hairless; WCE, whole-cell extract.

Figure 3

BOAT1 and ATXN1 inhibit Notch activity in mammalian cells. (A) Notch disrupts the interaction between CBF1 and BOAT1 or ATXN1 in mammalian cells. Western blot analysis, using the indicated antibodies, was performed on precipitated products from HEK293 cells expressing the indicated proteins; HDAC3 and MAML1 were used as controls. (B,C) BOAT1 and ATXN1 inhibit the transcriptional activity of Notch and CBF1. Reporter assays were performed on cell extracts prepared from HEK293 cells transfected with 4XCBS-luc, along with the indicated expression plasmids. 4XCBS-luc is a Notch-responsive reporter; CFP-NICD1 is a fusion protein formed by NICD1 and CFP; VP16-CBF1 is a fusion protein formed by CBF1 and VP16; the reporter activity was normalized with β-galactosidase activity; western blots show the levels of expressed proteins used in reporter assays; tubulin was used as a loading control. Error bars indicate s.d. (n=3). (D) Notch activity increases as a result of reduced BOAT1 expression. Reporter assays were performed on HEK293 cells transfected with a 4XCBS-luc reporter along with the indicated siRNA. Western blot shows the reduced expression of BOAT1 by the tested Boat1 siRNA; for our reporter assays, we used a mixture of two Boat1 siRNAs. Error bars indicate s.d. (n=3). ATXN1, ataxin-1; BOAT1, Brother of ataxin-1; HDAC3, histone deacetylase 3; HEK, human embryonic kidney; MAML1, Mastermind-like 1; NICD, Notch intracellular domain; scr, scrambled RNA; siRNA, short-interfering RNA.

Figure 4

ATXN1 and BOAT1 are recruited to the Hey1 promoter. (A) C2C12 cells express both BOAT1 and ATXN1. Cell extracts prepared from C2C12 cells grown in growth medium (marked as day 0) and in DM at three time points were analysed by western blots using the indicated antibodies. Tubulin is a loading control; MHC is a marker for myogenesis. (B) Hey1 expression is reduced in C2C12 cells that were cultured in DM. Reverse transcription–PCR was performed on total RNA isolated from the four indicated C2C12 cell populations; the level of Hey1 transcript, a direct target gene of CBF1/Notch, is normalized with the level of GAPDH transcript. Error bars indicate s.d. (n=3). (C) BOAT1 and ATXN1 bind to the Hey1 promoter in differentiated C2C12 cells. Schematic diagram shows the Hey1 promoter region that harbours two CBF1-binding sites. Primers used for PCR are marked with arrows. Chromatin prepared from the indicated C2C12 cell populations was immunoprecipitated with the marked antibodies or the control IgG; SMRT antibody was used as a positive control and acetylated-histone H3 antibody was used as a marker for open chromatin and gene activation; the intensity of PCR products correlates with the amount of proteins recruited to (or the modifications at) the Hey1 promoter. (D) A proposed model for the roles of BOAT1 and ATXN1 in the Notch signalling pathway. Both BOAT1 and ATXN1 function as transcriptional corepressors of CBF1. When Notch signalling is not activated, BOAT1 and ATXN1, along with their associated SMRT and HDAC3, are recruited to the Hey1 promoter through their interactions with CBF1. When Notch is activated, upon the formation of the CBF1–NICD complex, BOAT1 and ATXN1 are dissociated from CBF1 and coactivators, such as MAML, are recruited by CBF1. ATXN1, ataxin-1; BOAT1, Brother of ataxin-1; DM, differentiating medium; HDAC3, histone deacetylase 3; Hey1, Hairy/enhancer-of-split related YRPW motif protein 1; IgG, immunoglobulin G; MAML, Mastermind-like protein; MHC, myosin heavy chain; NICD, Notch intracellular domain.