DOI: 10.5061/dryad.05qfttfbf
title: Data from: Oct4 redox sensitivity potentiates reprogramming and differentiation

description: The transcription factor Oct4/Pou5f1 is a component of the regulatory
 circuitry governing pluripotency and is widely used to induce pluripotency
 from somatic cells. Here we use domain swapping and mutagenesis to study
 Oct4’s reprogramming ability, identifying a redox-sensitive DNA binding
 domain cysteine residue (Cys48) as a key determinant of reprogramming and
 differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition
 of DNA binding activity and promotes oxidation-mediated protein
 ubiquitylation. Pou5f1C48S point mutation has little effect on
 undifferentiated embryonic stem cells (ESCs), but upon retinoic acid (RA)
 treatment causes retention of Oct4 expression, deregulated gene expression
 and aberrant differentiation. Pou5f1C48S ESCs also form less
 differentiated teratomas and contribute poorly to adult somatic tissues.
 Finally, we describe Pou5f1C48S(Janky) mice, which in the homozygous
 condition are severely developmentally restricted after E4.5. Rare animals
 bypassing this restriction appear normal at birth but are sterile.
 Collectively, these findings uncover a novel Oct4 redox mechanism involved
 in both entry into and exit from pluripotency.
description: This raw dataset includes: 1)    Reprogramming
 data. GFP+ colonies with iPSC morphology were counted by microscopic
 observation. Viral titer data was collected in parallel. Titer was
 obtained using p24 levels or conserved viral RNA sequences.
 2)    ESC western blot quantification data. Intensities were
 collected and normalized using Image J. 3)    EMSA
 quantification data. Intensities were collected and normalized using Image
 J. 4)    Parental and Oct4<sup>C48S</sup>
 mutant ESC pluripotent marker expression in the pluripotent state and in
 RA differentiation. Intensities were collected and normalized using Image
 J. 5)    Parental and Oct4C48S mutant differential gene
 expression in the pluripotent state and in RA differentiation. mRNA
 expression levels were obtained using RNA sequencing, and normalized using
 methods described in teh materials and methods section of the Shen et al.
 manuscript. 6)    Parental and Oct4C48S mutant ESC
 teratoma formation. Differentiated structures were scored in the blanded
 manner by a clinical pathologist. 7)   
 Oct4<sup>C48S</sup> mouse line generation and defects in the
 homozygous Jky/Jky mouse. Genotypes were obtained using endpoint
 PCR.
description: # Data from: Oct4 redox sensitivity potentiates reprogramming and
 differentiation
 [https://doi.org/10.5061/dryad.05qfttfbf](https://doi.org/10.5061/dryad.05qfttfbf) This raw dataset includes: 1\) Reprogramming data. GFP+ colonies with iPSC morphology were counted by microscopic observation. Viral titer data was collected in parallel. Titer was obtained using p24 levels or conserved viral RNA sequences. 2\) ESC western blot quantification data. Intensities were collected and normalized using Image J. 3\) EMSA quantification data. Intensities were collected and normalized using Image J. 4\) Parental and Oct4C48S mutant ESC pluripotent marker expression in the pluripotent state and in RA differentiation. Intensities were collected and normalized using Image J. 5\) Parental and Oct4C48S mutant differential gene expression in the pluripotent state and in RA differentiation. mRNA expression levels were obtained using RNA sequencing, and normalized using methods described in teh materials and methods section of the Shen et al. manuscript. 6\) Parental and Oct4C48S mutant ESC teratoma formation. Differentiated structures were scored in the blanded manner by a clincal pathologist. 7\) Oct4C48S mouse line generation and defects in the homozygous Jky/Jky mouse. Genotypes were obtained using endpoint PCR. For procedures and results please see research paper Materials and methods section. ## Description of the data and file structure Among the Yamanaka factors, Oct4 cannot be replaced by Oct1 and other paralogs. To investigate which part of Oct4 has important role during reprogramming, we generated Oct1/4 chimeric constructs via swapping/mutating different domains/residues and monitored the emergence of GFP+ iPSC colonies, identifying a DNA binding domain cysteine (Cys 48) as a key reprogramming determinant. We then tested the effects of Oct4 C48S mutation using overexpression and purified recombinant proteins, finding that this mutation promotes ubiquitylation and degradation under conditions of oxidative stress using EMSA assays and immunoblotting. After generating embryonic stem cells with an Oct4 C48S mutation using CRISPR-mediated mutagenesis, we found that mutant ESCs are essentially normal in the undifferentiated condition (quantification of immunoblots for Oct4, Sox2, etc) but following RA treatment Oct4 expression is abnormally retained, and differentiation occurs abnormally (RNA sequencing, cell counts). Further, cardiomyocyte differentiation revealed a failure to form beating cardiomyocytes (quantification of microscopic observation). Teratoma formation data using the same cells indicates that mutant ESCs generate teratomas with less differentiated structures (quantification of sections scored by a clincal pathologist in a blinded fashion). The same cells also contribute poorly to adult animals in chimerism assays (quantification of dark coat and eye color in albino C57BL/6 animals). Finally we generated a germline mutant mouse allele through CRISPR, showing a severe developmental lethality after the blastocyst stage. For more detailed information: refer to the methods section of the manuscript for specific assay methodologies. Tab 1 (Fig.1C) values indicate GFP+ colonies number for each construct. Column A indicates constructs. Columns B-D show values for each biological replicate. Column E is average data and column F is the calculation of standard deviation (SD). Tab 2 (Fig.1D&Fig.S2A) indicates GFP+ colonies number for each construct at different time points. Column A indicates the construct and timepoint assessed. Columns B-D indicate biological replicates. Column E calculates the average data and column F is SD. Tab 3 (Fig.2A) indicates GFP+ colonies number for each construct at D12. Column B-D indicates biological replicates. Column E is average data and column F is SD. Tab 4 (Fig.3B) indicates a quantification of WT and C48S Oct4 protein binding to DNA with different concentrations of diamide. Row 2 is micromolar diamide treatment concentration. Column B-E shows quantified band intensity using lysates from cells expressing mutant protein, with no diamine set to 100 and other values shown as a percentage. Column J-M is the same calculation made using cells expressing wild-type protein. Rows 3-5 show biological replicates. Row 6 is SD and Row 7 is average. Tab 5 (Fig.3D) indicates Purified WT and C48S Oct4 protein stability with different concentrations of diamide. Row 2 is micromolar diamide treatment concentration. Column B-E shows quantified band intensity using purified mutant protein. Column J-M shows similar calculation using wild-type cells. Rows 3-5 show biological replicates. Row 8 is SD and Row 7 is average. Tab 6 (Fig.4C) indicates relative Oct4 intensities (normalized to beta-actin intensity for the same sample) in 4 repeats of parent and Oct4C48S ESCs lysates. Row 1 indicates the title, row 2 indicates the subtitles and row 3 indicates headers. Column A rows 4-7 indicates titles for 4 biological repeats. Column B and C rows 4-7 indicate BL/6 parent strain and C48S#46 strain Oct4 intensities respectively. Column E rows 4-7 indicates titles for 4 biological repeats. Columns F and G rows 4-7 indicate BL/6 parent strain and C48S#46 strain b-actin intensities respectively. Columns I and J rows 4-7 indicate BL/6 parent strain and C48S#46 strain Oct4/b-actin normalization respectively. Columns F and G rows 4-7 indicate BL/6 parent strain and C48S#46 strain b-actin intensities respectively. Column L rows 4-7 indicate BL/6 parent strain Oct4/b-actin normalized to 1 and column M rows 4-7 indicate C48S#46 strain Oct4/b-actin normalized to corresponding BL/6 strain. Tab 7 (Fig.4G) indicates relative Oct4 intensities (normalized to beta-actin intensity for the same sample) in 3 repeats of parent and Oct4C48S ESCs and RA treated day 1 to day 3 cells lysate. Row 1 indicates the title, rows 2, 13 and 24 indicate subtitles for 3 biological repeats and rows 3, 14 and 25 indicate headers. Column A indicates names of samples, and column B rows 4-11, 15-22 and 26-33 indicate Oct4 intensities for 3 repeats of BL/6 and C48S#46 ESC and RA treated cells respectively. Column C rows 4-11, 15-22 and 26-33 indicate b-actin intensities for 3 repeats of BL/6 and C48S#46 ESC and RA treated cells respectively. Column E indicates names of samples, and column F rows 4-11, 15-22 and 26-33 indicate normalized Oct4 intensities (normalized to BL/6 parent ESC) for 3 repeats of BL/6 and C48S#46 ESC and RA treated cells respectively. Column G rows 4-11, 15-22 and 26-33 indicate normalized b-actin intensities (normalized to BL/6 parent ESC) for 3 repeats of BL/6 and C48S#46 ESC and RA treated cells respectively. Column I indicate names of samples, and column J rows 4-11, 15-22 and 26-33 indicate normalized Oct4/normalized b-actin for 3 repeats of BL/6 and C48S#46 ESC and RA treated cells respectively. Tab 8 (Fig.5B) indicates cell number counts in 3 repeats of parent and Oct4C48S ESCs as well as RA treated day 1, 3, 6, 9, 12. Row 1 indicates the title, row 2 indicates the subtitle, and row 3 and column A indicates headers. Columns B, C and D indicate 3 biological repeats for BL/6 parent cell number counting and columns E, F and G indicate 3 biological repeats for C48S#46 cell number counting. Rows 4-9 indicate ESCs with 0, 1, 3, 6, 9, and 12 days of RA treatment respectively. Tab 9 (Fig.5E) indicates DCFDA intensities of WT BL/6 undifferentiated ESCs and RA treated day 3 cells. Row 1 indicates the title, row 2 indicates the subtitle, and row 3 indicates the header. Rows 4-6 indicate 3 biological repeats for DCFDA mean fluorescence intensity measurement. Columns A and B indicate BL/6 ESC and BL/6 ESC with 3 days of RA treatment respectively. Tab 10 (Fig.5F) indicates WT ES and Mutant ES (Oct4 C48S) beating events counted upon cardiomyocyte differentiation. Column A is the specific cell line. Column B-AK shows raw counts of beating events/well. Column AF is P-value. Row 13-15 is average. Row 17-19 is SD. Tab 11 (Fig.6A) indicates the percentage of immature components in day 15 teratomas formed by parent and Oct4C48S ESCs (G4-N strain) injection into mice. Row 1 indicates the title, row 2 indicates the subtitle, and row 3 indicates the header. Rows 4-8 indicate 5 biological repeats for teratoma immature component scoring. Column A indicates G4-N parent ESC formed teratoma and column B indicates Oct4C48S G4-N ESC formed teratoma. Tab 12 (Fig.6C) indicates the percentage of immature components in day 15 teratomas formed by parent and Oct4C48S ESCs (BL/6 strain) injection into mice. Row 1 indicates the title, row 2 indicates the subtitle, and row 3 indicates the header. Rows 4-8 indicate 5 biological repeats for teratoma immature component scoring. Column A indicates BL/6 parent ESC formed teratoma and column B indicates Oct4C48S BL/6 ESC formed teratoma. Tab 13 (Fig.6D) indicates chimerism generated by parental control and derived Oct4C48S ESC lines of 3 different strains. Row 1 indicates the title, row 2 indicates the subtitle, and row 3 indicates the header. Column A indicates ESC strain, column B indicates donor blastocyst, column C indicates ESC clone numbers, column D indicates successfully transferred embryo number, column E indicates pup number generated and column F indicates chimerism formed. Rows 4-7 suggest BL/6 strain 3 mutant clones and 1 WT clone respectively, rows 8-11 suggest G4-N strain 3 mutant clones and 1 WT clone respectively, and rows 12-16 suggest C4 strain 4 mutant clones and 1 WT clone respectively. Tab 14 (Fig.6E) indicates the number of offspring generated by different combinations of WT and +/Jky mating. Row 1 indicates the title, rows 2, 7, 12, and 17 indicates the subtitles. Row 3, 8, 13 and 18 are headers. Column A indicates litter sizes or dams, column B indicates WT offspring number, column C indicates heterozygous offspring number, column D indicates homozygous offspring number, column E indicates total offspring number, column F indicates chi-square p values and column G indicates mean litter sizes. Rows 4, 9, 14 and 19 indicates offsprings from WT x heterozygous, heterozygous x WT, heterozygous x heterozygous and heterozygous x heterozygous embryos respectively. Rows 5, 10, 15 and 20 are expected offspring or embryo numbers for p value calculation. Tab 15 (Fig.S1A) indicates GFP+ colonies number of Oct4 and Flag-Oct4(Oct4 N-terminal with Flag) constructs at different time point. Column A is different construct. Column B-C biological replicates. Column E is average. Column F is SD. Tab 16 (Fig S2B) indicates virus titer using P24 levels. Row 1-6 indicate standard curve of P24 protein concentration: column A-B is biological repeats of absorbance. Column D is standard P24 concentration. Column E is average absorbance. Column K-L is standard curve. The standard curve trendline equation is y=0.0084x+0.1187y means absorbance, x means p24 concentration. Rows 9 to 14 are samples absorbance: Column A-B is samples absorbance biological replicates. Column D-E is samples p21 concentration after dilution. Column F is constructs. Column H-I is samples original P21 concentration. Columns J-K is virus titer according to manufacture instruction. Rows 19-24 are average titers and SD: Row 19 is constructs. Row 20 is the average titer of samples Row 21 is SD. Row 23-24 is the original sample titer. Tab 17 (Fig.S3B) indicates fluorescence intensity of different GFP+ colonies generated by different construct. Column A lists the construct. Row 1 is the title. Row 2 (between column B-G) is colony fluorescence intensity. Row 3-7 is background fluorescence. Row 2 (between column I-J) is colony net fluorescence intensity. Same apply to other colonies. Each construct has three replicates. Column M-S is average intensity of all constructs and SD. Tab 18 (Fig.S4A) indicates GFP+ colonies number generated by different construct at different time point. Column A is construct. Column B-D is the biological replicate. Column E is average and F is SD. Column L-R is average of all constructs and SD. Tab 19 (Fig. S4B) indicates the raw number of iPSC colonies generated using different constructs as measured by alkaline phosphatase staining at D12. Column A is the construct. Column B-D is the biological replicate. Column E is the average and F is the SD. Tab 20 (Fig. S4C) indicates viral titer using a qPCR method. Row 6-17 shows standard curve through cycles and viral RNA level. Row 1 to 10 (between column H-J) is average cycles and viral RNA concentration and its log value. Row 33 to 35 indicates samples biological repeats cycles. Row 37 is average cycles and row 38 is SD. Row 40-42 is samples log value of RNA levels. Row 44-46 is samples raw copy number. Row 48-50 is samples virus copies/ml. Row 54 is constructs. Row 55-57 is samples IFU/mL. Row 59 is sample average IFU/mL and row 60 is SD. Row 62-64 is IFU/mL/105. Row 67 is average of row62-64 and 68 is SD. Tab 21 (Fig.S9D) indicates Oct4, Sox2 and Nanog expression levels of WT ESC and Mutant ESC (Oct4C48S, #2). Row 1-7 is Nanog, Row 13-19 is Oct4 and Row 29-35 is Sox2.Row 1-2 is cell lines and name. Nanog: Row 3-5 is biological replicates. Row 6 is average and 7 is SD. Row 8-10 is x axis for superimposed individual value in plot. Oct4: Row 15-17 is biological replicates. Row 18 is average and 19 is SD. Row 21-23 is x axis for superimposed individual value in plot. Sox2: Row 31-33 is biological replicates. Row 34 is average and 35is SD. Row 37-39 is x axis for superimposed individual value in plot.