Author response: PLZF targets developmental enhancers for activation during osteogenic differentiation of human mesenchymal stem cells (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the ZBTB16 locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the ZBTB16/PLZF locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (NNMT) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation. Introduction Human mesenchymal stem cells (hMSCs) possess self-renewal and multi-lineage differentiation potential toward osteogenic, chondrogenic and adipogenic specification (Pittenger et al., 1999; Prockop, 1997). Therefore, hMSCs represent a promising resource for regenerative medicine. However, for treatment efficiency and safety, it is instrumental to obtain a thorough understanding of basic molecular mechanisms that control the orchestrated activation of lineage-specific genes, determining cell fate of hMSCs and factors that ensure maintenance of the terminal differentiated state(s). The adipogenic differentiation of MSCs have been extensively studied and have revealed that it consists of a cascade of transcriptional events that are driven by a series of transcription factors (Rosen and Spiegelman, 2014). However, the knowledge regarding osteogenic differentiation of MSCs is mainly limited to the activity of the key transcription factors RUNX2 and SP7 (osterix), and markers representing the different stages of differentiation such as proteins involved in matrix formation and mineralization (Karsenty and Wagner, 2002; Neve et al., 2011). Although it is well established that Polycomb (PcG)- and Trithorax (Trx)-group protein complexes are intimately linked to cell specification through gene repression and activation, respectively (Piunti and Shilatifard, 2016; Schuettengruber et al., 2017), very limited information regarding these complexes are available in naive hMSCs and cells undergoing osteogenic lineage specification. Moreover, there is a considerable lack in understanding of the chromatin changes associated with enhancer activation that leads to lineage-specific gene transcription during differentiation of mesenchymal stem cells. Enhancers are cis-acting DNA regulatory elements that function as transcription factor (TF) binding platforms, increasing the transcriptional output of target genes through chromatin topological changes involving enhancer-promoter looping (Calo and Wysocka, 2013; Plank and Dean, 2014). Binding of lineage-specific TFs to enhancers is key to cell specification during stem cell differentiation and organism development (Spitz and Furlong, 2012). However, the enhancer landscape that becomes active and regulates lineage-specific gene expression during osteogenic commitment has not been fully explored. The ZBTB16 gene locus encodes a BTB/POZ domain and zinc finger containing TF known as PLZF (Li et al., 1997). Targeted deletion of Zbtb16 in mice disrupts limb and axial skeleton patterning (Barna et al., 2000; Fischer et al., 2008), and although PLZF has been shown to be involved in osteogenic differentiation (Djouad et al., 2014; Ikeda et al., 2005), the underlying mechanisms of its function is only partly understood. PLZF has been described to have a dual function in transcription 1) as a gene repressor through interaction with HDAC1, mSin3a, SMRT and NCOR (David et al., 1998; Hong et al., 1997; Melnick et al., 2002; Wong and Privalsky, 1998), and PcG proteins (Barna et al., 2002; Boukarabila et al., 2009), 2) as a gene activator due to its positive impact on transcription (Doulatov et al., 2009; Hobbs et al., 2010; Labbaye et al., 2002; Xu et al., 2009). These studies have been performed in other tissues and cell types than MSCs, such as hematopoietic and germline cells. In these cell types, PLZF is already expressed at the stem cells stage and found to be required for the maintenance of the stem cell pool. However, in MSCs, PLZF is expressed only in differentiating MSCs and not in naive cells (stem cells), and its molecular function is so far unknown in these cells. Through the use of genome-wide ChIP-sequencing and expression analysis, we now present evidence for a novel function of PLZF at developmental enhancers directing osteogenic differentiation of hMSCs. Interestingly, we find that the ZBTB16 (PLZF) locus is bound and repressed by PcG proteins and extensively H3K27 tri-methylated (H3K27me3) in naive hMSCs. Upon osteoblast commitment of progenitor cells (pre-osteoblast stage), H3K27me3 demethylation takes place through JMJD3 (also known as KDM6B) recruitment to the ZBTB16 locus, followed by extensive H3K27 acetylation and PLZF expression. Interestingly, upon expression, PLZF binds to a subset of enhancers, which gain H3K27 acetylation and correlates with induced expression of nearby genes important for osteogenic differentiation and function. Intriguingly, an initially PcG-repressed enhancer within the ZBTB16 locus becomes active in pre-osteoblasts, gaining PLZF, p300 and Mediator binding. By use of Chromosome Conformation Capture combined with high-throughput sequencing (4C-seq) (van de Werken et al., 2012b), we show that this ZBTB16 intragenic enhancer ‘EnP’ physically contacts the promoter of the nicotinamide n-methyltransferase gene (NNMT) and regulate its expression during osteogenic differentiation. Taken together, our data reveal that the activation of the ZBTB16 (PLZF) gene locus is an important event during osteogenic differentiation. PLZF regulates osteogenic differentiation of hMSCs through binding at gene enhancers and thereby it positively affects transcription of lineage-specific genes. The enhancer, EnP, localized within the ZBTB16 locus is an example of a latent developmental enhancer that becomes active upon differentiation and regulates NNMT expression in a PLZF-dependent manner through Mediator and RNA-PolII recruitment and enhancer-promoter looping. Results The ZBTB16 locus is activated during early osteogenic differentiation To understand the transcriptional events that control the osteogenic differentiation of hMSCs, we performed RNA-seq analysis for global expression levels and ChIP-seq analysis for histone marks known to be associated with transcriptionally repressed (H3K27me3) and active (H3K4me3, H3K27ac) gene loci. We looked for the changes taking place at an early time point of differentiation (10 days) as compared to naive hMSCs (day 0). Figure 1A shows the timeline of osteogenic differentiation of hMSCs (Kulterer et al., 2007; Neve et al., 2011; Qi et al., 2003). The time point chosen for our analyses was based on prior microarray analyses (Supplementary file 1, Figure 1—figure supplement 1B), and an in situ calcium staining assay and western blots, which revealed day 10 as a timepoint where progenitor cells have committed to the osteoblast lineage, but cells are still far from the mineralization stage that takes place between day 15 and day 21 (Figure 1—figure supplement 1A–C). We will refer to the cells at this stage as immature osteoblasts (day 10) (Figure 1A). Two days after adding the osteogenic differentiation medium, the hMSCs are still proliferating (data not shown), which is supported by the expression of cyclin E and the presence of hyperphosphorylated pRB2 (p130) as revealed by western blotting (Figure 1—figure supplement 1A). Cells at this stage can be referred to as osteogenic committed progenitor cells (pre-osteoblasts) (Figure 1A) (Neve et al., 2011). While cells are still proliferating to some extent at day 5 (Cyclin E expression), they were completely arrested 10 days after induction of osteogenic differentiation (immature osteoblasts). Figure 1 with 2 supplements see all Download asset Open asset Epigenome and transcriptome profiling in hMSCs during osteogenic differentiation. (A) Schematic presentation of osteogenic (Ost) differentiation of hMSCs. (B) Scatter diagram of base mean (X-axis) and log2 fold differences in H3K27me3 levels (Y-axis) in immature osteoblasts relative to naive hMSC. Values were quantified from two replicates within ± 5 kbp of each TSS, and genes with a significant gain or loss were colored green or orange, respectively. ChIP-seq signal for H3K27me3 (biological replicates) in naive hMSCs and immature-osteoblasts (day 10 of osteogenic differentiation) were quantified at 8,822 TSS (±5 kbp), and analyzed by Deseq2. Green dots represent gene loci with gain of H3K27me3, while orange dots represent gene loci with loss of H3K27me3 (see details in Materials and methods and Supplementary file 3). As indicated, the largest loss of H3K27me3 was observed at the ZBTB16/PLZF locus in immature osteoblasts. (C) ChIP-seq tracks for histone marks and RNA-seq tracks for transcripts are shown for naive hMSCs (day 0) and immature osteoblasts for the ZBTB16/PLZF gene locus. (D) Upper part is a schematic presentation of the ZBTB16 locus (an upstream region; exons 1–7; coding exons 2–7) showing the position of primers used for ChIP-QPCR (marked A-F). Lower panel represents ChIP-QPCR validation of H3K27me3 loss, and H3K27ac gain. The data shown represents three biological replicates shown as average of triplicate values from QPCR ± SD. (E) Loss of Polycomb (SUZ12) binding and increased JMJD3 enrichment after 2 days of osteogenic differentiation analyzed by ChIP-QPCR. The data shown represents three independent experiments and are averages of triplicate samples from QPCR ± SD. General IgG was used as control in ChIP-QPCR. Figure 1—source data 1 Validation of PLZF antibody in ChIP by PLZF kncokdown. https://cdn.elifesciences.org/articles/40364/elife-40364-fig1-data1-v2.xlsx Download elife-40364-fig1-data1-v2.xlsx By analyzing the genome wide data revealing the most pronounced collective changes in histone marks and gene transcription, we observed a correlation between the loss of H3K27me3, a gain in H3K4me3 and increased gene expression at several genomic loci (Figure 1—figure supplement 1D–H, Supplementary file 2). The GO-terms for these groups of genes were associated to bone formation such as ‘extracellular matrix’, ‘calcium’ and ‘osteogenesis’ among others (Figure 1—figure supplement 1I). Examples of induced genes include 1) BMP4, BMP6, IGF2, IGFBP2; gene products known to promote osteogenic differentiation (Hamidouche et al., 2010; Lavery et al., 2008), 2) Leptin, COMP, PPL; genes encoding proteins involved in matrix formation (Ishida et al., 2013; Turner et al., 2013), 3) ZBTB16, HIF3α, examples of transcription factors involved in bone development (Ikeda et al., 2005; Zhu et al., 2014). Examples of ChIP-seq tracks and aligned RNA-seq data are shown in Figure 1C and Figure 1—figure supplement 1J. In contrast to differentiation of other cell types such as neuronal lineage, where a substantial number of gene loci showed dynamic changes in Polycomb repression (Prezioso and Orlando, 2011), H3K27me3 levels were surprisingly stable in differentiating hMSCs, and only few genes have significant changes in H3K27me3 levels at their TSS (Figure 1B, Supplementary file 3). The most striking loss of H3K27me3 was found at the ZBTB16 locus including intragenic exons and 3’UTRs (Figure 1B and C). Interestingly, the loss of H3K27me3 was accompanied by strong gain of H3K27ac across the whole ZBTB16 locus and gain of H3K4me3 at the TSS, which was furthermore confirmed by ChIP-QPCR (Figure 1C and D). PLZF is expressed in osteoblast committed progenitor cells The ZBTB16 gene encodes the transcription factor PLZF, which has been shown to be critical for limb and axial skeleton patterning (Barna et al., 2000; Fischer et al., 2008). Intriguingly, our gene expression and histone modification data revealed that the ZBTB16 gene locus was highly transcribed in response to osteogenic commitment of hMSCs, which is consistent with the loss of H3K27me3 in immature osteoblasts (day 10 of differentiation) (Figure 2A,B, and Figure 1—figure supplement 2C). Observing earlier timepoints, we found that the expression of ZBTB16 was strongly induced (≥ 4000 fold analyzed by QPCR) already at day 2 of differentiation which corresponds to pre-osteoblasts. We therefore analyzed the ZBTB16 locus for the presence of PcG proteins and H3K27me3 levels at day 2 of osteogenic differentiation. Interestingly, we observed a loss of H3K27me3 and binding of the PRC2 subunit SUZ12 already in pre-osteoblasts (Figure 1D and E and data not shown). Furthermore, recruitment of the H3K27me3 demethylase JMJD3 was observed at the locus correlating to the loss of H3K27me3 (Figure 1E). These data revealed that derepression and activation of the ZBTB16 locus and high expression of the PLZF (Figure 2A and B) was an early event in osteogenic differentiation of hMSCs corresponding to the transition of stem cells to osteoblast committed progenitor cells (pre-osteoblasts). Figure 2 with 2 supplements see all Download asset Open asset PLZF induction and binding to enhancers during osteoblasts commitment of progenitor cells. (A) PLZF expression during early osteogenic differentiation of hMSCs shown by (A) RT-QPCR (ZBTB16) and (B) western blot. GAPDH was used as a loading control. (C) Average tag density plot for PLZF peaks from ChIP-seq in naive hMSCs or immature-osteoblasts (day 10). Peak finding revealed 2,282 specific PLZF peaks in immature-osteoblasts (Supplementary file 4). See Supplementary Methods for details regarding peak finding analyses. (D) Pie chart showing the percentage of PLZF peaks (n = 2,282) that overlapped with different genomic regions; promoter (−1 kb to TSS, n = 70); upstream (−10 kb to TSS excluding promoters, n = 195); downstream (+10 kb from the end of the gene, n = 110); exon (n = 75); intron (n = 792); and intergenic (> ± 10 kb from TSS or end of the gene, n = 1,040). (E) GO term analyses of genes nearby to PLZF peaks (n = 2,282) observed by DAVID (The Database for Annotation, Visualization and Integrated Discovery) (Huang da, Bailey et al., 2009). The key words enriched are displayed in the plot. The Y-axis represents the Benjamini Hochberg corrected –LogP value. Grey parts of the bars are above a 0.05 cut-off. (F) Heat-maps at PLZF peaks (n = 2,282) clustered according to local densities of the H3K27me3, H3K4me3, H3K27ac and H3K4me1 histone marks in immature-osteoblasts. ChIP-seq signals from each antibody were normalized to library sizes and visualized at PLZF peaks ± 5 kb. Only PLZF peaks specific for day 10 of osteogenic differentiation were included, and peaks with PLZF signal at day 0 were excluded (for threshold and genomic peak positions please see Supplementary file 3, and Supplementary methods). (G) Heat maps showing the changes in H3K27ac and H3K4me1 at PLZF peaks (n = 2,282), and expression of the nearest gene in naive hMSCs (day 0) and immature osteoblasts. The distribution of normalized H3K27ac or H3K4me1 ChIP seq signal in naïve hMSCs or immature osteoblasts is shown in the heat maps, centered at PLZF peaks identified in immature osteoblasts (± 5 kb). The ratiometric heatmap depicts the changes in H3K27ac and H3K4me1 levels in immature-osteoblasts compared to naive hMSCs (log2fc day10/naïve) and all heatmaps were sorted according to the H3K27ac ratio with the highest day10/naive H3K27ac ratio at the bottom. Gain is colored red (log2 fold ≥ 1, corresponds to 403 PLZF peaks), no change is colored green (log2 fold>-1 and < 1, n = 1,844 PLZF peaks) and loss is colored blue (log2 fold ≤ 1, n = 35 PLZF peaks). The RNA-seq based heat-map shows the log2fc in expression (day10/naive) of nearest transcript mapped to the center of individual PLZF peaks. Transcripts with induced expression are colored red, while blue coloring reflects decreased expression between naïve hMSCs and immature osteoblasts. Examples of genes with increased expression correlating with increased H3K27ac and PLZF binding at nearest genomic region (enhancer) are indicated on the right side. COL11A1, Collagen11A1; SAMHD1, SAM domain and HD domain1; C6, Complement Component 6; NFATC2, nuclear factor of activated T-cells; ALPL, Alkaline Phosphatase; LEPROT, Leptin Receptor Overlapping Transcript; ZBTB16, Zinc Finger and BTB Domain Containing Protein 16. (H) Average normalized ChIP-seq pile-up signal from PLZF or IgG control in naive hMSCs or immature-osteoblasts,± 1 kb around midpoints of enhancers (defined by FANTOM5) transcribed in mesenchymal stem cells (naive hMSCs) and osteoblasts. Pile-up signal was normalized to the average base pair signal across all FANTOM5 enhancers (n = 43,011). Several markers that are expressed during osteogenic differentiation of hMSCs have been described. To understand how the induction of PLZF expression was timed relative to these, we investigated our RNA-seq data for known markers of osteogenesis. As summarized in Figure 1—figure supplement 2A, we observed that RUNX2, often referred to as the master regulator of bone formation (Liu and Lee, 2013; Long, 2011) was expressed already in proliferating naive hMSCs and that the expression was quite stable throughout the time course. Importantly, SOX9 an essential factor for chondrocyte specification and known to antagonize RUNX2 transcriptional activity (Loebel et al., 2015; Zhou et al., 2006), was highly expressed in naive hMSCs, but was strongly reduced at day 2 of osteogenic differentiation. This down regulation of SOX9 likely reflects the transition to osteoblast committed progenitor cells. Furthermore, FOXO1, previously shown to be involved in osteogenic differentiation (Teixeira et al., 2010) was induced at day 2. Importantly, transcription factors of other lineage fates such as MYOD1 (myocytic commitment) and SOX5/8 (chondrocytic commitment)(Chimal-Monroy et al., 2003) were repressed and marked by H3K27me3 in naive hMSCs and still at day 10 of osteogenic differentiation (Figure 1—figure supplement 2A). In addition, factors known to mark the transition from the pre-osteoblast stage to immature osteoblasts such as ALPL (alkaline phosphatase) and SPP1 (osteopontin) (Aubin et al., 1995; Huang et al., 2007) were strongly induced at day 10 of osteogenic differentiation (Figure 1—figure supplement 2A,B), while SP7 (osterix) and BGLAP (osteocalcin), markers of the mineralization stage (Aubin et al., 1995; Harada and Rodan, 2003) were still repressed and marked by H3K27me3 in immature osteoblasts (Figure 1—figure supplement 2A,B,C and data not shown). In conclusion, our analyses position PLZF expression as a very early event during osteogenic differentiation and likely implicated in the transition from naive hMSCs to osteoblast committed progenitor cells (pre-osteoblasts). PLZF is recruited to distal regulatory regions and affects expression of osteogenic-specific genes during differentiation of hMSCs To understand the function of PLZF in osteogenic differentiation of hMSCs, we performed ChIP-seq against PLZF in naive proliferating (day 0) and immature osteoblasts (day 10 of differentiation). We identified 2,282 PLZF peaks that were observed only in immature osteoblasts (Figure 2C, Supplementary file 4). The analysis revealed that the majority of PLZF-binding sites localized at intergenic (45.5%) and intronic regions (34.7%) combined representing 1,830 out of 2,282 PLZF-binding sites (Figure 2D). Very few PLZF-binding sites were observed at gene promoters (± 1 kb of TSS: 3.0%) and upstream (−10 kb from TSS: 8.5%) or downstream (+ 10 kb from TSE: 4.8%) regions (Figure 2D). Discriminative de novo motif analysis of the DNA sequences within the PLZF peaks, compared to sequences from negative control regions, revealed a significant occurrence of a TACAGC motif (E = 1.1 × 10−81), which is highly similar to the TAC(T/A)GTA PLZF motif identified by Ivins et al. (Figure 2—figure supplement 1A and B) (Ivins et al., 2003). Gene ontology analyses of closest genes to PLZF bound regions enriched for terms highly relevant for osteogenic differentiation such as ‘cytoskeleton’ and ‘extracellular matrix’ among others (Figure 2E). The specificity of the PLZF antibody in ChIP was confirmed by PLZF knockdown in hMSCs followed by induction of differentiation and ChIP-QPCR at selected loci (Figure 2—figure supplement 1C–E). PLZF binds to active chromatin regions in immature osteoblasts To study the relationship of PLZF binding at genomic sites and histone modifications, we performed a cluster analysis relating PLZF-binding sites to ChIP-seq data for H3K27me3, H3K4me3, H3K4me1 and H3K27ac histone marks in immature osteoblasts. It has previously been suggested that PLZF functions as a repressor of gene transcription and has a link to PcG recruitment. Therefore, we were surprised to observe a very limited of PLZF with and PRC2 (SUZ12) as well as the H3K27me3 mark by PRC2 (Figure and Figure 2—figure supplement 2A). In there was a of PLZF with H3K27ac and histone marks active gene enhancers et al., 2010) and promoters However, the number of gene promoters bound by PLZF was very (Figure that the majority of regions represent active gene PLZF is recruited to gene enhancers in immature osteoblasts transcription factors often histone to H3K27 acetylation as part of the gene activation (Spitz and Furlong, 2012). To PLZF during osteogenic differentiation to increased H3K27ac at its binding we analyzed the levels of H3K27ac and after PLZF recruitment genome As in the heat (Figure of the 2,282 genomic sites binding PLZF, gained H3K27ac (≥ 2 Supplementary file upon osteogenic differentiation of regions their H3K27ac subset of regions showed in H3K27ac 2 In the H3K4me1 mark was at the regions for the majority of sites (n = immature osteoblasts to naïve hMSCs (Figure Interestingly, the PLZF bound enhancers that gained H3K27ac to that H3K27ac levels and it to the expression of nearest we observed a strong correlation between gain of H3K27ac and increased expression of genes. Gene analyses revealed that of these genes were to panel in Figure and examples of ChIP-seq tracks in Figure 2—figure supplement and Supplementary file This suggested that a subset of enhancers active for the regulation of nearest gene that to an in Furthermore, some of the PLZF-binding sites with levels of showed increased expression of nearest gene upon induction of osteogenic differentiation. This be to other chromatin for other histone at individual enhancers (Figure (Spitz and Furlong, 2012). PLZF to active enhancers enhancers enhancer which can be by global end sequencing In the FANTOM5 this was used to an of active enhancer regions across cell types cell types and cell and tissues et al., 2014). the PLZF-binding sites in immature osteoblasts with the enhancer we observed that out of 2,282 PLZF-binding sites localized within ± 1 kb of a FANTOM5 enhancer, corresponding to enhancers (Supplementary file Interestingly, enhancers in our hMSCs ChIP-seq data were highly enriched for enhancers that were expressed in of naive hMSCs < and osteoblasts < (Figure be that the time point 10 days of osteogenic differentiation, immature not in the FANTOM5 Interestingly, of enhancers identified in our ChIP-seq analysis in immature osteoblasts were positive in naive hMSCs or in osteoblasts out of and out of and out of or of these enhancers are in between naive hMSCs and osteoblasts. This that 1) in naive hMSCs, where PLZF is other TFs a of active enhancers that on during osteogenic differentiation is bound by PLZF and 2) that PLZF likely to be bound to a significant number of such enhancers in terminal differentiated osteoblasts. Moreover, the data that there a number of lineage-specific enhancers that active and gain PLZF binding in the process of differentiation (Figure 2—figure supplement PLZF affects histone H3K27ac at bound enhancers and an osteogenic gene expression To the relationship between PLZF recruitment to enhancers and changes in H3K27ac genome we performed PLZF knockdown in naïve hMSCs followed by induction of osteogenic differentiation, and H3K27ac We three different PLZF (Figure supplement three showed significant knockdown of PLZF compared to control We for it is not to a stable knockdown of PLZF for 10 days of differentiation we analyzed the changes in H3K27ac at day 2 of differentiation where PLZF knockdown was still pronounced (Figure of the data revealed a highly in H3K27ac in pre-osteoblasts, which was reduced PLZF expression was by (Figure Figure with 1 supplement see all Download asset Open asset PLZF-dependent H3K27 acetylation and osteogenic-specific gene expression. (A) of PLZF expression and after knockdown in naïve and hMSCs by western blot. GAPDH was used as loading control. (B) heat maps showing the changes in H3K27ac at PLZF peaks (n = 2,282) in control or the of PLZF knockdown right in hMSCs. The distribution of normalized H3K27ac ChIP seq signal in naive or in osteogenic hMSCs is centered at PLZF peaks (± were clustered according to the to average values for all in each of the three biological replicates for each The regions that H3K27ac upon induction of osteogenic differentiation are colored in blue while regions that gain H3K27ac are marked changes are in (C) tracks representing examples of genomic regions that gain H3K27ac in a PLZF-dependent manner upon induction of osteogenic differentiation (day 2). (D) induced in osteoblast progenitor cells reduced expression in the of PLZF, as observed by representing the of genes from RNA-seq of PLZF down or control hMSCs in three biological replicates) at 2 days of osteogenic are log2 normalized to the average signal of all samples and PLZF from naive and osteogenic induced The is similar to the clustered heatmap from genes from microarray analyses shown in Figure 1—figure supplement 1B (n = (E) the log2 fold between PLZF down or control hMSCs at day 0 and day 2. shows the average values from RNA-seq performed in three biological and the is a (F) The mean normalized expression of selected osteogenic lineage-specific genes in hMSCs after induction of osteogenic differentiation days) in control or PLZF hMSCs. The values are averages from three biological replicates ± SD. SAMHD1, SAM and HD domain containing COL11A1, 1 1 Receptor Receptor Figure data 1