Supplementary figures and tables

Mature erythrocyte membrane homeostasis is compromised by loss of

the GATA1-FOG1 interaction

Atsushi Hasegawa, Ritsuko Shimizu, Narla Mohandas and Masayuki Yamamoto

Figure S1. Macroscopic appearance of V205GR embryos at E18.5. V205GR-A (upper right)

and V205G-B (lower right) embryos at E18.5 were shown with their wild-type littermates,

respectively. Scale bars correspond to 5.0 mm.

Figure S2. Expression of GATA1 target genes in the rescued spleens. Semi-quantitative

RT-PCR was performed using V205GR-A and wild-type (WT) spleens at the age of 2 (P2) and 3

(P3) day-old pups. Sequences of the primers used were

5’-TGCACAATGTTAACAGGCCACT-3’ and 5’-ATGTCCATGTCTGTCCAGTGG-3’ for

Gata2, 5’-TCGCCGGAGACGCAGGCT-3’ and 5’-CCCAGTCCTTGTGCAGGA-3’ for Eklf,

5’-CTGTTGCTGACGGTTCTGG-3’ and 5’-GACAAGGCTGTTCTCATAGG for EpoR, and

5’-TCAGCAGAACAGGAACAGGT-3’ and 5’-GCTTTGACACTGGTATAGCT for Nf-e2 p45.

The other primers used in this study were described in supplemental Table 1.

Figure S3. Transient reporter assays for GATA1 on Slc4a1 and Aqp1 gene expression. (A, B)

Schematic illustration of reporter constructs. Reporter constructs were generated by inserting

minimal-promoter sequence (gray boxes) derived from pGL4.28 vector (Promega) into the

multiple cloning site of pGL4.10 vector (Promega), followed by the insertion of the region

encompassing GATA motifs in Slc4a1 (A) and Aqp1 (B) genes (filled boxes). Arrowheads

indicate GATA motifs. (C, D) Enhancer activity of GATA1-binding loci in Slc4a1 (C) and Aqp1

(D) genes. Reporter constructs (open bars) or reporter constructs whose GATA motifs were

mutated (filled bars) (both 260 ng) were transfected into 1×105 of HEK293T cells together with

0, 10, 20, 30 ng of pEF-GATA1 expression constructs using Lipofectamine 2000

(Invitrogen-Life Technologies) according to the manufacturer’s protocol. 10 ng of pRL-TK

(Promega) was co-transfected in every well to normalize the transfection efficiency. Total

amount of DNA transfected in each well was adjusted to 300 ng. Luciferase assays were

performed using Dual-Luciferase reporter assay system (Promega) according to the

manufacturer’s protocol. Luciferase activity of samples without pEF-GATA1 construct was set

to one (**: P < 0.001).

Figure S4. Spherocytic erythrocytes in adult V205GR-B mice. Scanning electron microscopic

analysis of erythrocytes prepared from V205GR-B (right panel) and wild-type littermate (left

panel) at the age of 12 months. Scale bars correspond to 1 µm.

Figure S5. Expression of erythroid membrane proteins and genes. (A) Expression of erythroid

membrane proteins. Coomassie brilliant blue-stained SDS-PAGE profile of red blood cell ghosts.

Three wild-type and four V205GR-B mice (age of 6 to 9 months) were used. Loading volume

was adjusted by the original number of erythrocytes utilized for preparation of cell ghosts. (B)

Expressions of Spna1, Slc4a1, Aqp1 Alas2 and Hbb-b1 genes were analyzed by quantitative

RT-PCR in TER119+ erythroblasts derived from adult borne marrows of wild-type (open bars)

and V205GR-B (filled bars) mice at the age of 3 months. The values were represented as a mean

of three independent age-matched and genotype-matched mouse samples. The mean value of the

transcripts in corresponding wild-type mice was set to one. Primer sequences are described in

supplemental Table 2 (**: P < 0.001).

Figure S6. Characteristics of mature erythrocytes in V205GR mice. (A) Osmotic fragility

curves of erythrocytes taken from V205GR-B (black line) and wild-type (gray line) mice. 3

adult mice from each group were utilized for this experiment. Percent hemolysis was calculated

for each solution based on the absorbance value obtained from 0.2 % concentration. (*: P <

0.05; **: P < 0.01) (B) ROS levels in erythrocytes of wild-type (dotted line) and V205GR-B

(solid line) mice without (left panel) or with (right panel) addition of exogenous H2O2 (50 µM).

Table S1. Primer sequences for semi-quantitative RT-PCR. Gene Forward primer sequence Reverse primer sequence Gata1 5’-GCTGAATCCTCTGCATCAAC-3’ 5’-TAGGCCTCAGCTTCTCTGTA-3’ Zfpm1 (Fog1) 5’-CTCCCTGTGCAGGAACCAGT-3’ 5’-GGGTTTCTCTTCCGTCGCCG-3’ Spna1 (Spectrin-α1) 5’-ATCAGAGGTGCAAGCCAAAT-3’ 5’-CTTGGGTCAACTCCAAGAGC-3’ Spnb1 (Spectrin-β1) 5’-GGATCAAGGCCTTGGCAGATGA-3’ 5’-GGAATCGAAGGATGATGGTCCAG-3’ Ank1 (Ankyrin1) 5’-GCCGATGCTGCTACCAGCTTTC-3’ 5’-CTGAGACTGGGCATTGACATTGG-3’ Slc4a1 (Band3) 5’-CTCAGCCAGTCACAGAG-3’ 5’-GCTCCACATAGACCTGACC-3’ Epb41 (Band4.1) 5’-TCAGGAAGAACACAGAGAGGACCC-3’ 5’-TCTTTGGCAGAATCCAGCCATG-3’ Epb42 (Band4.2) 5’-GTGCTCCAACCCACACATTTCTG-3’ 5’-TGTGCGTTCAGCCTCGTTCTG-3’ Gypa (Glycophorin A) 5’-CTGTGGTGGCTTCAACTGTA-3’ 5’-CGATAATCCCTGCCATCACG-3’ Aqp1 (Aquaporin1) 5’-GTGTGTGGGAGCCATCGT-3’ 5’-CAGTACCAGCTGCAGAGTGC-3’ Hba-a1 (α-globin) 5’-GATTGGTGGCCATGGTGCTG-3’ 5’-ATGCACCGCGGGGGTGAAAT-3’ Hbb-b1 (β-major) 5’-TGGTTGTCTACCCTTGGACC-3’ 5’-GGTACTTGTGAGCCAGGGCA-3’ Alas2 5’-GTCCTGTGGAGGAATTGTGT-3’ 5’-GTTTTCCATCATCTGAGGGC-3’ Alad 5’-AAGGAGCCTGAGAGAGTGGGAGCA-3’ 5’-AGCTGCAGAGCCCTGTTCATCCTT-3’ Pbgd 5’-ACAACAGATCCTATTACAGCTTTT-3’ 5’-AAGGTTTCCAGGGTCTTTCCAATA-3’ Uros 5’-CTCTCCTTCCAACATCAC-3’ 5’-CTGATCTTCATAGTAGCTCT-3’ Urod 5’-GCTCCGTGGACCCTAATGACATA-3’ 5’-GCAAAGATGATCATGGGCACTGG-3’ Ppox 5’-TGGAGTCTGAAGTCTTGCCTGTC-3’ 5’-GGTGGGTTTGTTCAGCTTGGAAG-3’ Cpox 5’-TAGATGGCGTTGCCGACTTCACT-3’ 5’-GTGTGTGTTACCGTCAGCTTCCT-3’ Fech (Ferrochelatase) 5’-CCGACTGGTTTGGCAGTCCAAG-3’ 5’-GAAGGATTTAGTCTTCCTGCAGA-3’ G6pdx 5’-GGTGATGCCTTCCACCAAGCTG-3’ 5’-CCAGGTAGAATAGACGGTTGGCCTG-3’ Pklr (Pyruvate kinase) 5’-GCTTTAAGTGGGGCTCCAGGAG-3’ 5’-TCATGGGAGCCATGGGAGAAG-3’ Hprt 5’-GCTGGTGAAAAGGACCTCT-3’ 5’-CACAGGACTAGAACACCTGC-3’

Table S2. Primer sequences for quantitative RT-PCR Gene Forward primer sequence Reverse primer sequence Spna1 (Spectrin-α1) 5’-ATCAGAGGTGCAAGCCAAAT-3’ 5’-CTTGGGTCAACTCCAAGAGC-3’ Slc4a1 (Band3) 5’-TATGGGGTCGCCCACATCTAT-3’ 5’-AGGCCGAATCTGATCCTCGTA-3’ Aqp1 (Aquaporin1) 5’-GTGTGTGGGAGCCATCGT-3’ 5’-CAGTACCAGCTGCAGAGTGC-3’ Alas2 5’-GCAGCTATGTTGCTACGGTC-3’ 5’-GATGGGGCAGCGTCCAATAC-3’ Hbb-b1 5’-GGGAAAGGTGAACGCCGAT-3’ 5’-GAGGCTGTCCAAGTGATTCAG-3’ Gapdh 5’-GTCGTGGAGTCTACTGGTGTCTT-3’ 5’-GAGATGATGACCCTTTTGGC-3’

Table S3. Primer sequences used for ChIP-PCR assay Gene Forward primer sequence Reverse primer sequence Slc4a1 (Band3) 5’-TGCCTGGACCTGATAAGAGC-3’ 5’-ACCTCCTGGGCTCCAGAT-3’ Aqp1 (Aquaporin1) 5’-AGCCAGGCTCCCCACTCT-3’ 5’-CGCCCTGACACCTCTTATCT-3’