DOI: 10.1038/ncb2153 Figure S1 Ectopic expression of HAUSP up-regulates REST protein. (a) Immunoblotting showed that ectopic expression of HAUSP increased REST protein levels in ENStemA NPCs. (b) Immunofluorescent staining confirmed that ectopic expression of HAUSP up-regulated REST protein levels in nuclei. Differentiated ENStemA cells were transfected to express HA-HAUSP for 48 hours and then fixed for immunofluorescent staining with anti-ha (HA-HAUSP) and anti-rest specific antibodies. The cells expressing HA-HAUSP (green) displayed increased nuclear REST levels (red). The cells expressing HA-HAUSP with increased REST are indicated by arrows. Nuclei are counterstained with DAPI (blue). www.nature.com/naturecellbiology 1
Figure S2 REST knockdown in NPCs phenocopied HAUSP knockdown in neuronal differentiation. (a) Immunoblotting showed that reduced REST protein by shrna to the levels caused by HAUSP knockdown induced similar expression of the neuronal marker TUJ1. 17231 NPCs were infected with lentiviruses expressing shhausp, shrest or the non-targeting (NT) shrna for 72 hours, and harvested for immunoblotting. (b) Immunofluorescent staining confirmed that REST knockdown in NPCs induced neuronal differentiation similar to that induced by HAUSP knockdown. 17231 NPCs cultured on the coated cover glasses were infected with lentiviruses expressing shhausp, shrest or NT shrna for 5 days, and analyzed by the immunofluorescent staining with specific antibodies against Nestin (an NPC maker, in red) and TUJ1 (a neuronal differentiation marker, in green). (c) Quantified data from b indicated that HAUSP knockdown and REST knockdown in the NPCs induced similar fractions of cells expressing the neuronal marker TUJ1. Data are means ± s.d. [n = 3 (200 cells/ experiment)]. 2 www.nature.com/naturecellbiology
Figure S3 HAUSP knockdown reduced self-renewal potential of ENStemA NPCs, which was rescued by REST overexpression. (a) Targeting HAUSP with specific shrna reduced neurosphere formation of NPCs. ENStemA NPCs were infected with lentiviruses expressing shhausp or nontargeting (NT) shrna for 48 hours and allowed to form neurospheres for 9 days. HAUSP knockdown reduced neurosphere size and increased the attachment of neurospheres when cultured in suspension conditions. (b) Reduced HAUSP expression in ENstemA NPCs with two specific shrna (B2 and B5) significantly decreased neurosphere number. NPCs were targeted with shhausp or the control non-targeting (NT) shrna through lentiviral infection for 48 hours and subjected to neurosphere formation assay. The number of neurospheres were counted and compared between cells targeted with shhausp and control non-targeting (NT) shrna. Data are means ± s.d. (n = 4). (c, d) Ectopic expression of REST rescued the reduced neurosphere formation capacity caused by shhausp. 17231 NPCs were transfected with Flag-REST or vector control through lentiviral infection, infected with lentiviruses expressing shhausp (B5 clone) or control non-targeting (NT) shrna for 96 hours, and allowed to form neurospheres for 9 days. REST overexpression restored neurosphere size reduced by HAUSP knockdown (c) and significantly rescued the reduced neurosphere number caused by HAUSP knockdown (d). Data are means ± s.d. (n = 3). www.nature.com/naturecellbiology 3
Figure S4 HAUSP interacts with REST and stabilizes REST protein through deubiquitination. (a) Interaction of overexpressed HA-REST and Flag- HAUSP in 293T cells. Cell lysates of 293T cells expressing HA-REST and Flag-HAUSP were co-immunoprecipitated with anti-rest specific antibody and then immunoblotted with anti-flag (Flag-HAUSP) and anti- REST antibodies. (b) Reciprocal co-immunoprecipitation with anti-ha (HA-HAUSP). The lysate of 293T cells expressing HA-HAUSP and Flag- REST was immunoprecipitated with anti-ha (HA-HAUSP) antibody and then immunoblotted with anti-flag (Flag-REST) and anti-hausp specific antibodies. Anti-HA (HAUSP) pulled down Flag-REST, indicating interaction of the expressed HAUSP and REST. (c) HAUSP knockdown increased REST ubiquitination in NPCs. 15167 NPCs were infected with lentiviruses expressing HAUSP-targeting shrna or non-targeting (NT) control shrna for 48 hours, treated with the proteasome inhibitor MG132 for 6 hours, and immunoprecipitated (IP) with an anti-rest specific antibody or the control IgG, and immunobloted with anti-ubiquitin specific antibody. HAUSP knockdown increased REST ubiquitination in NPCs. (d) Ectopic expression of HAUSP suppressed REST ubiquitination. 15167 NPCs were transfected with Flag-HAUSP or vector control (V) through lentiviral infection for 36 hours and then subjected for analysis of REST ubiquitination. Forced expression of Flag-HAUSP reduced REST ubiquitination and increased REST protein levels. 4 www.nature.com/naturecellbiology
Figure S5 Potential conserved binding sequences (P/AXXS) on human, monkey and mouse REST proteins for the HAUSP-mediated deubiquitination are shown (red letters). The real consensus binding site on human REST (310-PYSS-313) required for the HAUSP-mediated REST deubiquitination was identified and indicated by an arrow. www.nature.com/naturecellbiology 5
Figure S6 HAUSP and β-trcp regulate REST ubiquitination. (a) Increased REST ubiquitination results in decreased REST protein levels during neuronal differentiation. ENStemA NPCs were induced by all-trans retinoic acid (RA) for two days. NPCs and differentiated cells were treated with MG132 for 6 hours and harvested for ubiquitination assessment. Cell lysates were immunoprecipitated with an anti-rest antibody (Rabbit) and immunoblotted with anti-ubiquitin and the anti-rest antibodies (mab). (b) HAUSP deubiquitinase counteracts the β-trcp ubiquitin E3 ligase to regulate REST ubiquitination. 293T cells were transfected to express Myc-REST and HA-ubiquitin in combination with HAUSP, Flag-β-TrCP or HAUSP + Flag-β-TrCP as indicated for 48 hours, treated with MG132 for 6 hours, and harvested for ubiquitination assessment. The cell lysates were immunoprecipitated with an anti-ha (HA-Ubiquitin) antibody and immunoblotted with an anti-rest specific antibody. Expression of HAUSP reduced REST polyubiquitination (lanes 1, 2 in top panel). Overexpression of β-trcp increased REST polyubiquitination (lanes 3, 2) but overexpression of HAUSP with β-trcp together abolished the increased REST ubiquitination induced by β-trcp overexpression (lanes 4, 3), indicating that both HAUSPmediated deubiquitination and the β-trcp-mediated ubiquitination play opposite roles to regulate REST protein at post-translational level. 6 www.nature.com/naturecellbiology
Figure S7 Lentiviral infection efficiency in neural stem/progenitor cells (NPCs) was determined with GFP-expressing lentiviruses. The infection efficiency in 15167 NPCs was examined with the lentiviruses expressing GFP. The majority (>93%) of NPCs were infected by the viruses and expressed GFP, when MOI of 3 was used for the infection. The uninfected cell without GFP expression is indicated by an arrow. www.nature.com/naturecellbiology 7
Figure S8 Uncropped images of immunoblots shown in the main figures (1a, 2a, 2b, 3e, 5a-f, 6c, 6d and 7b-7d). 8 www.nature.com/naturecellbiology