Supplementary information

TH2A is phosphorylated at meiotic centromere by Haspin

Masashi Hada, Jihye Kim, Erina Inoue, Yuko Fukuda, Hiromitsu Tanaka, Yoshinori Watanabe, and Yuki Okada

Supplemental figure legends

Fig. S1

Effect of Aurora kinase B/C and CDK1 inhibitor on pTH2A. (a, b) Immunofluorescent staining analysis of oocytes treated with AZD1152 (Aurora B/C inhibitor) for 4 hours (a) or Ro3306 (CDK1 inhibitor) for 6 hours (a). Indicated drugs were added to culture medium right after GVBD. Oocytes were prepared by chromosome spread (a) or whole mount (b). Representative images are shown (left panel). Relative immunofluorescent intensities are quantified as pTH2A/ACA (right panels). White arrowhead indicates magnified kinetochore (b).Drugs, time after GVBD, scale bars, antibodies, sample size number of oocytes, and p-values are as indicated.

Fig. S2

Uncropped images of western blot analysisshown in Fig. 3a (a) and 3c (b), respectively. The cropped area are shown by a red dashed box.

Fig. S3

In vitro kinase assay using recombinant proteins. Phosphorylated histones are distinguished as shifted bands in Phostag gel analysis (upper panel). Antibodies used for western blotting (lower panels) and lane number are as indicated.

Fig. S4

In vitro kinase assay using TH2A-HHK mutant and Bub1. Amino acid sequences of substrates are aligned (upper panel) and red line indicates epitope of anti-H2A-pT120 antibodies [1]. Western blot analysis is shown (lower panel) and bands corresponding to FLAG-tagged histone, endogenous H2A, phosphorylated, and unphosphorylated form of FLAG-tagged histone are indicated.

Fig. S5.

Validation of off-target effects.The potential off-target loci were searched with BLAST. The three genes with the highest sequence similarities are indicated by red lines (upper panel). These genes were amplified from the genomic DNA of Th2aTA/TA mice and sequenced (lower panel). Similarities to reference sequences obtained from NCBI database are indicated. Red and green shading indicate nucleotides matching the gRNA1 and gRNA2 sequences, respectively.

Fig. S6

Th2aTA/TA mice show no obvious defect for spermatogenesis. (a)Immunofluorescent staining of seminiferous tubules in Stage I-III with peanut agglutinin (PNA). Mouse genotype, antibodies used and scale bars are as indicated. Roman numerous, spermatogenic stage in seminiferous tubule; white boxes, magnified area; white dashed line, periphery of seminiferous tubule. (b) Comparison of testis sizebetween Th2a+/+ (black bars) and Th2aTA/TA mice (red bars). Mean testicular size in each group, sample size, andp-values are as indicated (not significant [n.s.])..

Fig. S7

Reduction of Rec8 in aged oocytes. Images in upper panels were obtained from young oocytes, those in lower panels from aged oocytes. Antibodies used, time postGVBD, and scale bars are as indicated.

Fig. S8

pTH2A is dependent on expression level of Haspin. One μg or four μg plasmids encoding 3HA-hHaspin was transfected in FLAG-TH2A overexpressing HEK293T cells. Star indicates bands corresponding endogenous hHaspin. Abbreviation; TA: TH2A-T127A mutant.

Fig. S9

Procedure for quantifying whole-mount immunostaining data for oocytes. The region of interest (ROI), corresponding to a square measuring approximately 1.19 x 1.19 μm, is set to the “peak ACA signal” (red) within each slice image, and the mean signal intensity is obtained. The mean background signal intensity, taken manually next to ACA (blue), is then subtracted (IACA-back). Using the same ROI, the mean signal intensity obtained from each antibody (green) is quantified (Iantibodies-back). At least, 36 kinetochores were quantified in each individual oocyte. The mean value for all quantified kinetochores is plotted as the signal intensity of one oocyte.

Supplementary reference

1.Kawashima SA, Yamagishi Y, Honda T, Ishiguro K-i and Watanabe Y (2010) Phosphorylation of H2A by Bub1 Prevents Chromosomal Instability Through Localizing Shugoshin. Science 327:172-177. doi: 10.1126/science.1180189

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