Autism-associated protein POGZ controls ESCs and ESC neural induction by association with esBAF

ES cell culture

Mouse embryonic stem cells (mESCs) R1 were maintained in Dulbecco’s Modified Eagle Medium (DMEM, BI, 01–052-1ACS) containing 20% knockout serum replacement (KSR, Gibco, 10,828,028), 1 mM sodium pyruvate (Sigma, S8636), 2 mM L-Glutamine (Sigma, G7513), 1,000 U/mL leukemia inhibitory factor (LIF, Millipore, ESG1107), and penicillin/streptomycin (Gibco, 15,140–122) at 37 °C with 5% CO2. Cells were routinely propagated by trypsinization and replated every 3–4 days, with a split ratio of 1:6.

Embryoid body formation

ESCs differentiation into embryoid bodies (EBs) was performed in attachment or suspension culture in medium lacking LIF and knockout serum replacement (KSR), as described previously [30].

Generation of Pogz−/− ESCs

Pogz−/− mESCs were generated by CRISPR/Cas9 technology. Briefly, we designed gRNAs on exon 2 of the Pogz gene by using the online Web site http://crispr.mit.edu/. The gRNA sequence is 5′-CGACCTGTTTATGGAATGTGAGG-3′. The guide sequence oligos were cloned into pSpCas9(BB)-2A-puro containing Cas9 and the sgRNA scaffold [31]. The plasmid without addition of the gRNA was used as control. The plasmids containing the sgRNA sequence or control plasmids were transfected into mESCs using Neon transfection system according to the manufacturer’s instructions. 48 h later, transfected mESCs were selected with 1 µg/mL puromycin for 4–5 days. Most of cells died within 4 days, and the remaining cells were allowed to proliferate. After 10–15 days selection, the single colony was picked up and passaged into 24-well plates.

DNA from single colonies from the passaged cells was extracted and used for genotyping by PCR. The PCR primer was designed to amplify 200–300 bp on either side of the sgRNA sites. The PCR products were ligated into pGEM-T vector and sequenced to determine the genotype of each single colony. We have successfully generated three mutant alleles (1 bp deletion, 7 bp insertion, and 284 bp insertion in exon 2 of the Pogz gene). Three homozygous Pogz−/− ESC lines were established: Mutation 1 (Mut1, 1 bp deletion), Mutation 2 (Mut2, 284 bp insertion), and Mutation 3 (Mut3, 7 bp insertion). One heterozygous ESC line (same allele as Mut1) was also included.

Generation of 3 × Flag-POGZ restoring Pogz−/− mESC cell lines

The full-length Pogz cDNA (NM_172683.4) was amplified by PCR and then cloned into pCMV-3 × Flag vector. The full-length Pogz cDNA sequence containing N-terminal 3 × Flag sequence was subcloned into pCAG-IRES-Puro vector. To make stable transgenic cells, Pogz−/− mESCs were transfected with pCAG-IRES-Puro-3 × Flag-Pogz vector using Lipofectamine 2000 (Gibco, 11,668,019). 48 h later, cells were selected by 1 μg/mL puromycin. After 4–5 days drug selection, cells were expanded and passaged. Western blot assay was performed to confirm the transgenic cell line using FLAG antibodies.

RNA preparation, RT-qPCR and RNA-seq

Total RNA from mESCs and ESC-derived EBs was extracted with a total RNA kit (Omega, R6834-01). A total of 1 μg RNA was reverse-transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China, AT341). Quantitative real-time PCR (qRT-PCR) was performed using the TransStart® Tip Green qPCR SuperMix (TransGen Biotech, China, AQ-141). All experiments were repeated at least three times. The relative gene expression levels were calculated based on the 2−∆∆Ct method. Data were shown as means ± S.D. The Student’s t test was used for the statistical analysis. The significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

For RNA-seq, control and Pogz mutant ESCs, and ESC-derived EBs were collected in tubes preloaded with Trizol (TransGen). RNAs were quantified by a NanoDrop instrument and were submitted to BGI Shenzhen (Wuhan, China) for mRNA enrichment, library construction, and sequencing (BGISeq 50SE). RNA-seq was performed in at least two technical repeats and two biological replicates, for control and mutant ESCs. P < 0.05 and a Log2 fold change > 1 was deemed to be differentially expressed genes (DEGs).

Protein extraction and Western blot analysis

For protein extraction, ESCs or HEK293T cells were harvested and lysed in TEN buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Na deoxycholate, supplement with Roche cOmplete Protease Inhibitor). The lysates were quantified by the Bradford method, and equal amount of proteins were loaded for Western blot assay. Antibodies used for WB were POGZ (Abcam, ab171934), anti-BRG1 (Proteintech, 21,634–1-AP), anti-HP1gamma (Proteintech, 11,650–2-AP), anti-CHD4 (Proteintech, 14,173–1-AP), anti-OCT4 (Proteintech, 60,242–1-Ig), anti-FLAG (F1804/F3165, Sigma, 1:1000), anti-MYC antibody (TransGen Biotech, HT101), and anti-HA (Abbkine, A02040, 1:1000). Briefly, the proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% (w/v) non-fat milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with the primary antibodies. Then, the membranes were incubated with a HRP-conjugated goat anti-rabbit IgG (GtxRb-003-DHRPX, ImmunoReagents, 1:5000) and a HRP-linked anti-mouse IgG (7076S, Cell Signaling Technology, 1:5000) for 1 h at room temperature. The GE ImageQuant LAS4000 mini luminescent image analyzer was used for photographing. Western blot experiments were repeated at least two times.

Quantification of Western blot bands was performed by ImageJ software, according to the Web site: https://imagej.nih.gov/ij/docs/guide/146-30.html. Briefly, the rectangle tool was selected and used to draw a box around the lane, making sure to include some of the empty gel between lanes and white space outside of the band. All lanes were selected one by one. Once all lanes are defined, go to Plot lanes to generate histograms of each lane. Then, the relative values were calculated by dividing each value by the control lane. The value of the control bands was set at 1.

Co-immunoprecipitation assay (co-IP)

Co-IPs were performed with the Dynabeads Protein G (Life Technologies, 10004D) according to the manufacturer’s instructions. Briefly, 1.5 mg Dynabeads was conjugated with antibodies or IgG overnight at 4 °C. Antibodies were used are: 10 μg IgG (Proteintech, B900610), or 10 μg anti-POGZ antibody, or 10 μg anti-FLAG antibody (Sigma, F3165/F1804), or 10 μg anti-HA antibody (Abbkine, A02040) or 10 μg anti-MYC antibody (TransGen Biotech, HT101). The next day, total cell lysates and the antibody-conjugated Dynabeads were incubated overnight at 4 °C with shaking. After three times of washing with PBS containing 0.1% Tween, the beads were boiled at 95 °C for 5 min with the 6 × protein loading buffer and the supernatant was collected for future WB analysis.

Immunofluorescence assay

ESCs or ESC-derived cells were collected and fixed with 4% paraformaldehyde for half an hour at room temperature. Then, the cells were washed with PBST (phosphate-buffered saline, 0.1% Triton X-100) for three times, each for 15 min. Following the incubation with blocking buffer (5% normal horse serum, 0.1% Triton X-100, in PBS) for 2 h at room temperature, the cells were incubated with primary antibodies at 4 °C overnight. Antibodies used were POGZ (Abcam, ab171934), OCT4 (Proteintech, 60,242–1-Ig), NANOG (Bethyl, A300-397A), GATA6 (Abcam, ab175349), HP1 (Abcam, ab213167), and PH3 (CST, #9701). After three times of wash with PBST, the cells were incubated with secondary antibodies (1:500 dilution in blocking buffer, Alexa Fluor 488, Life Technologies) at room temperature for 1 h in the dark. The nuclei were counterstained with DAPI (Sigma, D9542, 1:1000). After washing with PBS for twice, the slides were mounted with 100% glycerol on histological slides. Images were taken by a Leica SP8 laser scanning confocal microscope (Wetzlar, Germany). About 10 images were taken for each slide. Quantification of IF staining was performed using ImageJ [30].

Immunoprecipitation in combination with mass spectrometry

After Co-IP, the IP samples (immunoprecipitated by IgG or POGZ antibody or FLAG antibody) were run on SDS-PAGE gels and stained with the Coomassie Blue. Then, the entire lanes for each IP samples were cut off and transferred into a 15-mL tube containing deionized water. The mass spectrometry was done by Genecreate Biological Engineering Company (Wuhan, China). Briefly, the samples were digested into peptides by trypsin treatment overnight, followed by C18 column for desalination. Next, the peptides were dissolved and subjected to mass spectrometry analysis by AB SCIEX Triple TOF™ 5600 plus (USA), followed by identification ProteinPilot database search engine. With confidence level ≥ 95%, unique peptides ≥ 1, an average of 260 proteins were identified.

Chromatin Immunoprecipitation (ChIP) and ChIP-seq

ChIP experiments were performed according to the Agilent Mammalian ChIP-on-chip manual as described [32]. Briefly, 1 × 108 ES cells were fixed with 1% formaldehyde for 10 min at room temperature. Then, the reactions were stopped by 0.125 M glycine for 5 min with rotating. The fixed chromatin was sonicated to an average of 200–500 bp (for ChIP-Seq) or 500–1000 bp (for ChIP-qPCR) using the S2 Covaris Sonication System (USA). For ChIP-seq, chromatin was sheared for 15 min with peak power 135; duty factor 5.0; and cycles/burst 200. Then, Triton X-100 was added to the sheared chromatin solutions to a final concentration of 0.1%. After centrifugation, 50 μL of supernatants was saved as input. The remainder of the chromatin solution was incubated with Dynabeads previously coupled with 10 μg ChIP grade antibodies (POGZ, Abcam, Ab171934; FLAG, Sigma, F1804; H3K4me3, Abcam, ab1012; H3K27ac, Millipore, MABE647) overnight at 4℃ with rotation. Next day, after seven times of washing with the wash buffer, the complexes were reverse-cross-linked overnight at 65 ℃. DNAs were extracted by hydroxybenzene–chloroform–isoamyl alcohol and purified by a Phase Lock Gel (Tiangen, China). For ChIP-seq, the ChIPed DNA was dissolved in 15 μL distilled water. Library constructions and deep sequencing were done by the BGI Shenzhen (Wuhan, China). All ChIP-seq experiments were repeated at least two times using control and mutant ESCs.

For ChIP-PCR, the ChIPed DNA was dissolved in 100 μL distilled water. Quantitative real-time PCR (qRT-PCR) was performed using a Bio-Rad qPCR instrument. The enrichment was calculated relative to the amount of input. All experiments were repeated at least three times. The relative gene expression levels were calculated based on the 2−∆∆Ct method. Data were shown as means ± S.D. The Student’s t test was used for the statistical analysis. The significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

CUT and tag

The cleavage under targets and tagmentation (CUT&Tag) experiments were performed using the 3 × Flag-POGZ restoring Pogz−/− mESCs by the Frasergen Company (Wuhan, China) [33]. Aliquots of cells (500,000 cells) were washed twice in 1.5 mL wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1 × protease inhibitor cocktail) by gentle pipetting. Concanavalin A-coated magnetic beads (Bangs Laboratories) were prepared, and 10 µL of activated beads was added per sample and incubated at RT for 15 min. The supernatant was removed, and bead-bound cells were resuspended in 50–100 µL Dig-Wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1 × protease inhibitor cocktail; 0.05% digitonin) containing 2 mM EDTA and 5 µg FLAG antibodies (Sigma, F1804), rotating overnight at 4 °C. Next day, the primary antibody was removed using the magnet stand. To increase the number of protein A binding sites for the bound antibody, the pig anti-rabbit IgG antibody (Novusbio, NBP1-73,654) was diluted 1:50 in 50–100 µL of Dig-Wash buffer and cells were incubated at RT for 30 min. Cells were washed using Dig-Wash buffer to remove unbound antibodies. A 1:200 dilution of pA-Tn5 adapter complex (~ 0.04 µM) was prepared in Dig-med Buffer (0.05% digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitor cocktail). After removing the liquid on the magnet stand, 50–100 µL was added to the cells with gentle vortexing, which was incubated with pA-Tn5 at RT for 1 h. Next, the cells were resuspended in 50–100 µL tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. To stop tagmentation, 2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS, and 0.5 µL of 20 mg/mL proteinase K were added to 50 µL of sample, which was incubated at 55 °C for 30 min, and then at 70 °C for 20 min to inactivate proteinase K. To extract the DNA, 122 µL Ampure XP beads were added to each tube with vortexing, quickly spun, and held 5 min. Beads were washed twice in 1 mL 80% ethanol. After allowing to dry ~ 5 min, 30–40 µL of 10 mM Tris pH 8 was added. To amplify libraries, 21 µL DNA was mixed with 2 µL of a universal i5 and a uniquely barcoded i7 primer33, using a different barcode for each sample. A volume of 25 µL NEBNext HiFi 2 × PCR Master Mix was added and mixed. The sample was placed in a thermocycler with a heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s; and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 8 °C. Post-PCR clean-up was performed by adding 1.1 × volume of Ampure XP beads (Beckman Counter), and libraries were incubated with beads for 15 min at RT, washed twice gently in 80% ethanol, and eluted in 30 µL 10 mM Tris pH 8.0.

ATAC-seq experiments

50,000 control ESCs and Pogz−/− ESCs in LIF-FBS medium were used for ATAC-seq assay. The experiments were performed in two technical replicates and two biological replicates (− 1 and + 284 bp homozygous). Library preparation and ATAC-seq experiments were done by the BGI Shenzhen (Wuhan, China). Libraries were paired-end-sequenced (2 × 75 bp) using an Illumina NextSeq 500 device.

Protein–protein interaction assay using a rabbit reticulocyte lysate system

Protein–protein interaction assay using a rabbit reticulocyte lysate system has been described previously [32]. Tagged-POGZ, Tagged-POGZ mutants, Tagged-BRG1, Tagged-BRG1 mutants, and Tagged-BAF15 were synthesized using the TNT coupled reticulocyte lysate system according to the manual (Promega, L5020, USA). Briefly, 1 μg of circular pCS2 version of plasmids was added directly to the TNT lysates and incubated for 1.5 h at 30 °C. 1 μL of the reaction products was subjected to WB assay to evaluate the synthesized protein. For protein–protein interaction assay, 5–10 μl of the synthesized HA or FLAG tagged proteins was mixed in a 1.5-ml tube loaded with the 300 μl TEN buffer, and the mixture was shaken for 30 min at room temperature. Next, IP or pull-down assay was performed using Dynabeads protein G coupled with anti-FLAG or anti-HA antibodies as described above.

Apoptosis analysis by flow cytometry

Annexin V-EGFP/PI (Propidium Iodide) Apoptosis Detection Kit (Yeason, 40303ES20) was used to detect apoptotic cells according to the manual. The late apoptosis and necrotic cells will be AnnexinV+/PI+. Briefly, ESCs were treated with trypsin without EDTA and washed twice by DPBS. Then, cells were washed with 1 × Binding Buffer and incubated with Annexin V-EGFP for 5 min at room temperature in the dark, followed by treatment with PI Staining Solution. Next, apoptotic cells were detected by flow cytometry (BD, AccuriC6). The apoptosis analysis was supported by Wan Yan from the Analysis and Testing Center of Institute of Hydrobiology, Wuhan.

Quantification and statistical analysis

Data are presented as mean values ± SD unless otherwise stated. Data were analyzed using Student’s t test analysis. Error bars represent s.e.m. Differences in means were statistically significant when p < 0.05. Significant levels are: *p < 0.05; **P < 0.01, and ***P < 0.001.

BioinformaticsRNA-seq analysis

The RNA-seq data were processed through standard experimental and analytical pipelines. Raw.fastq files were first assessed for quality using FastQC software. Technical sequences and sequencing adapters were trimmed using Trimmomatic tool. Each sample produced more than 20 M clean reads and were mapped to the mm10 reference genome using HISTA. Then, Cufflinks was used to generate Fragments Per Kilobase of transcript per Million mapped reads (FPKM) table. DESeq2 was performed to calculate differentially expressed genes with fold change > 2 and P < 0.05. Volcano plots were drew with EnhancedVolcano package or ggplot2 package.

ChIP-seq analysis

ChIP-seq raw data were filtered with SOAPnuke filter -l 15 -q 0.5 -n 0.01 -Q 2 -c 21. After filtering, the clean data were mapped to mm10 genome by SOAPaligner/SOAP2 (version: 2.21t) with Parameter: soap_mm_gz -p 4 -v 2 -s 35. Peaks were called with macs2 -g 2.73e9 -s 50 -p 1e-5 -m 10 30 -broad -B -trackline. MAnorm software was used for finding the differential peaks. The P value for each peak was calculated based on the Bayesian model, and the significant regions were picked up if |M|≧1 and p value≦10−5. The bedtools (version 2.30.0) was used for calculating the differential peaks from two MAnorm results. BigWig files were generated using deepTools (version 3.3.2). Heat maps were generated from the average of replicates using the deepTools. Peak centers were calculated based on the peak regions identified by MACS2.

To investigate the co-occupancy of POGZ and BRG1, we consulted previously published ChIP-seq data sets for BRG1 (GSE87820; GSM2808653) [24, 34]. To analyze POGZ at gene promoters and enhancers, we consulted previously published ChIP-seq data sets for H3K4me1 (GSM2575694), H3K27Ac (GSM2575695) [35]. KLF4 (GSM4072779), ESRRB (GSM4087822), OCT4 (GSM2341284), NANOG (GSM2521520), MED1 (GSM4060038), SS18 (GSM2521508), BAF250a (GSM3318684), and BAF155 (GSE69140). ChIP-seq data were also downloaded for this work.

CUT-tag analysis

Technical sequences and sequencing adapters were trimmed using Trimmomatic tool. Raw data were evaluated using FastQC. Each sample was aligned in Bowtie2 with parameters -I 10 -X 1000 –dovetail –no-unal –very-sensitive-local –no-mixed –no-discordant. Non-aligning and multiple mappers were filtered out with Picard tools. Peaks were called using MACS2 (version 2.1.1) with the parameters -f BAMPE -B –call-summits –keep-dup all. The position of each peak in the peak total collection, the background value, and signal value (RPKM) of the peak were extracted for each sample, and the signal value was divided by the background value to obtain the enrichment fold of the peak. Then, the DESeq2 software was used to perform between-group different peak analysis, by setting the filtering threshold to: |log2FoldChange| ≧1, P value≦ 0.05. BigWig files were generated using deepTools (version 3.3.2). Heat maps were generated from averaged replicates using the deepTools.

ATAC-seq analysis

ATAC-seq raw data were filtered out with SOAPnuke filter -l 5 -q 0.5 -n 0.1 -Q 2 -5 1 -c 25. The clean data were mapped to mm10 genome by Bowtie2 (version: 2.2.5) with parameters: bowtie2 -q –phred64 –sensitive –dpad 0 –gbar 99,999,999 –mp 1,1 –np 1 –score-min L,0,-0.1 -I 1 -X 1000 -p 16 -k 200. Peaks were calling with macs2 (version: 2.1.0) with peak calling parameter: macs2 -g mm -s 50 -B –trackline –nomodel –shift -100 –extsize 200. BigWig files were generated using the deepTools (version 3.3.2). The MAnorm software was used for finding differential peaks. The P value for each peak was calculated based on the Bayesian model, and the significant regions were picked up if ≧1 and p≦10−5. The bedtools (version 2.30.0) was used for calculating the differential peaks from two MAnorm results. BigWig files were generated using deepTools (version 3.3.2). Heat maps were generated from averaged replicates using the deepTools.

For comparison analysis of POGZ and BRG1, we downloaded the previously published ATAC-seq data sets for Brg1 KD ESCs (GSM1941485-6), Brg1 KO ESCs (GSM2341280), and OCT4 KO ESCs (GSE87819) [24, 35].

Combined analysis of ATAC-seq and RNA-seq

BETA was used for the combined analysis of ATAC-seq and RNA-seq data [36].

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