Online Supplemental Material
Supplementary Table 1. Knock-down of Cdx2 reduces the level of Cdx2, ATRX protein, and H3K9me3. The Western blot data in the Figure 2j was analyzed by the densitometry (NIH ImageJ program). Data are the mean of two experiments.
Cont / shCdx2 / shCdx2Cdx2 level / 1 / 0.75 / 0.41
ATRX level / 1 / 0.34 / 0.16
H3K9me3 level / 1 / 0.34 / 0.17
Protein expression (fold change)
Supplementary Figures
Supplementary Figure 1. ATRX immunoreactivity is elevated in the brain of HD (R6/2) mice. a, ATRX immunoreactivity is increased in cortex, striatum, and hippocampal regions including dentate gyrus (DG), CA1, and CA2. ATRX immunohistochemistry was performed using coronal sections from wild-type littermate (WT) and HD (R6/2) mice at 3 months of age. Scale bar: 30 mm. b, The colocalization of ATRX with H3K9me3 is increased in the nuclear foci of HD (R6/2) mice. c, Cyclohexamide (CHX) chase and Western analysis of ATRX protein. d, The CHX chase study shows an increase in the stability of ATRX protein between control (Q7/Q7) and HD (Q111/Q111) cells. CHX (10 mg/ml) was chased for the indicated period of time. Data are average ± SE of three experiments.
Supplementary Figure 2. ATRX promoter contains cis-elements for Cdx and Sp transcription factors. a, Alignment of human and mouse ATRX promoter sequence represents a homology in the proximal region containing the consensus Cdx binding element (Cdx BE) (TAAAT/CGTA). The occupancy of Sp1 (b) and Sp3 (c) to DNA of ATRX promoter is slightly increased in HD (R6/2) mice (n=3) compared to WT but there was no significant difference (n=3).
Supplementary Figure 3. ATRX deletion mutants show different cellular localizations and cellular effects. a, GFP-ATRX fusions containing different regions of ATRX (1-745, 1201-2492, and NLS1916-2492) were transfected into human neuroblastoma (SH-SY5Y) cells, and the cellular localization of the GFP-ATRX proteins was examined by fluorescence microscopy. N-terminal fragment of ATRX including PH domain is the dominant signal for the nuclear localization. Fusion of SV40 NLS to ATRX helicase domain leads to nuclear localization. The nucleus was counter stained with DAPI. b, Increased helicase activity by overexpressing the C-terminal helicase domain of ATRX leads to abnormal heterochromatin condensation and nuclear damage in mouse striatal cells. GFP-ATRX helicase domain (GFP-ATRX NLS1916-2492) including an SV40 NLS was transfected in Q7/Q7 and Q111/Q111 cells for 24hr. Arrows (white) indicate the spots of nuclear deformation and hollow structure. Cells were fixed and immunostained with anti-GFP (green) and H3K9me3 (red). The nucleus was counter stained with DAPI.
Supplementary Figure 4. Knock-down of ATRX reduces the H3K9me3 foci and pericentromeric condensation in the nucleus of Q111/111 cells as well as in Q7/Q7 cells. SiRNA for control (siCont) and siRNA for ATRX (siATRX) (each 400 ng/ml) were co-transfected with EGFP vectors for 48hr. Cells were fixed and immunostained with anti-H3K9me3 (red). The GFP-positive cells (green) were analyzed by confocal microscopy. The nucleus was counter stained with DAPI (blue).
5
Lee et al.