EMBO/SourceData huggingface dataset:
Figure 2Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both.
Figure 3Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel.Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017).
Figure 4Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty).
Figure 5Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638.Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes).
Figure 6Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs.Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).
Figure 7tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b. MEF, Reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines. Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows. Color corresponds to log10 of transcript counts for each individual cell.Bar graphs indicating number of cells expressing Tet1, Tet2, Cdh1 or Nanog from the reprograming population (Day 3, 6, 9 and 12). Number of cells per reprogramming population is highlighted within legend.Scheme for deriving Tet1 and Tet2 double knockout pre-iPSC lines. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl MEFs were treated with retroviruses containing OSKM. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl pre-iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre-iPSCs with Tet1KO/KO Tet2fl/fl, Tet1KO/KO Tet2KO/KO, Tet1WT/WT Tet2fl/fl, and Tet1WT/WT, and Tet2KO/KO. Scale bar, 50 μm.Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment.
Figure 1RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c-/- using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel.SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either anti-Myc or the SPPL2c antiserum (C-terminal epitope) were employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively.Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control.N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treatedSubcellular fractionation of postnuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulfide isomerase (PDI), Cathepsin D and CD44 were detected as organelle marker proteins.
Figure 2HEK293 cells were transiently transfected with murine N-terminally HA-tagged heme oxygenase 1 (HA-mHO-1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Substrate levels were determined by Western blot analysis with anti-HA. For detection of SPP, an antiserum recognizing both endogenous human (h) and overexpressed murine (m) SPP was employed. Murine SPPL2c was visualised using the antiserum generated against the C-terminus of the protein. Actin was detected to control for equal protein loading.Immunofluorescence analysis of HeLa cells transiently expressing murine HO-1 (HA-mHO-1) alone or together with active or inactive (D/A) SPPL2c-myc. Substrate and proteases were visualised using the HA and Myc epitopes, respectively.The TA protein Ube2J1 is not proteolysed by co-expressed SPP or SPPL2c in HEK293 cells. Western blot analysis was performedDifferential cleavage of CYB5a, RAMP4-2 and RAMP4 by SPPL2c and SPP. Substrates and proteases were co-expressed in HEK293 cells followed by Western blot analysis as described above. Densitometric quantification from at least n=3 independent experiments was performed. Substrate levels were normalised and compared to cells without protease-overexpression.HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate.Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.
Figure 3Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate.SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control.The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight.Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype.SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c.Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm.Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second.Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively.
Figure 4Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes.HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading.Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts.HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A.Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease.
Figure 5Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly.Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype.Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR.Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate.Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples.Median forward scatter (FSC) was controlled in the 1C-co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype.Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated.Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids.
Figure 1 A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)  B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%) a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-testsD a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel fo human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests
Figure 2Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-testhuman neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test C Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test
Figure 3 A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m
Figure 4 A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 hours, and then imaged by 2P-FLIM before and 30 minutes after addition of insulin. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of 3 independent assays B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays  C Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test
Figure 5A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit ­mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent aC WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±