a. RNF8-deficient testes contained less condensed late spermatids. PAS-hematoxylin staining of testes from 3-month old wild type (WT) and RNF8-deficient (KO) mice were performed. Stages of seminiferous epithelium cycle were determined according to the morphology of spermatocytes and round spermatids. Spermatids at different stages are shown. Pl, preleptotene; L, leptotene; Z, zygotene; P, packytene; D, diplotene; RS, round spermatids; ES, elongating spermatids. b. Defect of DNA condensation during step 13-16 of spermatid development was observed in RNF8-deficient testes. Typical electron microscopic images of spermatids at different steps are shown. See also Supplementary Figure S4.

a. Protamines failed to replace histones in RNF8-deficient sperm. Typical immunostaining images of sperm from wild type (WT) and RNF8-deficient (KO) mice are shown. Antibodies used are indicated. Prm1: protamine 1; Prm2: protamine 2. b. mRNA levels of transition proteins and protamines were similar in wild type and RNF8-deficient mice. Semi-quantitative RT-PCR was performed using testes cDNA from wild type (WT) and RNF8-deficient (KO) mice. Tnp1: transition protein 1; Tnp2: transition protein 2. GAPDH was used as internal control. c. Incorporation of transition proteins into chromatin was defective in RNF8-deficient mice. Western blots using testes from wild type (WT) and RNF8-deficient (KO) mice are shown. Both NETN-soluble and HCl-released chromatin-bound fractions were used. Antibodies used are indicated, and Plk1 and H3K27me3 were used as soluble and chromatin-bound fraction loading controls respectively. See also Supplementary Figure S6.

a. ub-H2A, ub-H2B and H4 K16 acetylation were decreased in RNF8-deficient MEFs. Western blots of chromatin-bound proteins from wild type (WT) and RNF8-deficient (KO) MEFs are shown. Antibodies used are indicated, and H4 was used as loading control. b. Depletion of RNF8 affects not only H2A and H2B ubiquitination, but also H4K16 acetylation. Western blots of chromatin-bound proteins from control siRNA or RNF8 siRNA-treated HeLa cells are shown. c. MOF is enriched in elongating spermatids. Typical immunostaining images of seminiferous tubules at similar stages from wild type (WT) and RNF8-deficient (KO) mice are shown. SC: spermatocytes; ES: elongating spermatids. Antibodies used are indicated. d.-e. Chromatin-associated MOF was decreased in both testes and MEFs from RNF8-defiecient mice. Western blot using wild type (WT) and RNF8-deficient (KO) testes (d) and MEFs (e) are shown. Both NETN-soluble and chromatin-bound fractions were used. Antibodies used are indicated. Plk1 and H3K27me3 were used as soluble and chromatin-bound fraction loading controls, respectively. f. Depletion of MOF affects H4K16 acetylation only, but not H2A and H2B ubiquitination. Western blots of chromatin-bound proteins from control siRNA or MOF siRNA-treated HeLa cells are shown.

a.-d. Ubiquitinated H2A and ubiquitinated proteins (FK2) were absent in the XY body of RNF8-deficient spermatocytes, while γH2AX and BRCA1 were intact. Typical immunostaining images of spermatocytes at pachytene stage are shown. SCP3 represents synapsed chromosomes and γH2AX marks the XY body. e. RNA polymerase II remained excluded from the XY body in RNF8-deficient spermatocytes. Typical immunostaining images of spermatocytes at pachytene stage are shown. f. Sex chromosome inactivation was maintained in RNF8-deficient mice. Real time PCR results of expression levels of several genes on sex chromosomes in purified spermatocytes are shown. The levels in WT mice were arbitrarily set to 1 and those in RNF8-deficient mice were normalized accordingly. See also Supplementary Figure S3.

a. Number of sperm was decreased in RNF8-deficient mice. Sperm from 3-month old wild type (WT) and RNF8-deficient (KO) mice were counted. Sperm from both cauda epididymis were included. 6 mice of each genotype were used, p<0.001. b. Abnormal rounded sperm heads were observed in RNF8-deficient mice. Upper, typical morphologies of sperm from wild type (WT) and RNF8-deficient (KO) mice with hematoxylin staining are shown; Lower, quantification of sperm with abnormal rounded head is shown. c. Majorities of sperm from RNF8-deficient mice were immotile. Sperm mobility of wild type (WT) and RNF8-deficient (KO) mice are compared. d. H&E staining of paraffin sections of cauda epididymis from wild type (WT) and RNF8-deficient (KO) mice are shown. On right are images with higher magnification showing details of sperm heads. e. Sperm were less condensed and contained residue cytoplasm. Typical electron microscopic images of sperm heads from wild type (WT) and RNF8-deficient (KO) are shown. See also Supplementary Figure S5.

a.-d. ub-H2A, ub-H2B and K4 K16 acetylation were decreased in elongating spermatids of RNF8-deficient mice. Typical immunostaining images of testes seminiferous tubules at similar stages from wild type (WT) and RNF8-deficient (KO) mice are shown. SC: spermatocytes; ES: elongating spermatids. Antibodies used are indicated, FK2: antimono/poly-ubiquitinated proteins. e.-h. Typical immunostaining images of individual elongating spermatids from wild type (WT) and RNF8-deficient (KO) mice are shown. i. ub-H2A, ub-H2B and H4 K16 acetylation were decreased in testes from RNF8-deficient mice. Western blots of chromatin-bound proteins using testes from wild type (WT) and RNF8-deficient (KO) mice are shown. Antibodies used are indicated, and H4 was used as loading control.