Cell Reports, Volume 20

Supplemental Information

CFP1 Regulates Histone H3K4 Trimethylation

and Developmental Potential in Mouse Oocytes

Chao Yu, Xiaoying Fan, Qian-Qian Sha, Hui-Han Wang, Bo-Tai Li, Xing-Xing Dai, LiShen, Junping Liu, Lie Wang, Kui Liu, Fuchou Tang, and Heng-Yu Fan

Supplementary information

SUPPLEMENTAL FIGURES

Figure S1. Levels of methylation on histone H3 at lysine-4 (H3K4) in mouse oocytes and

preimplantation embryos, Related to Figure 1. (A) Pattern of H3K4me3 (green) in oocytes

and preimplantation embryos. GV, germinal vesicle; NSN, non-surround nucleus; SN,

surrounded nucleus; MII, metaphase II; PB, polar body; ICM, inner cell mass. PN, pronucleus.

For each stage, at least 15 oocytes or embryos were observed with similar results. Scale bar,

10 μm. (B-C) Levels of mono- and di-methylated Histone H3 at lysine 4 (H3K4me1, B;

H3K4me2, C) in oocytes and preimplantation embryos. At least 12 oocytes or embryos were

observed for each stage. Scale bar, 10 μm.

Figure S2. Expression pattern of CFP1 in oocytes and preimplantation embryos, Related

to Figure 1. (A) Immunohistochemistry (IHC) showing expression pattern of CFP1 in

oocytes of different stages. Arrow heads indicate the nuclei of oocytes. Scale bar, 30 μm. (B)

Immunofluorescent staining results of CFP1 in oocytes and preimplantation embryos. For

each stage, at least 15 oocytes or embryos were observed with similar results. Scale bar, 10

μm. (C) Quantitative RT-PCR results showing Cxxc1mRNA levels in oocytes,

preimplantation embryos and some somatic tissues. The expression level in brain was set as

“1”. Error bars, S.E.M. (D) Western blot results showing CFP1 levels in oocytes and

preimplantation embryos. ERK1/2 was blotted as a loading control.

Figure S3. The effects of maternal CFP1 deletion on histone modification, Related to

Figure 2. (A-B) Immunofluorescent staining of H3K4me3 in WT and CFP1-deleted oocytes

with NSN (A) or SN (B) chromatin configurations. CFP1-deleted oocytes were injected with

mRNAs encoding for CFP1-GFP at 12 h before immunofluorescent staining. Scale bars, 10

μm. (C) Quantification of the H3K4me3 immunofluorescence signals by ImageJ in oocytes

shown in (A-B). Error bars, S.E.M. ** P<0.01 and *** P<0.001 by two-tailed Student’s t

tests. (D-F) Immunofluorescent staining of H3K4me1 (D), H3K4me2 (E) and H3K9me3 (F)

in GV oocytes derived from WT and Cxxc1fl/fl;Zp3-Cre females. Scale bars, 10 μm. For each

staining, at least 15 oocytes were observed with similar results. (G) Western blotting of

indicated proteins in GV oocytes derived from WT and Cxxc1fl/fl;Zp3-Cre females.

Figure S4. RNA-seq results of WT and CFP1-deleted samples, Related to Figure 3. (A)

Heatmap of spearman correlation coefficients between WT and CFP1-depleted oocytes and

zygotes. (B) Venn diagram summarizing the genes showed different expression patterns from

GV oocyte to the zygote stage in the WT samples. Blue circles reflect genes selected from the

FPKM results and pink circles indicate the genes selected by evaluated absolute mRNA copy

number results.

Figure S5. Maternal, but not zygotic CFP1-deletion, is crucialfor zygotic genome

activation, Related to Figure 4. (A) Illustration of the difference between maternal versus

zygotic Cxxc1 knockout. Green shades indicates the expression of mRNA and protein in

specific cell types. (B) Immunofluorescent staining of CFP1 in embryos derived from

Cxxc1+/– parents. For each stage, at least 35 embryos were analyzed. Scale bar, 10 μm. (C)

Quantification of the phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2)

immunofluorescence signals by ImageJ in embryos shown in Figure 4F. Error bars, S.E.M.

*** P<0.001 by two-tailed Student’s t tests. (D) Quantification of the H3K4me3

immunofluorescence signals by ImageJ in oocytes shown Figure 4I. Error bars, S.E.M. **

P<0.01 and *** P<0.001 by two-tailed Student’s t tests. (E) Quantification of the γH2AX

immunofluorescence signals by ImageJ in embryos shown in Figure 7C. Error bars, S.E.M. **

P<0.01 and *** P<0.001 by two-tailed Student’s t tests.

Figure S6. CFP1 is required for proper distributions of cytoplasmic organelles in

oocytes, Related to Figure 5. (A) Transmission electron microscopy (TEM) results of WT

and CFP1-deleted oocytes at MII stages. Scale bars, 2 μm. (B) Western blotting results

showing levels of indicated proteins in HeLa cells before and after CFP1 deletion. (C)

Distribution of indicate cytoplasmic organelle markers in HeLa cells before and after CFP1

deletion. Scale bar, 10 μm.

Table S1. Quality control of RNA-seq results (WT and CFP1-deleted oocytes and

zygotes), Related to Figure 3.

Table S2. Primer sequences, Related to Experimental Procedures.

Primer

name

Genes

targeted Application Sequences (5’-3’)

Cxxc1-F Cxxc1 Real-time PCR (219bp)

5’-GAACGACAGTGATGACACAGATCT-3’

Cxxc1-R 5’-ACCTGGAGCCGCTTACAATA-3’

Tle6-F Tle6 Real-time PCR (204bp)

5’-CTGTTCAACAGCAGGAAGAGG-3’

Tle6-R 5’-CTCAGTCATGTCGAAGCATCTG-3’

Nlrp5-F Nlrp5 Real-time PCR (196bp)

5’- GCATTGTCATTGGCCATCTC-3’

Nlrp5-R 5’- TGACAAACTCAACTTCCTCCAG-3’

Khdc3-F Khdc3 Real-time PCR (236bp)

5’- CGTGGAGGCCACTGATACTAG-3’

Khdc3-R 5’- GCTGAACTATGACATAGCTGCC-3’

Ooep -F Ooep Real-time PCR (213bp)

5’-CTCAGAAATAGAGTGGATGTGCC-3’

Ooep -R 5’-GAGGAGGCACGAATTTTCAA-3’

Padi6-F Padi6 Real-time PCR (241bp)

5’-AGTGTATCAGCCTGAACCGC-3’

Padi6-R 5’-AGGTGCCATTGATTTTGGG-3’

Gapdh -F Gapdh Real-time PCR (181bp)

5’- ACACTGAGGACCAGGTTGTCTC-3’

Gapdh -R 5’- TACTCCTTGGAGGCCATGTAG-3’

Cxxc1-1

Cxxc1

Genotyping

WT: 199bp; fl: 230bp;

null: 330bp

5’- CGAGAGATGAAGAGGAGCCA-3’

Cxxc1-2 5’- CTGAATGGTCCTAGAACCTC-3’

Cxxc1-3 5’- CTCACCGAGGAAGGGAAGTAC-3’

Cre-1 Zp3-Cre;

Genotyping (370bp)

5’-AGCTGTGTCTGCGTGGGACTGA-3’

Cre -2 5’-AGCTGTGTCTGCGTGGGACTGA-3’

Table S3. Antibody information, Related to Experimental Procedures.

Protein

name

Manufacture

(catalogue

number)

Applications

(working

dilution)

Website Link*

H3K4me3 Abcam

(ab8580)

IF (1:400)

WB (1:1000)

http://www.abcam.com/histone-h3-tri-methyl-k4-antibody-chip-gra

de-ab8580.html

H3K9me3 Abcam

(ab8898)

IF (1:400) http://www.abcam.com/Histone-H3-tri-methyl-K9-antibody-ChIP-

Grade-ab8898.html

FITC-α-T

ubulin

Sigma

(F2168)

IF (1:500) http://www.sigmaaldrich.com/catalog/product/sigma/f2168?lang=z

h&region=CN

RNA

PolII

(pS2)

Abcam

(ab5095)

IF (1:40000) http://www.abcam.cn/rna-polymerase-ii-ctd-repeat-ysptsps-phosph

o-s2-antibody-chip-grade-ab5095.html

CNOT7 Santa Cruz

(sc-101009)

WB (1:100) http://www.scbt.com/datasheet-101009-cnot7-18w-antibody.html

ERK1/2 Santa Cruz

(sc-94)

WB (1:1000) http://www.scbt.com/datasheet-94-erk-1-k-23-antibody.html

p-ERK1/2 Cell Signaling

(9101)

IF (1:400) http://www.cellsignal.com/products/primary-antibodies/phospho-p

44-42-mapk-erk1-2-thr202-tyr204-antibody/9101

BTG4 Abcam

(ab206914)

WB (1:500); http://www.abcam.com/btg4-antibody-eprzju-21-ab206914.html

CFP1 Abcam

(ab198977)

WB (1:500);

IHC (1:100):

IF (1:100)

http://www.abcam.com/cgbp-antibody-epr19199-ab198977.html

HSP60 Abcam

(ab46798)

IF (1:100) http://www.abcam.com/hsp60-antibody-ab46798.html

TOP2B Abcam

(ab109524)

IF (1:200) http://www.abcam.com/Topoisomerase-II-beta-antibody-EPR5377-

ab109524.html

CREST Fitzgerald

Industries

IF (1:100) https://www.fitzgerald-fii.com/crest-antibody-70r-21494.html

International

(70R-21494)

MVH Abcam

(ab13840)

IHC(1:500) http://www.abcam.com/ddx4-mvh-antibody-primordial-germ-cell-

marker-ab13840.html

DDB1 Epitomics

(3821-1)

WB (1:10000) http://www.epitomics.com/products/product_info/2283

5mC Calbiochem

(NA81)

IF (1:500) http://www.emdmillipore.com/US/en/product/Anti-5-Methylcytosi

ne-Mouse-mAb-(162-33-D3),EMD_BIO-NA81

5hmC Active motif

(39769)

IF (1:400-800) https://www.activemotif.com/catalog/details/39769/5-hydroxymeth

ylcytidine-5-hmc-antibody

H3K4me1 Cell Signaling

(5326P)

IF (1:200) http://www.cellsignal.com/products/primary-antibodies/mono-meth

yl-histone-h3-lys4-d1a9-xp-rabbit-mab/5326

H3K4me2 Cell Signaling

(9725P)

IF (1:200) http://www.cellsignal.com/products/primary-antibodies/di-methyl-

histone-h3-lys4-c64g9-rabbit-mab/9725

GM130 Abcam

(ab52649)

IF(1:200) http://www.abcam.com/gm130-antibody-ep892y-cis-golgi-marker-

ab52649.html

Calnexin Cell Signaling

(2679P)

IF (1:100) http://www.cellsignal.com/products/primary-antibodies/di-methyl-

histone-h3-lys4-c64g9-rabbit-mab/9725

pH2AX Cell

Signalling

(9718S)

IF(1:200);

WB(1:1000) http://www.cellsignal.com/products/primary-antibodies/phospho-hi

stone-h2a-x-ser139-20e3-rabbit-mab/9718

*The validation of using these antibodies in mouse is provided on the manufacturer’s website.

SUPPLEMENTARY EXPERIMENTAL PROCEDURES

Animals. Mice were maintained in a controlled environment of 20oC‒22oC, with a 12 h/12 h

light/dark cycle, 50%‒70% humidity, and food and water provided ad libitum. All mice used

were handled with care and according to the Animal Research Committee guidelines of

Zhejiang University. Cxxc1fl/fl mouse strain has been engineered and described previously

(Cao et al., 2016). They were bred over the well characterized Zp3-Cre transgenic mice,

which allow CRE-mediated recombination specifically in the growing oocytes shortly after

primordial follicle activation (Yu et al., 2013). All mice have a C57BL/6J genetic

background. Primers used for genotyping are listed in Table S2. Sample size in each

experiment was indicated in related figure legends.

In vitro oocyte maturation. Fully grown oocytes were obtained from the ovaries of

3–4-week-old female mice 48 h after intraperitoneal injection of 5 IU of pregnant mare serum

gonadotropin (PMSG, Ningbo Sansheng Pharmaceutical Co., Ltd., China). GV oocytes were

released by puncturing antral follicles with a fine needle on the stage of a dissecting

microscope. For in vitro maturation, oocytes were washed and cultured in IBMX-free M16

medium (Sigma-Aldrich) for various periods of time at 37 oC in 5% CO2 atmosphere.

To obtain MII oocytes, 5 IU of human chorionic gonadotropin (hCG, Ningbo Sansheng

Pharmaceutical Co., Ltd., China) was administered 48 h after PMSG injection. Mice were

humanly sacrificed at 16 h after hCG injection, and oocytes were collected from the oviducts

and released into a hyaluronidase/M2 solution for removal of the cumulus cells.

In vitro fertilization. Adult WT males (3-6 months old) were sacrificed and epididymides

were dissected into prewarmed human tubal fluid (HTF) and then transferred to a 200-μl HTF

drop covered with mineral oil and capacitated for 1 h in the incubator before adding oocytes.

The cumulus-oocyte complexes (COCs) were added directly to the sperm suspension and

incubated for 6 h at 37 oC, 5% CO2 in HTF.

Embryo collection and culture. Mice were superovulated and fertilized by WT males, and

checked for the presence of vaginal plugs. E0.5 embryos (zygotes) were collected from the

oviducts and released into a hyaluronidase/M2 solution for dissociation. E1.5 (2-cell)

embryos were flushed out of the oviducts, and E3.5 embryos (blastocysts) were flushed out of

the uteri. In some experiments the obtained embryos were further cultured at 37 oC, 5% CO2

in KSOM (Millipore).

Immunofluorescent staining. Oocytes were fixed in 4% paraformaldehyde in PBS for 30

min at room temperature and permeabilized for 15 min in 0.2% Triton X-100 in PBS.

Antibody staining was performed using standard protocols described previously (Yu et al.,

2015). The antibodies used are listed in Table S3. Imaging of embryos following

immunofluorescence was performed on a Zeiss LSM710 confocal microscope. The signal of

fluorescence is quantified by ImageJ.

DNase I sensitivity. GV oocytes were pre-extracted in ice-cold solution (50 mMNaCl, 3 mM

MgCl2, 0.5% Triton X-100, 300 mM sucrose in 25 mM HEPES, pH 7.4) for 5 min. The

oocytes were then incubated with different concentrations (0.1, 0.01 or 0.001 U/μL) of DNase

I (NEB) for 5 min at 37 oC in the same buffer without Triton X-100 and fixed for 10 min in

2% PFA/PBS at room temperature. TUNEL assay was done using Click-iT TUNEL Alexa

Fluor Imaging Assay Kit (Life Technologies, C10245) according to manufacturer’s

instructions.

In vitro mRNA synthesis and microinjection. The cDNA sequence of human histone H3.1,

H3.2, H3.3, and H2AX was subcloned into pGEM-T Easy Vector (Promega) and Flag-epitope

tag was added at N-terminal tail by PCR amplification. In vitro mRNA synthesis and

microinjections were performed as described previously (Yu et al., 2016). Briefly, plasmids

were liberalized by appropriate restriction enzymes. 5’-capped mRNAs were synthesized

using Sp6 or T7 mMessage mMachine kit (Thermofisher) and poly (A) tails were added by a

Poly (A) Tailing Kit (Thermo fisher). Finally, the DNA template was removed by Turbo

DNase treatment and synthesized mRNA was extracted by phenol/chloroform extraction

followed by isopropanol precipitation. Microinjection was performed under an inverted

microscope (Eclipse TE200; Nikon) using amicromanipulator and microinjector (Eppendorf).

Approximately 10 pl synthetic RNA (~200 μg/ml) was microinjected into the cytoplasm of

oocytes or zygotes.

Histological analysis. Ovaries were collected and fixed in formalin overnight, processed, and

embedded in paraffin using standard protocols. Ovaries were serially sectioned at 5 μm and

stained with hematoxylin and eosine (H&E). IHC was performed using standard protocols.

The antibodies used are listed in Table S3.

Chromosome spreading and immunofluorescence. Zona pellucida-free oocytes were fixed

in a solution containing 1% paraformaldehyde, 0.15% Triton X-100, and 3 mM DTT on glass

slides for 30 min and air dried. Immunofluorescent staining was performed as in oocytes

described above.

Transmission electron microscopy. Ovaries were fixed in 2.5 % glutaraldehyde and 1%

osmic acid. After that, the ovaries were dehydrated and incubated in the embedding medium

overnight. The next day, they were embedded in the embedding medium and heated at 65 °C

for 2–3 days. The samples were cut into sections of 70–90 nm on an ultramicrotome (Leica).

The sections were stained by a lead citrate solution and analyzed under an electron

transmission microscope (HT7700, Hitachi).

Detection of transcription in oocytes and zygotes. In order to detect general transcription,

oocytes or early embryos were transferred into medium containing 1mM of 5-ethynyl uridine

(EU, Life Technologies) 6 hours after insemination. Zygotes were collected at 10 hours after

insemination. EU staining was done according to manufacturer’s instruction.

Western blotting. 100 oocytes or embryos were lysed in 2-mercaptoethanol containing

loading buffer and heated at 95°C for 5 minutes. SDS-PAGE and immunoblots were

performed following standard procedures using a Mini-PROTEAN Tetra Cell System

(Bio-Rad, Hercules, CA). The antibodies used are listed in Table S3. The signal of Western

blotting results is quantified by ImageJ.

Real-time RT-PCR. Total RNA was extracted from oocytes using the RNeasy Mini kit

(Qiagen) according to the manufacturer’s instruction, followed by reverse transcription (RT)

using Superscript RT kit (Bio-Rad). Quantitative RT-PCR was performed using a Power

SYBR Green PCR Master Mix (Applied Biosystems, Life technologies) with ABI 7500

Real-Time PCR system (Applied Biosystems) using primers listed in Table S2.

RNA-seq library preparation and gene expression level analysis. Oocytes were collected

from 3-week-old and 3-month-old WT and Cxxc1fl/fl;Zp3-Cre female mice. Zygotes were

collected from WT and Cxxc1fl/fl;Zp3-Cre mice after superovulation and mating with WT

males (60 oocytes or zygotes per sample). After picking the cells into 350μl lysis buffer

supplied in RNeasy micro kit (QIAGEN), 3*107 ERCC molecules were added into the lysis

buffer. Total RNA was extracted with the RNeasy micro kit (QIAGEN) following the manual.

Libraries were prepared with NEBNext® Ultra RNA Library Prep Kit for Illumina. The

libraries were sequenced on illumina platform with 150bp pair-end reads. We used a

perlscript to filter out low quality reads and then the clean reads were mapped to mouse

genome mm10 with TopHat (Version 2.0.6). Gene expression levels were calculated and

normalized to fragments Per Kilobase of transcript per Million mapped reads (FPKM) using

cuffquant and cuffnorm.

Absolute mRNA copy number calculation with ERCC molecules. The ERCC mix

(External RNA Controls Consortium, life) consists of 92 kinds of RNA transcripts with

different concentrations for each RNA transcript. When mapping the high through-put

sequencing reads, we added these RNA sequences to the reference mm10 genome. After

normalizing expressions for all genes, linear regression was used to analyze between each

log10FPKM and the corresponding log10(ERCC transcripts copy number) in each sample.

Only ERCC transcripts detected with FPKM more than 0.1 were taken for regression analysis.

The absolute total mRNA copy number was calculated by applying the total FPKM to the

regression function for each sample. In the same way, the absolute copy number for each gene

was calculated with the regression function and genes with FPKM=0 were manually set to 0

copy.

Expression levels comparison between WT and CFP1-deleted zygotes. The genes which

were at least detected in all samples with FPKM>0.1 were arranged according to their mean

expression in wild-type GV oocyte samples. And then these genes were divided into 10 or 5

equal-sized bins. The averaged mRNA copy number of each gene within replicates was

calculated and then the relative expressions for each gene were calculated as copy number in

each replicate to the average number of WT_GV oocytes. Afterwards, the embryo samples

were plotted with boxplot in R. In the same way, the relative expression ratio of zygote to GV

oocyte for each filtered gene in the bins was plotted. We used edgeR in R (version 3.2.5) to

search for genes differentially expressed between WT and CFP1-deleted oocytes and

identified genes up-regulated and down-regulated in CFP1-deleted oocytes as

FC[wild-type/CFP1-deleted]<=0.5 or >=2, p-value<0.05, FDR<0.05.

Category of genes during oocyte-to-zygote development and GSEA analysis. We firstly

find out the differentially expressed genes (DEGs) between WT GV oocytes and zygotes

using edgeR. Genes with FC[zygote/GV] <=0.5, p-value<0.05, FDR<0.05 were recognized as

GV-Zygote decreased genes, while those with FC[zygote/GV] >=2, p-value<0.05, FDR<0.05

were GV-Zygote increased genes. GV-Zygote stable genes were those with log2(FC

[wild-type/CFP1-deleted]) between -0.5 to 0.5. The table of converted mRNA abundance

which contains both WT and Cxxc1fl/fl;Zp3-Cre GV oocyte and zygote samples were uploaded

to the GSEA software, and the GV-Zygote decreased genes were uploaded as gene set

database. We set phenotype as permutation type, so the significant FDR cutoff could be 0.25

according to the GSEA user guide.

Statistical analysis. The experiments were randomized and were performed with blinding to

the conditions of the experiments. No statistical method was used to predetermine sample

size. Informed content was obtained from all subjects. Results are given as means ± S.E.M.

Each experiment included at least three independent samples and was repeated at least three

times. Results for two experimental groups were compared by two-tailed unpaired Student’s t

tests. Statistically significant values of P<0.05, P<0.01, and P<0.001 are indicated by asterisks

(*), (**), and (***), respectively.

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