PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation

Abstract Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.


Generation of gene knock-in hESCs by CRISPR/Cas9
To generate PAU-PAKI hESC lines, gRNA targeting PAUPAR listed in Supplemental Table S2 was designed using the CRISPR DESIGN website (http://crispr.mit.edu/). The PGK promoter-puro segment and 3×polyA segment were cloned into the pLB vector (TIANGEN) and integrated into the transcription start site (TSS) of PAUPAR with the designed sgRNA respectively.
To generate NSD1-3×myc hESC lines, gRNA targeting the C-terminus of NSD1 (Supplemental Table S2) was used. The 3×myc tag segment and a stop codon with an EF1A1 promoter-driven neomycin cassette segmented between two LoxP sequences were cloned into the pLB vector and integrated into the end of NSD1. After individual colonies were selected by G418, the Cre recombinase driven by the EF1α promoter was transfected into hESCs through electroporation.
To generate inducible PAX6a/dCas9-overexpression hESCs lines, the PAX6a/dCas9 fusion protein driven by the pTREtight promoter and rtTA driven by the CAG promoter were integrated into one allele of the AAVS1 locus, and gRNA targeting the SOX1 locus (Supplemental Table S2) modified as previously described (2) and driven by the U6 promoter was integrated into another allele through electroporation with TALEN.

Western blotting
The cells were collected, washed with PBS, and then lysed in 1× SDS lysis buffer with protease inhibitor cocktail (Roche). Lysates were separated from cell extracts by 10-12% Bis-Tris SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Blots were blocked in 3% BSA/TBST at room temperature for 1 h and incubated with primary antibody at 4°C overnight and secondary antibody at room temperature for 1 h. The signals were visualized by using enhanced chemiluminescence (ECL). The antibodies used in this study are listed in Supplemental   Table S1.

Quantitative RT-PCR
Total RNA was extracted from the cells using RNAiso (Takara) according to the manufacturer's instructions. A total of 500 ng of total RNA was reverse transcribed into cDNA using a PrimeScript TM RT reagent kit (Takara). The resultant cDNA was used for QRT-PCR on an Mx3000 instrument (Agilent) with relative expression levels calculated using the 2 -ΔΔ Ct method(3) by normalizing against GAPDH expression; results are presented as the fold change relative to the control. Each experiment was performed in triplicate and repeated three times. The primer sequences used in this study are listed in Supplemental Table S2.

Fluorescent in situ hybridization (FISH)
FISH assays were performed as previously described (4). Cells cultured on coverslips were fixed with 4% paraformaldehyde at 4°C for 15 min and washed three times with PBS. The samples were subsequently incubated with Pre-Hybridization Buffer at 37℃ for 30 min and Hybridization Buffer with FISH probes at 37°C overnight in the dark using a Ribo TM Fluorescent In Situ Hybridization Kit (RiboBio). The next day, the coverslips were washed three times with wash buffer I (4× SSC with 0.1% Tween-20), once each with wash buffer II (2× SSC) and wash buffer III (1× SSC) at 42°C in the dark for 5 min and once with PBS at room temperature. Then, the cells were stained with Ho 33342 in the dark for 10 min. PAUPAR-Cy3 FISH probes (RiboBio) were designed and synthesized by RiboBio Co., Ltd. Human U6 FISH probes (RiboBio) and Human 18S FISH probes (RiboBio) were used as nuclear and cytoplasmic controls, respectively. All images were captured by fluorescence microscopy (Nikon).

Chromatin isolation by RNA purification (ChIRP)
ChIRP was performed as previously described with modifications (6,7). Antisense probes were designed using an online probe designer (singlemoleculefish.com). Cells were crosslinked with 1% glutaraldehyde (Sangon Biotech) for 10 min and quenched with 0.25 M glycine for 5 min at room temperature. The cell pellet was lysed in nuclei lysis buffer (50 mM Tris (pH 7.0), 10 mM EDTA and 1% SDS) with the addition of dithiothreitol, protease inhibitor cocktail, PMSF and RNaseOUT (Invitrogen). Then, each sample was sonicated to generate 100-500 bp fragments. Chromatin fractions were diluted in two volumes of hybridization buffer (50 mM Tris (pH 7.0), 750 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100 and 15% formamide (Sigma) with dithiothreitol, protease inhibitor cocktail, PMSF and RNaseOUT) supplemented with 100 pmol of probes by end-to-end rotation at 37°C for 4 h. Then, 100 μl of prewashed streptavidin magnetic C1 beads (invitrogen) was added and incubated at 37°C by end-to-end rotation for an additional 0.5-1 h. The beads were then washed five times with wash buffer (2× SSC and 0.1% SDS supplemented with dithiothreitol and fresh PMSF). For RNA elution, beads were resuspended in RNA pK buffer (10 mM Tris (pH 7.0), 100 mM NaCl, 1 mM EDTA, 0.5% SDS) and Proteinase K. Then, samples were incubated at 65°C for 45 min with end-to-end shaking, followed by boiling at 95°C for 10 min. Immunoprecipitated RNAs were extracted using RNAiso (Takara) and analyzed by QRT-PCR. For DNA elution, beads were washed once with SDS elution buffer (50 mM Tris, 75 mM NaCl, 5 mM MgCl2, 1% SDS) at 37°C for 30 min, followed by one wash with elution buffer (50 mM Tris, 75 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100) at 37°C for 5 min. Then, DNA was sequentially eluted by elution buffer supplemented with RNaseH and RNaseA and by SDS elution buffer at room temperature for 2 min. The combined eluents were treated with proteinase K, 150 mM NaCl and 10 mM EDTA at 65°C for 50 min. DNA was then purified with phenol-chloroform (Sangon Biotech) and subjected to QRT-PCR analysis.
The primer sequences and the probe sequences used in this study are listed in Supplemental Table   S2.

Coimmunoprecipitation (Co-IP)
Co-IP assays were performed as previously described with modifications (8). A mixture of Ezview Red Protein A affinity gel (Sigma) and Ezview Red Protein G affinity gel (Sigma) (1:1) was incubated with antibodies or control normal IgG overnight at 4°C. Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche) on ice. The cells were sonicated for 30 s (2 s interval) at 25 Amps (Qsonica Sonicators), after which antibodies were added and incubated for 4-6 h at 4°C. The protein-bead complex was washed with RIPA buffer 3 times and subjected to western blotting analysis. The antibodies used in this study are listed in Supplemental Table S1.

MS2bp-YFP RNA pulldown
The MS2bp-YFP RNA pulldown assay was performed as described previously (9). A total of 5×10 6 for 30 min on ice. The proteins were immunoprecipitated using control IgG (CST) or anti-GFP antibody (Abcam), which was able to recognize the YFP protein. The RNA and RNA-bound protein complexes were treated with RNAiso (Takara) to purify the RNA or SDS lysis buffer for western blotting analysis.

Chromatin immunoprecipitation (ChIP)
ChIP assay was performed as described previously (4). Cells were crosslinked with 1% formaldehyde (Sigma) for 10 min, and the crosslinking reaction was then quenched with 0.25 M glycine for 5 min at room temperature. Cell pellets were lysed in cell lysis buffer (5 mM PIPES (pH and TE buffer (10 mM Tris (pH 8.1) and 1 mM EDTA). Each wash was performed on a rotator over 15 min at 4°C. Beads were treated with 300 μl of elution buffer (100 mM NaHCO3 and 1% SDS). 30 μl of 5 M NaCl was added to the eluted samples except input. All samples were reverse crosslinked overnight at 65°C. After RNaseA and proteinase K treatment, samples were purified with phenolchloroform (Sangon Biotech). Immunoprecipitated DNA and input DNA were used as templates for QRT-PCR analysis. The antibodies used in this study are listed in Supplemental Table S1. The primer sequences used in this study are listed in Supplemental Table S2.

RNA-seq
RNA-Seq library generation and sequencing were performed as previously described (10).
Quality control and adapter trimming of sequencing raw data were completed by fastqc (11) and Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). All RNA-seq reads were aligned to the human genome (hg19) using TopHat (v2.0.12) with default parameters (12). Gene expression levels were measured as FPKM using Cufflinks (v2.2.1) to eliminate the effects of sequencing depth and transcript length (13). For each comparison, differentially expressed (DE) genes with a GFOLD value larger than 1 (fold change larger than 2) were found using GFOLD (v1.1.3) (14). For the following analysis, FPKMs were log2 transformed after adding a pseudocount of 1. Functional annotation was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID 6.7) bioinformatics resource (15). GO terms for each functional cluster were summarized as a representative term, and P-values were plotted to show the significance.

ChIP-seq
ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology). DNA libraries were constructed by following Illumina library preparation protocols. All ChIP-seq reads were aligned to the human genome (hg19) using Bowtie2 (v2.2.9) with default parameters (16). The reads signal for each sample were generated using MACS2 (v2.1.1.20160309) and normalized to 1 million reads for visualization with the parameter -SPMR (17). The normalized reads signal were used to calculate the average signal of each sample around the TSS in different groups of genes. The binding motif of PAX6 was predicted by the MEME method (18). The antibodies used in this study are listed in Supplemental Table S1.

Supplemental Tables S2. Primers used for this study.
Primers used for gRNA.

Primers used in QRT-PCR of gene expression assays.
PAUPAR Primers used in QRT-PCR of ChIP, ChIRP and in vitro triplex pulldown assays.