Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

Benjamin L Kidder; Gangqing Hu; Zu-Xi Yu; Chengyu Liu; Keji Zhao

doi:10.1128/MCB.00692-13

. 2013 Dec;33(24):4793–4810. doi: 10.1128/MCB.00692-13

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

Benjamin L Kidder ^a,^✉, Gangqing Hu ^a, Zu-Xi Yu ^b, Chengyu Liu ^c, Keji Zhao ^a,^✉

PMCID: PMC3889548 PMID: 24100015

Abstract

Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process.

INTRODUCTION

Embryonic stem (ES) cells have the unique ability to self-renew indefinitely and differentiate into the hundreds of cell types that exist in the mammalian developmental repertoire. Epigenetic regulation of transcription is critical to achieve defined cellular states that persist in development. ES cell self-renewal versus differentiation is regulated in part by external stimuli that signal to transcription factors (TFs) and chromatin modifiers to regulate the underlying chromatin structure. ES cells express high levels of TFs, such as Oct4, Sox2, Nanog, and Tbx3, that regulate pluripotency by associating with specific DNA sequences to drive expression of a network of pluripotency-related genes and to repress developmentally regulated genes (1–3). Disruption of these core regulatory factors results in a compromised self-renewal state leading to differentiation (4). While the functions of many TFs have been evaluated in ES cells, few studies have focused on the roles of chromatin modifiers in ES cell pluripotency (5–7). Chromatin regulation by way of posttranslational modification of histone tails creates an environment that is conducive or repressive for transcriptional activity, which is critical for propagating expression of networks of genes that maintain self-renewal or promote differentiation.

The trithorax group (trxG) complex regulates methylation of lysine 4 of histone H3 (H3K4me), which is predominantly associated with active genes (8). H3K4me3 is highly enriched at transcriptional start sites (TSS) of highly active genes (9–12) where it is important for RNA polymerase II binding and activation of target genes (13–15). In mammals, methylation of H3K4 is administered by the homolog of trxG, which includes members of the Set/MLL histone methyltransferase (HMT) family. Wdr5 is a core subunit of the MLL complex that is indispensable for development (16), ES cell self-renewal (5), and cellular reprogramming (5). Demethylation of H3K4me3 is facilitated by the lysine demethylase 5 (KDM5/JARID1) family of jumonji (J) C-containing protein complexes, of which there are four members in mammals (17). KDM5 demethylases antagonize H3K4 methylation, and as such they are thought to be transcriptional corepressors (18–20). KDM5B catalyzes the demethylation of tri-, di-, and monomethylation states of H3K4. Kdm5b is mainly expressed during development and in adult tissues, such as testis, thymus, brain, spleen, and eye (21, 22). Kdm5b is also highly expressed in stem cells, including ES cells (23–25), neural progenitors (23, 24), trophoblast stem (TS) cells (26), and blood lineages (27).

Kdm5b is important for early embryonic development, where Kdm5b null embryos (deletion of exon 1) fail to develop beyond the preimplantation stage (embryonic day 4.5 [E4.5]), while deletion of exon 6 results in major neonatal lethality due to many developmental defects (28, 29), and derivation of ES cells from Kdm5b null blastocysts was unsuccessful (29). Previous reports have described contrasting roles for Kdm5b as essential (25) or dispensable (24) for ES cell self-renewal and differentiation. These results suggest that Kdm5b regulates chromatin in a way that is conducive for ES cell pluripotency, although the specific role for Kdm5b in this process is not fully known.

Previous studies have suggested that chromatin modulators regulate reprogramming (5, 30), although the precise epigenetic regulators that mediate this process remain largely unknown. We reasoned that epigenetic “rewiring” of a differentiated genome to a state of pluripotency, by way of Oct4, Sox2, Klf4, and c-Myc overexpression, must involve the regulation of H3K4 methylation, because these marks are enriched at active genes. Since a loss of Wdr5 has been attributed to a decreased reprogramming efficiency (5), it is plausible that depletion of the H3K4 demethylase, Kdm5b, enhances reprogramming.

Therefore, to clarify how the control of H3K4 methylation states by histone demethylase Kdm5b contributes to ES cell function and reprogramming, we investigated the role of Kdm5b in the self-renewal and differentiation of ES cells and in induced pluripotent stem (iPS) cell formation. We found that depletion of Kdm5b in ES cells resulted in decreased self-renewal and impaired differentiation. Kdm5b short hairpin RNA (shRNA) knockdown (shKdm5b) ES cells or Kdm5b conditional knockout ES cells exhibited an inability to deactivate pluripotency regulators and fully differentiate. We also observed extended self-renewal of Kdm5b knockdown ES cells in the absence of LIF, demonstrating that KDM5B is required to exit self-renewal and undergo differentiation. Moreover, we found that depletion of Oct4 does not rescue the differentiation defect of Kdm5b-depleted ES cells. Using mouse embryonic fibroblasts (MEFs) containing doxycycline (dox)-inducible Oct4, Sox2, Klf4, and c-Myc (OSKM) transgenes (31), we found that a loss of Kdm5b accelerates the early stages of reprogramming, which is accompanied by downregulated expression of mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT). We also observed downregulation of EMT genes in the absence of Kdm5b prior to induction of OSKM and rapid deactivation of fibroblast-specific enhancers in the absence of Kdm5b during early reprogramming, demonstrating that Kdm5b is a barrier to reprogramming. Overall, our findings describe novel insights into the role for KDM5B in regulating ES cell differentiation and reprogramming.

MATERIALS AND METHODS

ES cell culture.

R1 ES cells were cultured as previously described, with minor modifications (32). Briefly, R1 ES cells were cultured on irradiated MEFs in Dulbecco's modified Eagle medium (DMEM) and 15% fetal bovine serum (FBS) medium containing LIF (ESGRO) at 37°C with 5% CO₂. For chromatin immunoprecipitation (ChIP) experiments, ES cells were cultured on gelatin-coated dishes in ES cell medium containing 1.5 μM CHIR9901 (glycogen synthase kinase 3 [GSK3] inhibitor) for several passages to remove feeder cells. ES cells were passed by washing with phosphate-buffered saline (PBS) and dissociating with trypsin. For self-renewal experiments in the absence of LIF, ES cells were cultured on gelatin-coated dishes in ES cell medium without LIF and without feeders. For embryoid body (EB) formation, ES cells were cultured in low-attachment binding dishes to promote three-dimensional (3D) formation in ES cell medium without LIF. For differentiation experiments in the absence of Oct4, ZHBTc4 ES cells (33) were infected with luciferase shRNA (shLuc) or shKdm5b lentiviral particles and selected in the presence of 2 μg/ml puromycin. shLuc and shKdm5b ZHBTc4 ES cells were cultured in the presence of 2 μg/ml doxycycline for at least 48 h to downregulate OCT4 expression.

Lentiviral infection.

shRNAs were cloned into the pGreenPuro vector (System Biosciences) according to the manufacturer's protocol. To generate lentiviral particles, 293T cells were cotransfected with an envelope plasmid (plpVSVG), packaging vector (psPAX2), and shRNA expression vector using Lipofectamine 2000. Twenty-four to 48 h posttransfection, the medium containing lentiviral particles was harvested and used to transduce ES cells. Twenty-four hours posttransduction, ES cells were stably selected in the presence of 1 to 2 μg/ml puromycin.

Generation of iPS cells and mouse chimeras.

MEFs containing dox-inducible Oct4, Sox2, Klf4, and c-Myc (OSKM) transgenes (4TF-MEFs) (31) were transduced with shLuc or shKdm5b lentiviral particles expressing green fluorescent protein (GFP) and cultured in MEF medium containing 10% FBS. To initiate overexpression of OSKM, 4TF-MEFs were cultured in ES cell medium containing 2 μg/ml doxycycline. Mouse chimeras were generated by injecting iPS cells into albino B6 blastocysts. All animals were treated according to Institutional Animal Care and Use Committee guidelines approved for these studies at the National Heart, Lung, and Blood Institute.

Teratoma formation.

ES cells were cultured on gelatin-coated dishes to remove feeder cells and dissociated into single cells, and 10⁶ ES cells were injected subcutaneously into SCID-beige mice. After 3 to 4 weeks, mice were euthanized and teratomas were washed and fixed in 10% buffered formalin. Teratomas were then embedded in paraffin. Thin sections were cut and stained with hematoxylin and eosin (H&E) using standard techniques.

qRT-PCR expression analysis.

RNA isolation and quantitative reverse transcription-PCR (qRT-PCR) were performed as previously described, with minor modifications (34). Total RNA was harvested from ES cells using an RNeasy minikit or miRNeasy minikit (Qiagen, Valencia, CA) and DNase treated using Turbo DNA-free (Ambion). Reverse transcription was performed using a Superscript III kit (Invitrogen, Carlsbad, CA). qRT-PCR was performed using TaqMan probes or custom 6-carboxyfluorescein (FAM)-labeled probes and primers and TaqMan universal PCR master mix reagents (Applied Biosystems). Primers used for qRT-PCR with Roche universal probes were designed using the universal probe library assay design center (Roche).

RNA-Seq analysis.

RNA was harvested from ES cells and EBs as described above. mRNA was purified using a Dynabeads mRNA purification kit (Invitrogen). Double-stranded cDNA was generated using a SuperScript double-stranded cDNA synthesis kit (Invitrogen). cDNA was end repaired using the End-It DNA end repair kit (Epicentre), followed by addition of a single A nucleotide and ligation of paired-end (PE) adapters (Illumina) or custom indexed adapters. PCR was performed using Phusion high-fidelity PCR master mix. RNA-Seq libraries were sequenced on an Illumina GAIIX or HiSeq platform according to the manufacturer's protocol.

The RPKM measure (read per kilobases of exon model per million reads) proposed previously (35) was used to quantify the mRNA expression level of a gene from RNA-Seq data sets. Differentially expressed genes were identified using EdgeR (false discovery rate [FDR], <0.001; fold change [FC], >2) (36). Genes with an RPKM of <3 in both conditions were excluded from this analysis.

ChIP-Seq.

ChIP-Seq experiments were performed as previously described, with minor modifications (9). The polyclonal H3K4me3 antibody (CS200580) was obtained from Millipore. The polyclonal H3K4me2 (ab32356) antibody was obtained from Abcam. Briefly, 4TF-MEFs were harvested and chemically cross-linked with 1% formaldehyde (Sigma) for 5 to 10 min at 37°C and subsequently sonicated. Sonicated cell extracts were used for ChIP assays. ChIP-enriched DNA was end repaired using the End-It DNA end repair kit (Epicentre), followed by addition of a single A nucleotide and ligation of PE adapters (Illumina) or custom indexed adapters. PCR was performed using Phusion high-fidelity PCR master mix. ChIP libraries were sequenced on an Illumina HiSeq platform according to the manufacturer's protocol.

Sequence reads were mapped to the mouse genome (mm8) by bowtie (37) with settings eliminating reads mapped to multiple genomic sites. ChIP-Seq read-enriched regions were identified by SICER (38) with a window size setting of 200 bp, a gap setting of 400 bp, and an FDR setting of 0.001.

Microarray data accession number.

The sequencing data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE46893.

RESULTS

Kdm5b-depleted ES cells exhibit reduced self-renewal.

To identify the key regulators of H3K4 methylation in ES cells, we evaluated the absolute expression profile of ES cell transcripts using microarray data (GEO microarray accession number GSE26087) (32) and RNA-Seq data (data not shown). Key regulators of pluripotency, such as Oct4, Sox2, Nanog, and Tbx3, were among the factors highly expressed in ES cells (data not shown). Kdm5b was found to be one of the most highly expressed histone demethylases in ES cells, and as such it was chosen as a target for these studies. To study the loss-of-function phenotype of Kdm5b in ES cells, we designed five shRNAs directed against Kdm5b and infected ES cells with lentiviral particles encoding shRNA sequences. Kdm5b knockdown (shKdm5b) ES cells and control Luciferase-shRNA (shLuc) ES cells were stably selected in the presence of 1 to 2 μg/ml puromycin, which resulted in GFP-positive colonies (Fig. 1A). Kdm5b-shRNA-1 (shKdm5b) resulted in the greatest knockdown of Kdm5b mRNA, as evaluated by qRT-PCR (see Fig. S1A in the supplemental material), and was used for subsequent studies. Kdm5b-shRNA-5 (shKdm5b-5) was used as a second shRNA to confirm the Kdm5b knockdown phenotype. Notably, knockdown of Kdm5b resulted in an altered ES cell colony morphology (Fig. 1A, shKdm5b-5 ES cells; also see Fig. S1B) and decreased proliferation (Fig. 1B). The doubling time of shKdm5b ES cells increased ∼1 to 2 h compared to shLuc ES cells (Fig. 1B). Morphological changes observed following knockdown of Kdm5b were also accompanied by decreased alkaline phosphatase (AP) staining (Fig. 1C), which is indicative of differentiation. Quantification of changes in colony morphology and AP staining levels demonstrated a reduced shape factor, or reduction in colony form, relative to a perfect circle, decreased intensity of AP staining, and a reduced colony size, all of which indicate decreased self-renewal (Fig. 1D). To confirm the phenotype of Kdm5b-knockdown ES cells, we infected a second ES cell line, ES10 (40, 41), with shKdm5b or shLuc lentiviral particles. ES10 ES cells have been shown to exhibit robust differentiation, as demonstrated by high levels of contribution to mouse chimeras via normal blastocyst injection (41) (see Fig. S2A in the supplemental material) or tetraploid complementation (40), which are stringent tests for ES cell pluripotency. shLuc and shKdm5b ES10 ES cells were stably selected in the presence of 1 to 2 μg/ml puromycin (Fig. 1E). Similar to our results observed with R1 ES cells, shKdm5b ES10 ES cells also exhibited an altered colony morphology relative to shLuc ES10 ES cells (Fig. 1E; also see Fig. S2B). shKdm5b ES10 ES cells also demonstrated a decreased proliferation rate relative to shLuc ES10 ES cells (Fig. 1F), suggesting that depletion of Kdm5b results in slightly reduced ES cell self-renewal.

Fig 1 — shKdm5b and Kdm5b^−/− ES cells have reduced self-renewal. (A) R1 ES cells infected with shLuc (control) or shKdm5b lentiviral particles and stably selected with puromycin. The inset shows GFP expression of a selected colony. (B) shKdm5b ES cells have a reduced proliferation rate relative to shLuc ES cells. shKdm5b and shLuc ES cells were passaged several times and replated at equivalent densities to evaluate cell doubling times. (C) Alkaline phosphatase (AP) staining of shLuc and shKdm5b ES cells. Volocity software was used to identify and pseudocolor ES cell colonies. (D) shKdm5b ES cells have reduced shape factor, AP staining, and size relative to shLuc ES cells. (E) ES10 ES cells infected with shLuc or shKdm5b lentiviral particles and selected in the presence of puromycin. (F) shKdm5b ES10 ES cells have a reduced proliferation rate compared to shLuc ES10 ES cells. (G and H) Kdm5b^−/− ES cells (knockout) have a reduced proliferation rate compared to Kdm5b^F/F ES cells (control). (I) Scatter plot of gene expression measured by RPKM between shLuc and shKdm5b ES cells. Log₂-adjusted differentially expressed genes are plotted (greater than 2-fold; RPKM, >1). UCSC genome browser view (J) and qRT-PCR expression (K) of self-renewal genes in shLuc and shKdm5b ES cells. (L) GSEA of differentially expressed genes in Kdm5b knockdown ES cells relative to undifferentiated and retinoic acid differentiation ES cells. (M) Gene ontology (GO) functional annotation of differentially expressed genes was analyzed using DAVID. (N) Western blot of KDM5B, H3K4me3, and H3K4me2 in shLuc and shKdm5b ES cells.

To unambiguously determine the role of Kdm5b in ES cell self-renewal and differentiation, we utilized Kdm5b conditional targeted ES cells (Kdm5b^F/F), where Kdm5b can be efficiently deleted upon administration of doxycycline (24). To induce conditional deletion of Kdm5b, Kdm5b^F/F ES cells were cultured in ES cell medium containing doxycycline for at least 72 h. Following deletion of Kdm5b (Fig. 1G), we observed reduced proliferation of Kdm5b^−/− ES cells relative to Kdm5b^F/F ES cells (Fig. 1H). These findings are similar to our results from shKdm5b R1 ES cells and shKdm5b ES10 ES cells and demonstrate that depletion of Kdm5b results in slightly decreased ES cell self-renewal.

To identify genes that are differentially expressed following knockdown of Kdm5b, we performed RNA-Seq analysis. Using this approach, we found 1,776 genes that were differentially expressed at least 2-fold between shLuc and shKdm5b ES cells, including the pluripotency-related gene Tbx3 (Fig. 1I). Visualization of individual tracks on the UCSC browser (Fig. 1J) and qRT-PCR (Fig. 1K) showed substantial downregulation of Kdm5b transcripts and decreased levels of only a few self-renewal genes, such as Tbx3 (Fig. 1J to K) and Socs3, suggesting that Kdm5b positively regulates a subset of self-renewal genes. However, expression of other self-renewal genes, such as Nanog, Pou5f1, and Sox2, was only slightly decreased (Fig. 1K), and expression of other Jarid family members was unaltered in shKdm5b ES cells relative to shLuc ES cells (see Fig. S3A in the supplemental material). To identify additional regulators of ES cell self-renewal whose expression changes following knockdown of Kdm5b, we compared our RNA-Seq results to data from a previous study describing positive regulators of Oct4 expression and ES cell self-renewal (42). By evaluating the overlap between genes identified in our study and genes identified in this RNA interference (RNAi) screen (42), we identified 29 genes that were differentially expressed at least 1.5-fold between shLuc and shKdm5b ES cells (see Fig. S3B and C), including downregulation of genes such as Gale, Ppp4c, and Zfp771, further suggesting that depletion of Kdm5b results in some loss of ES cell self-renewal.

To understand the expression state of differentially expressed genes, we compared these genes to public gene expression data from undifferentiated and retinoic acid (RA)-differentiated ES cells (4) using gene set enrichment analysis (GSEA) (43). These findings show that differentially expressed genes are enriched in ES cells relative to differentiated cells (Fig. 1L). We then analyzed the gene ontology (GO) terms of genes that are differentially expressed upon knockdown of Kdm5b and identified overrepresented GO terms of genes associated with gene expression, cell cycle, blastocyst development, and differentiation (Fig. 1M). RNAi knockdown of Kdm5b was also accompanied by global increases in H3K4me3 and H3K4me2 levels (Fig. 1N), which is consistent with the role of KDM5B acting as an H3K4 demethylase. These results show that KDM5B regulates global levels of H3K4 methylation, and knockdown of Kdm5b results in a slight decrease in ES cell self-renewal.

KDM5B is important for ES cell differentiation.

We used EB formation as a tool to functionally evaluate the role of Kdm5b during ES cell differentiation. EB formation recapitulates early embryonic development and, as such, represents a stringent in vitro model to evaluate ES cell differentiation. To this end, ES cells were cultured in the absence of LIF for 24 h to 14 days on low-attachment dishes to promote differentiation into EB structures. While shLuc ES cells formed a typical combination of solid and cystic/cavitated EBs in the absence of LIF, the size and cavitation of shKdm5b EBs was significantly reduced (shKdm5b R1 EBs [Fig. 2A], shKdm5b-5 EBs [see Fig. S1C in the supplemental material], and shKdm5b ES10 EBs [see Fig. S2C in the supplemental material]), demonstrating that Kdm5b is important for ES cell differentiation. We also differentiated Kdm5b^F/F (control) and Kdm5b^−/− (knockout) ES cells into EBs as described above. Similar to our findings from shKdm5b ES cells, the size and cavitation of Kdm5b^−/− EBs was reduced compared to that of Kdm5b^F/F EBs (Fig. 2B), demonstrating that Kdm5b is important for ES cell differentiation. A further evaluation of EB differentiation using H&E staining showed that while shLuc ES cells readily form cavitated EBs that contain a primitive endoderm layer (Fig. 2C), shKdm5b ES cells failed to form this cellular layer and maintained a dense cellular mass at the center of the EB (Fig. 2C), suggesting a failure of shKdm5b ES cells to cavitate and fully differentiate. Teratoma formation was subsequently used to evaluate the potential of shKdm5b ES cells to differentiate in vivo into cells represented in the three germ layers. shLuc and shKdm5b ES cells were injected subcutaneously into immunocompromised SCID-beige mice and allowed to develop for 3 to 6 weeks. Teratomas were subsequently dissected and subjected to H&E histological analysis. While shLuc ES cell-derived teratomas contained cells represented in the three germ layers, including ectoderm (keratinized epithelium), mesoderm (muscle and mesenchymal cells, adipocytes), and endoderm (glandular epithelium) (see Fig. S4A in the supplemental material), shKdm5b ES cell-derived teratomas displayed less heterogeneity and differentiated into mainly endoderm (glandular epithelium), into mesoderm cells to a lesser extent, and into ectoderm cells to an even lesser extent (see Fig. S4B), further suggesting that knockdown of Kdm5b impairs differentiation of ES cells. Because shKdm5b ES cells have impaired differentiation, we tested whether shKdm5b ES cells maintain normal colony morphology under differentiation-inducing conditions. Our results demonstrate that shLuc ES cell colonies become flattened upon culture in the absence of LIF and feeder cells (MEFs) for 3 to 4 days (Fig. 2D), while shKdm5b ES cells maintain their three-dimensional structure, further suggesting that they are refractory to differentiation. These results indicate that KDM5B is required for efficient differentiation of ES cells both in vitro and in vivo.

Fig 2 — Kdm5b is important for ES cell differentiation. (A) Embryoid body differentiation is impaired in shKdm5b ES cells. ES cells were grown in the absence of LIF on low-binding culture dishes for 14 days. shLuc ES cells form a heterogeneous mixture of three-dimensional and cavitated/cystic and solid EBs, while shKdm5b ES cells mainly form solid EBs. Scale bar, 1 mm. (B) Impaired EB differentiation of Kdm5b^−/− EBs compared to that of Kdm5b^F/F EBs. EBs were cultured without LIF for 12 days to initiate differentiation. (C) H&E histological sections of shLuc and shKdm5b day 9 EBs. Note the presence of a primitive endoderm layer in shLuc EBs and absence in shKdm5b EBs. Cavitation is significantly diminished in shKdm5b EBs, suggesting an inability to fully differentiate. (D) ES cells were cultured in the absence of LIF for 3 to 4 days to induce differentiation. While shLuc ES cells lost their normal 3D dome-like colony morphology at day 3, shKdm5b ES cells maintained their normal ES cell colony morphology in the absence of LIF over the time course. (E) Schematic representation of the experimental design. ES cells were grown in the absence of LIF for 9 days to induce differentiation, trypsinized, and replated in ES cell medium containing LIF. Fewer ES cell colonies were recovered from day 9 shLuc-ESC-derived EBs than from shKdm5b-ESC derived EBs. (i) Bright-field microscopy of shLuc- and shKdm5b-recovered ES cells. (ii) Alkaline phosphatase (AP) staining of recovered ES cells. (F) shKdm5b EBs, but not shLuc EBs, grown in the absence of LIF for 46 days contain pluripotent cells. (i) Bright-field microscopy of shLuc- and shKdm5b-recovered ES cells. (ii) AP staining of ES cells recovered from day 46 differentiated EBs. (G) Number of AP-positive ES cell colonies following 9 days of EB differentiation.

KDM5B is important for silencing of pluripotency genes during ES cell differentiation.

We attempted to force the differentiation of shKdm5b ES cells by culturing under EB-inducing conditions (without LIF and feeders) for 9 days and subsequently investigating whether a self-renewing population of ES cells is maintained within the EB by dissociating and replating the cells in ES cell medium containing LIF. These results show that a significantly greater number of ES cell colonies were recovered from shKdm5b EBs than with shLuc EB (Fig. 2Ei and Eii and G), as assessed by morphology and AP staining. Surprisingly, ES cells could be recovered from EBs subjected to a longer duration of differentiation by culturing ES cells in the absence of LIF and feeders for 46 days and subsequently assaying, as described above, for the reemergence of ES cell colonies. In this case, while ES cells were unable to be recovered from shLuc EBs, shKdm5b EBs contained a substantial number of ES cells that were AP positive (Fig. 2Fi, Fii, and G). These results are striking because they demonstrate that knockdown of Kdm5b leads to an ES cell state that can be maintained long term in the absence of external self-renewing signals.

To evaluate genes that are differentially expressed during differentiation of shLuc and shKdm5b ES cells, we performed RNA-Seq analysis of undifferentiated and differentiated ES cells through a time course of 14 days. RNA was harvested at several time points from EBs cultured in the absence of LIF (Fig. 3A) and subjected to RNA-Seq and qRT-PCR analyses. K-means clustering (KMC) followed by hierarchical clustering analysis was used to identify major patterns of gene expression variability during EB differentiation (Fig. 3B). These patterns are also visible on a three-dimensional terrain plot (Fig. 3C). Notably, RNA-Seq data displayed on the UCSC browser (Fig. 3D; also see Fig. S5 in the supplemental material) and qRT-PCR (Fig. 3E) showed that pluripotency-related genes, such as Oct4, Sox2, Nanog, Tcl1, Fgf4, and Zfp42, were overexpressed in shKdm5b EBs, while differentiation genes, including Afp, Gata6, Sox17 (endoderm), Col1a1 (ectoderm), and Tbx5 (mesoderm), were underexpressed (Fig. 3D and E; also see Fig. S5 in the supplemental material), suggesting an inability of shKdm5b ES cells to fully escape self-renewal and differentiate. These results show that knockdown of Kdm5b leads to a perturbed regulation of self-renewal and differentiation genes during EB formation, which is consistent with the observed delay in differentiation (Fig. 2). These qRT-PCR results were confirmed by comparing shKdm5b-5 EBs to shLuc EBs (see Fig. S1D in the supplemental material), shKdm5b ES10 EBs to shLuc ES10 EBs (see Fig. S2D in the supplemental material), and Kdm5b^−/− EBs to Kdm5b^F/F EBs (see Fig. S6A and B in the supplemental material). In all cases, we found that self-renewal genes were overexpressed in Kdm5b-depleted EBs, while differentiation genes were underexpressed.

Principal component analysis (PCA) was used as a dimensionality reduction tool to determine the two-dimensional spatial proximity of Kdm5b knockdown versus control EB global expression patterns. PCA showed that shKdm5b EBs move through the first two components at a delayed rate relative to shLuc EBs (Fig. 3F), demonstrating that Kdm5b is required to undergo normal differentiation. We then categorized genes that were differentially expressed between shKdm5b and shLuc EBs based on their reported expression in the three germ layers, ectoderm, mesoderm, and endoderm (24, 44). Using this approach, we found that many ectodermal genes were underexpressed in shKdm5b EBs relative to shLuc EBs (Fig. 3G). Moreover, we also observed overexpression of germ line genes in shKdm5b EBs compared to shLuc EBs (Fig. 3G). Foxd3, whose expression is enriched in ES cells and mesoderm, was also overexpressed in shKdm5b EBs. We also observed several mesoderm and endoderm genes that were differentially expressed in shKdm5b EBs (Fig. 3G).

DAVID (45) was then used as a tool to functionally annotate differentially expressed genes at each time point during EB differentiation (Fig. 3H). These results revealed that a significant number of GO terms related to development were overrepresented in our data set (Fig. 3H), suggesting that Kdm5b regulates developmental genes during differentiation. Because many developmental genes are marked by activating H3K4me3 and repressive H3K27me3 modifications in ES cells (46), we further compared our data set to one for bivalently marked genes. To this end, we evaluated the percentage of bivalent genes that are differentially expressed (purple), and the percentage of differentially expressed genes that are bivalent (green), during differentiation of shLuc and shKdm5b ES cells (Fig. 3I). We observed an increase in the percentage of bivalent genes that are differentially expressed and an increase in the percentage of differentially expressed genes that are bivalent during EB differentiation, suggesting that depletion of Kdm5b results in misregulation of bivalent genes during differentiation. These findings implicate a role for Kdm5b in regulating the expression of bivalent genes during differentiation.

We also used GSEA to evaluate the expression state of differentially expressed genes during shLuc and shKdm5b ES cell differentiation (Fig. 3J). These findings show that during early EB formation, differentially expressed genes are enriched in ES cells. However, during later EB formation, differentially expressed genes are enriched in EBs, suggesting that developmental genes are misexpressed in shKdm5b EBs (Fig. 3J).

We also built a predictive model to determine the probability of differential expression due to chance or delayed differentiation following Kdm5b knockdown and EB formation. Expression patterns that represent delayed differentiation include genes that are not appropriately downregulated or upregulated during EB formation. Our results show that the percentage of upregulated and downregulated genes lagged behind the expected level (Fig. 3K), demonstrating that Kdm5b knockdown results in delayed differentiation. The x axis shows genes that are upregulated or downregulated from ES cells to EBs at 24 h or from ES cells to EBs at day 14. These findings provide further evidence that Kdm5b knockdown leads to gene expression changes that are consistent with delayed differentiation.

Depletion of Oct4 is insufficient to rescue the differentiation defect of Kdm5b-depleted ES cells.

To test whether the observed reduced differentiation of shKdm5 ES cells is caused by sustained expression of pluripotency factors, we employed an ES cell line (ZHBTc4) (33) that can be induced to deplete Oct4, in combination with shRNA-mediated knockdown of Kdm5b transcripts as described above. In the presence of LIF, the phenotype of shKdm5b ZHBTc4 ES cells relative to shLuc ZHBTc4 ES cells was similar to that of shRNA-depleted cells or conditionally deleted Kdm5b ES cells as described above, where shKdm5b ZHBTc4 ES cells have a slightly reduced colony size (Fig. 4A). Expression analysis revealed that Kdm5b transcript levels were downregulated at least 95% in shKdm5b ZHBTc4 ES cells (Fig. 4B). Moreover, we observed an expression pattern of ES cell regulators in shKdm5b ZHBTc4 ES cells relative to shLuc ZHBTc4 ES cells that was similar to the pattern we found when comparing shKdm5b ES cells to shLuc ES cells (Fig. 1K and 4B). Following 3 days of Oct4 depletion in the presence of doxycycline and 1 day in the absence of LIF, we observed differentiation of shLuc ZHBTc4 ES cells (Fig. 4C). However, shKdm5b ZHBTc4 ES cells remained in a more typical ES cell three-dimensional colony morphology (Fig. 4C), suggesting that depletion of Oct4 subsequent to downregulation of Kdm5b is not sufficient to induce normal ES cell differentiation. We next differentiated shKdm5b ZHBTc4 ES cells under EB-inducing conditions (without LIF or feeders) as described above. In the presence of Oct4 and absence of LIF, the size and cavitation of shKdm5b ZHBTc4 EBs was reduced compared to that of shLuc ZHBTc4 EBs (Fig. 4D, top). Moreover, in the absence of Oct4 and LIF, while shLuc ZHBTc4 EBs formed numerous cavitated EBs, shKdm5b ZHBTc4 EBs failed to undergo cavitation (Fig. 4D, bottom), demonstrating that depletion of Oct4 does not rescue the impaired differentiation of Kdm5b-depleted ES cells. Expression analysis revealed that while self-renewal genes were downregulated during differentiation in the absence of Oct4 and Kdm5b, lineage-specific genes were underexpressed in shKdm5b ZHBTc4 EBs relative to shLuc ZHBTc4 EBs (Fig. 4E). These results suggest that while depletion of Oct4 in shKdm5b ZHBTc4 ES cells leads to downregulation of several self-renewal genes that are targets of Oct4, it is not sufficient to induce normal differentiation; Kdm5b is required for downregulation of several pluripotency factors and activation of differentiation genes, which is required for efficient differentiation of ES cells.

Fig 4 — Depletion of Oct4 does not rescue the differentiation defect of Kdm5b-depleted ES cells. (A) Oct4-regulatable ES cells (ZHBTc4) infected with shLuc or shKdm5b lentiviral particles. (B) qRT-PCR expression of self-renewal genes in shLuc and shKdm5b ZHBTc4 ES cells. (C) shLuc and shKdm5b ZHBTc4 ES cells were cultured in the presence of doxycycline to downregulate Oct4 levels and induce differentiation in the absence of LIF. Note the presence of colonies with typical 3D colony morphology in shKdm5b ZHBTc4 ES cells relative to shLuc ZHBTc4 ES cells. (D) EB differentiation is impaired in shKdm5b ZHBTc4 ES cells in the absence of Oct4 and LIF. shLuc ZHBTc4 ES cells (−Oct4) form predominantly cavitated EBs, while shKdm5b ZHBTc4 ES cells (−Oct4) mainly form solid EBs. (E) qRT-PCR expression of self-renewal and differentiation genes in shLuc and shKdm5b ZHBTc4 ES cells and EBs.

Depletion of Kdm5b leads to accelerated reprogramming.

Generating induced pluripotent stem (iPS) cells by somatic cell reprogramming requires global epigenetic changes (47). While several studies have surveyed epigenetic states before and after reprogramming (44, 48), few studies have investigated the roles of epigenetic modifiers during reprogramming (5, 30). Therefore, to evaluate the role of Kdm5b in reprogramming, we used a genetically homogenous dox-inducible lentivirus system to overexpress Oct4, Sox2, Klf4, and Myc (OSKM) in MEFs (31). Because this system is genetically homogenous, expression of the reprogramming factors is less variable relative to lentiviral infection of a population of MEFs. Therefore, this model represents a robust system to study the role of Kdm5b in reprogramming. Equal numbers of MEFs containing dox-inducible OSKM transgenes (4TF-MEFs) were infected with lentiviral particles encoding either shLuc or shKdm5b RNA sequences, cultured in MEF medium for 2 days, and subsequently reseeded for 2 days in MEF medium (Fig. 5A). Twenty-four to 48 h postinfection, shLuc and shKdm5b 4TF-MEFs contained relatively equal numbers of GFP-positive cells. To initiate reprogramming, shLuc and shKdm5b 4TF-MEFs were cultured in ES cell medium containing doxycycline, LIF, and a small-molecule inhibitor(s), either GSK3β (CHIR99021; GSK3i), GSK3i/MEKi (two inhibitors), or GSK3i/MEKi/LSD1i (three inhibitors) (Fig. 5A). Addition of GSK3β, MEK, and LSD1 inhibitors has been shown to enhance the efficiency of generating iPS cell colonies (49, 50). Fluorescence and bright-field microscopy was used to visualize reprogramming of shLuc and shKdm5b 4TF-MEFs. Following 4 days of reprogramming, shKdm5b 4TF-MEFs contained slightly increased levels of GFP-positive cells compared to shLuc 4TF-MEFs (Fig. 5B). After 6 days, shKdm5b 4TF-MEFs exhibited an increased number of GFP-positive cells and increased reprogramming, as assessed by morphological changes, relative to shLuc 4TF-MEFs (Fig. 5B). Strikingly, shKdm5b 4TF-MEFs contained iPS colonies that were ready to be picked and subcloned at 6 days, demonstrating the ability of MEFs to rapidly convert to a primitive state in the absence of Kdm5b. After 11 days, shKdm5b 4TF-MEFs continued to rapidly proliferate, as determined by increased GFP-positive cells, relative to shLuc 4TF-MEFs (Fig. 5B). Several GFP-positive shLuc and shKdm5b iPS cell clones were picked and expanded beginning at day 7 for shKdm5b iPS cells and days 12 to 14 for shLuc iPS cells (Fig. 5C; also see Fig. S7A in the supplemental material). Several shLuc and shKdm5b iPS cell lines that morphologically resemble typical iPS cells are shown in Fig. S7B in the supplemental material. shLuc and shKdm5b iPS cell lines that morphologically resemble typical iPS cells are shown in Fig. 5C. shLuc and shKdm5b iPS cells morphologically resemble their ES cell counterparts, where shKdm5b iPS cells exhibit a slightly compromised colony morphology relative to shLuc iPS cells (Fig. 5C). This slight moderation in colony morphology was also observed between shLuc and shKdm5b ES cells (Fig. 1A).

We also visualized reprogramming in 6-well dishes using tiled wide-field confocal microscopy (Fig. 5D). This approach allows for imaging of GFP-positive cells in their entirety within a dish, representing a robust method to capture differential reprogramming efficiencies. These results show that shKdm5b 4TF-MEFs have a greater number of GFP-positive colonies than shLuc 4TF-MEFs (Fig. 5E). Quantitation of changes in the number of GFP-positive colonies demonstrated an increase in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs (Fig. 5F), suggesting that depletion of Kdm5b leads to enhanced reprogramming.

To test the efficiency of generating iPS cells upon knockdown of Kdm5b, we performed AP staining of shLuc and shKdm5b 4TF-MEFs after 18 days of reprogramming (Fig. 5G). These results reveal that shKdm5b 4TF-MEFs have a greater number of AP-positive colonies than shLuc 4TF-MEFs (Fig. 5H), demonstrating that the knockdown of Kdm5b results in greater reprogramming efficiency, and suggest that Kdm5b is a barrier to reprogramming.

We also investigated the expression of pluripotency and lineage-specific genes during early reprogramming in the absence of Kdm5b. qRT-PCR demonstrated that Kdm5b expression was substantially downregulated in shKdm5b 4TF-MEFs compared to shLuc 4TF-MEFs during early reprogramming (48 h) (Fig. 5I). While the transcription factors Pou5f1, Sox2, Klf4, and Myc were relatively equally upregulated in both shLuc and shKdm5b 4TF-MEFs (Fig. 5I), expression of Pou5f1 and Sox2 was slightly higher in shKdm5b 4TF-MEFs. Interestingly, mesenchymal master regulators, which are induced in EMT (Snai1, Snai2, Zeb1, and Zeb2), were downregulated to a greater extent during reprogramming of shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs (Fig. 5I). It was previously shown that a mesenchymal-to-epithelial (MET) transition, which involves the suppression of pro-EMT genes, is required to initiate and facilitate reprogramming (51). The decrease in expression of fibroblast genes during reprogramming in the absence of Kdm5b suggests that a loss of Kdm5b results in a transcriptional landscape that is more susceptible to reprogramming.

To further evaluate differentially expressed genes between control and Kdm5b knockdown MEFs prior to the induction of reprogramming and during reprogramming, we used RNA-Seq analysis to compare the expression profiles of shLuc and shKdm5b 4TF-MEFs (Fig. 6A) and shLuc and shKdm5b 4TF-MEFs following 48 h of OSKM overexpression (Fig. 6B). A comparison of shLuc and shKdm5b 4TF-MEFs demonstrated that many mesenchymal master regulators are downregulated in shKdm5b 4TF-MEFs prior to induction of OSKM, including Snai2, Twist1, Twist2, Zeb1, and Zeb2 (Fig. 6A). Moreover, expression of other fibroblast-enriched genes, including Cldn1, Dsp1, and Tgfβ2, was also downregulated in shKdm5b 4TF-MEFs (Fig. 6A). Following 48 h of OSKM overexpression, we observed continued downregulation of mesenchymal master regulators and fibroblast-enriched genes, including Twist2, Zeb2, and Tgfβ2, and upregulation of ES cell-enriched genes, such as Rest, Stat3, and Tcf3 (Fig. 6B). These results suggest that Kdm5b is important to maintain expression of mesenchymal genes in MEFs, depletion of Kdm5b leads to rapid MET transition upon OSKM overexpression, and a loss of Kdm5b leads to early activation of self-renewal genes during reprogramming.

Fig 6 — Kdm5b is important for iPS cell differentiation. Scatter plot of gene expression measured by RPKM between shLuc and shKdm5b 4TF-MEFs (A), shLuc and shKdm5b 4TF-MEFs following 48 h of OSKM overexpression (B), and shLuc and shKdm5b iPS cells (C). Log₂-adjusted differentially expressed genes (greater than 1.5-fold; RPKM, >1) are shown as black (A and B) or blue (C) points. In panel C, self-renewal genes are shown as red points. (D) qRT-PCR expression of self-renewal genes in shLuc and shKdm5b iPS cells and shLuc ES cells. (E) EB differentiation is perturbed in shKdm5b iPS cells. iPS cells were grown without LIF for 14 days. shLuc iPS cells form a heterogeneous population of cavitated and solid EBs, while shKdm5b ES cells mainly formed solid EBs. Scale bars, 500 μm and 1 mm. (F) H&E histological sections of shLuc and shKdm5b day 10 and day 13 EBs. Note the presence of a primitive endoderm layer in shLuc EBs and its absence from shKdm5b EBs. Cavitation is also decreased in shKdm5b EBs, demonstrating an inability to fully differentiate. (G) qRT-PCR expression of self-renewal and differentiation genes in undifferentiated ES cells and EB-differentiated shLuc and shKdm5b iPS cells for 14 days. (H and I) Teratomas generated from shLuc and shKdm5b iPS cells injected into SCID-beige mice. Tumors were harvested and evaluated as described above. Transmitted white-light microscopy of sectioned teratomas. (H) Heterogeneous differentiation of shLuc iPS cells into ectoderm, mesoderm, and endoderm. (I) Differentiation of shKdm5b iPS cells into a less heterogeneous mixture of cells. (J) shLuc 4TF-MEF-derived iPS cells were shown to be pluripotent by their contribution to a mouse chimera after injection into albino B6 blastocysts as indicated by coat color.

Kdm5b is important for iPS cell differentiation.

Representative shLuc and shKdm5b iPS cell colonies were picked and expanded for further characterization (Fig. 5C). We further compared the expression profiles of shLuc iPS cells and shKdm5b iPS cells using RNA-Seq analysis (Fig. 6C). Using this method, we found that many pluripotency-related genes, including the Pou5f1, Sox2, Nanog, Esrrb, Klf4, Nr5a2, Stat3, Tcf3, and Utf1 genes, were relatively similarly expressed between shLuc and shKdm5b iPS cells (Fig. 6C). These findings suggest that the expression profile of shKdm5b iPS cells is relatively similar to that of shLuc iPS cells. qRT-PCR analysis revealed that shLuc and shKdm5b iPS cells expressed self-renewal genes for Oct4, Sox2, Nanog, Klf4, Tbx3, and Rest at a level relatively similar to that of shLuc ES cells (within ∼2-fold) (Fig. 6D). Moreover, shKdm5b iPS cells expressed reduced levels of Kdm5b mRNA relative to shLuc iPS cells or shLuc ES cells (∼70%) (Fig. 6D), although the level of knockdown was not as pronounced as that for the original knockdown in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs (>90%) (Fig. 5I) or shKdm5b ES cells relative to shLuc ES cells (>95%) (Fig. 1I to K). This most likely is due to the absence of drug selection (puromycin) during the culture of shKdm5b and shLuc iPS cells.

We then used EB formation to evaluate the role of Kdm5b during iPS cell differentiation. shLuc and shKdm5b iPS cells were cultured in the absence of LIF for 14 days on low-attachment dishes to promote EB differentiation. Our findings from these experiments are similar to the results we observed following differentiation of shKdm5b ES cells. shLuc iPS cells formed a heterogenous mixture of solid and cavitated EBs in the absence of LIF, while shKdm5b iPS cells formed EBs of significantly decreased size and cavitation (Fig. 6E), showing that Kdm5b is important for iPS cell differentiation. H&E histological staining also showed that while shLuc iPS cells form cavitated EBs that contain a primitive endoderm layer (Fig. 6F), shKdm5b iPS cells formed EBs without a primitive endoderm layer and with less cavitation (Fig. 6F). These results suggest a failure of shKdm5b iPS cells to cavitate and fully differentiate. qRT-PCR was then used to evaluate differential expression of self-renewal and differentiation genes in undifferentiated ES cells and EB-differentiated shLuc and shKdm5b iPS cells for 14 days. (Fig. 6G). RNA was harvested from ES cells and EBs cultured in the absence of LIF (Fig. 6G) and subjected to qRT-PCR. Similar to our results from ES cells, pluripotency-related genes such as the Oct4, Sox2, and Nanog genes were overexpressed in shKdm5b EBs, while differentiation genes such as the Afp, Cdx2, Gata6, and Sox17 genes (endoderm), as well as the Hand1, Nkx2.5, and Tbx5 genes (mesoderm), were underexpressed (Fig. 6G), suggesting an inability of shKdm5b iPS cells to fully differentiate. These results are in alignment with the observed delayed differentiation of shKdm5b iPS cells and are similar to our findings from ES cells (Figs. 2 to 3), where depletion of Kdm5b leads to a misregulation of self-renewal and differentiation genes during EB differentiation.

Teratoma formation was then used to evaluate the in vivo differentiation potential of shKdm5b iPS cells. shLuc and shKdm5b ES iPS cells were injected into immunocompromised mice as described above and allowed to develop for 4 to 6 weeks. H&E histological analysis was subsequently performed on dissected teratomas. Our results show that shLuc iPS cell-derived teratomas contain a mixture of cells, including neuroectodermal cells, mesoderm lineages, including adipocytes and muscle, and glandular endodermal cells (Fig. 6H), while shKdm5b iPS cell-derived teratomas contain a less heterogeneous mixture of cells, including neuroectodermal cells, mesoderm lineages, such as adipocytes, muscle, and osteoclasts, and glandular endodermal cells (Fig. 6I). Overall, while these results show that shLuc and shKdm5b iPS cells are both pluripotent, teratomas derived from Kdm5b-depleted iPS cells have reduced cellular heterogeneity relative to shLuc iPS cells. These results are consistent with the perturbed differentiation of Kdm5b-depleted ES cells, suggesting that Kdm5b is important for normal differentiation.

Contribution to mouse chimeras represents a stringent test to evaluate pluripotency. Therefore, to evaluate the in vivo differentiation potential of control and Kdm5b knockdown iPS cells, 2-shLuc iPS cells and 7-shKdm5b iPS cells were injected into albino B6 host blastocysts and transferred to pseudopregnant female mice. Mice were allowed to develop to term, and chimerism was evaluated by coat color. Our results show that shLuc iPS cells are able to contribute to mouse chimeras (Fig. 6J). However, we did not observe live chimeric mice generated from shKdm5b iPS cells, which may result from defective differentiation or development in the absence of Kdm5b in ES or iPS cells. These findings are in alignment with the in vitro EB differentiation phenotypes of shKdm5b ES cells and Kdm5b^−/− ES cells, where depletion of Kdm5b leads to defective differentiation.

KDM5B regulates H3K4 methylation at MEF-specific promoters and enhancers during early reprogramming.

Because depletion of Kdm5b, which is highly expressed in both ES cells and MEFs (∼1- to 1.5-fold higher in ES cells than in MEFs), leads to accelerated reprogramming of MEFs, we evaluated the regulation of H3K4 methylation profiles during early reprogramming of MEFs. shLuc and shKdm5b 4TF-MEFs were cultured in MEF medium without LIF for 7 days, and doxycycline was added subsequently for 48 h to induce OSKM overexpression (Fig. 7A). Following 24 h of OSKM overexpression, shKdm5b 4TF-MEFs exhibited a spatially condensed cellular morphology (Fig. 7B), which is one of several cellular alterations that occurs during reprogramming, relative to shLuc 4TF-MEFs. We then performed ChIP-Seq using chromatin from shLuc and shKdm5b 4TF-MEFs treated for 48 h with doxycycline and antibodies specific to H3K4me3 and H3K4me2. Our results revealed modest global changes to the number of H3K4me3 (Fig. 7C)- and H3K4me2 (Fig. 7D)-enriched islands (fold change, >1.5; FDR, <0.001). However, upon inspection of genes with altered H3K4 methylation levels at their promoters, several fibroblast-enriched genes showed significant decreases in H3K4me3 levels (Fig. 7E). These results are also evident when evaluating promoter densities of H3K4me3 at fibroblast genes (Fig. 7F). Conversely, ES cell-specific genes, such as the Pou5f1 and Tbx3 genes, displayed increased H3K4me3 densities at their promoters in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs. These results suggest that Kdm5b is important in maintaining H3K4 methylation levels at genes expressed in fibroblasts, and in the absence of Kdm5b, MEFs are more susceptible to reprogramming by OSKM overexpression. We then analyzed the GO terms of genes with differential H3K4me3/2 levels following knockdown of Kdm5b during early reprogramming and identified overrepresented GO terms of genes associated with various developmental events (Fig. 7G and H), suggesting that Kdm5b regulates H3K4 methylation at developmental genes during reprogramming.

Fig 7 — Depletion of Kdm5b leads to accelerated deactivation of MEF enhancers during early reprogramming. (A) 4TF-MEFs were infected with shLuc or shKdm5b lentiviral particles, reseeded at a lower density, and grown in MEF medium for 7 days. To activate OSKM overexpression and induce reprogramming, 4TF-MEFs were grown in ES cell medium containing doxycycline for 48 h. (B) Bright-field microscopy of shLuc and shKdm5b 4TF-MEFs 24 h following OSKM overexpression. Note the condensed spatial morphology and increased ratio of nucleus to cytoplasm of shKdm5b MEFs, which are cellular alterations that occur during reprogramming. Also shown are changes in global levels of H3K4me3 (C) and H3K4me2 (D) in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs after 48 h of OSKM overexpression. (E) UCSC genome browser view of H3K4me3/2 marks at promoters of genes whose expression is enriched in MEFs (Snai, Msc1, and Rims2) after 48 h of OSKM overexpression to induce reprogramming of shLuc and shKdm5b 4TF-MEFs. shKdm5b 4TF-MEFs have decreased H3K4me3/2 levels at these promoters relative to 4TF-MEFs. (F) Normalized densities of H3K4me3 at representative promoters in shLuc and shKdm5b 4TF-MEFs after 48 h of reprogramming. (G) Gene ontology (GO) functional annotation of genes whose promoters have differentially enriched H3K4me3/2 was analyzed using DAVID. (H) Enrichment of developmental GO terms associated with genes exhibiting altered H3K4me3/2 levels between shLuc and shKdm5b 4TF-MEFs during early reprogramming (48 h of OSKM overexpression). CNS, central nervous system. (I) H3K4me3 levels at fibroblast-defined p300 marked enhancers during early reprogramming. Note the decrease in H3K4me3 levels at enhancers in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs.

We next investigated whether MEF enhancers are deactivated more rapidly in shKdm5b 4TF-MEFs than shLuc 4TF-MEFs during early reprogramming. To this end, we compared H3K4me3 densities at MEF-specific p300-bound regions (52) in shLuc and shKdm5b 4TF-MEFs 48 h after OSKM overexpression (Fig. 7I). These results show a marked decrease in H3K4me3 levels at enhancers in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs (Fig. 7I), suggesting that depletion of Kdm5b leads to accelerated deactivation of fibroblast-specific enhancers during early reprogramming.

Sustained knockdown of Kdm5b prior to OSKM overexpression allows for rapid reprogramming.

Because we observed accelerated reprogramming in the absence of Kdm5b, we reasoned that depletion of Kdm5b before OSKM overexpression allows for more rapid reprogramming. To test this hypothesis, we plated equal numbers of shLuc and shKdm5b 4TF-MEFs at low density in MEF medium without LIF for 7 days and subsequently initiated OSKM overexpression by addition of ES cell medium containing doxycycline, LIF, and small molecules (GSK3i, GSK3i/MEKi [two inhibitors], PDCA, LSD1i, or GSK3i/MEKi/LSD1i [three inhibitors]) for up to 21 days (Fig. 8A). Two studies have identified 2,4-pyridine-dicarboxylic acid (PDCA) as a small-molecule inhibitor of KDM5B (53, 54). Following 24 h of OSKM overexpression, we observed a higher density and compaction of shKdm5b 4TF-MEFs than shLuc 4TF-MEFs (Fig. 8B). Following 7 days of OSKM overexpression, we observed increased reprogramming of shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs (Fig. 8C), as determined by the formation of iPS cell colonies. Strikingly, a combined depletion of Kdm5b transcripts in 4TF-MEFs and chemical inhibition using PDCA resulted in iPS cell colonies within 6 days of OSKM overexpression (Fig. 8C), demonstrating that sustained depletion of Kdm5b in MEFs facilitates rapid reprogramming upon OSKM overexpression. We then tested the efficiency of generating iPS cells upon sustained knockdown of Kdm5b by performing AP staining of shLuc and shKdm5b 4TF-MEFs after 14 days of reprogramming (Fig. 8D). These results show an increase in AP-positive colonies in shKdm5b 4TF-MEFs compared to shLuc 4TF-MEFs, further demonstrating that knockdown of Kdm5b enhances reprogramming. Tiled confocal microscopy of 12-well dishes (Fig. 8E) also showed an increase in GFP-positive colonies after 13 days of reprogramming (Fig. 8F). AP staining also showed an increase in AP-positive shKdm5b 4TF-MEF colonies relative to shLuc 4TF-MEF colonies (Fig. 8G), further demonstrating that sustained depletion of Kdm5b enhances reprogramming efficiency.

Fig 8 — Rapid reprogramming after sustained knockdown of Kdm5b and treatment with small-molecule inhibitors. (A) Schematic of experimental design. 4TF-MEFs were infected with shLuc or shKdm5b lentiviral particles, reseeded at a lower density, and grown in MEF medium for 7 days. 4TF-MEFs were then grown in ES cell medium containing small molecules and doxycycline for up to 3 weeks to induce OSKM overexpression and reprogramming. (B and C) Bright-field microscopy of shLuc and shKdm5b 4TF-MEFs after 24 h (B) and 7 days (C) of reprogramming by OSKM overexpression and treatment with small molecules. Note the increased density of shKdm5b cells compared to that of shLuc cells during reprogramming and visibility of shKdm5b iPS cell colonies within 7 days of reprogramming. (D) AP staining of shLuc and shKdm5b 4TF-MEFs reprogrammed with OSKM overexpression and small molecules for 2 weeks. (E) Tiled wide-field confocal imaging of 12-well plates used to capture GFP from shLuc and shKdm5b 4TF-MEFs during reprogramming. (F) Confocal imaging of shLuc and shKdm5b 4TF-MEFs after 13 days of reprogramming. (G) AP staining of reprogrammed shLuc and shKdm5b 4TF-MEFs used for confocal imaging shown in Fig. 5E.

DISCUSSION

ES cells have the unique ability to self-renew indefinitely in the presence of appropriate signals and to differentiate into the myriad of cell types that exist in mammals. While many studies have focused on deconvoluting the signaling pathways and transcription factors that maintain ES cell self-renewal, few studies have focused on the roles of demethylases in ES cell differentiation and reprogramming. Our results demonstrate that Kdm5b is important for ES cell differentiation and is a barrier to the reprogramming process.

KDM5B regulates ES cell differentiation.

Our results demonstrate that KDM5B functions in ES cells to maintain self-renewal and to control differentiation. We found that Kdm5b knockdown and knockout ES cells have reduced proliferation and colony morphology compared to control ES cells. These changes are also accompanied by differential gene expression of several genes associated with self-renewal, such as the Tbx3 gene. While we observed a slight reduction in self-renewal of shKdm5b ES cells and Kdm5b^−/− ES cells compared to control ES cells, the colony morphology was not as compromised as that presented in a previous report (25). This contrasting result may be explained in part by the use of transient transfection, which may have led to the differentiated or cytotoxic phenotype. However, our results are more comparable to those of another study which showed that depletion of Kdm5b has little effect on ES cell self-renewal (24). Our findings also support a role for Kdm5b in regulating expression of developmental genes during differentiation. Interestingly, we found that Kdm5b-depleted ES cells failed to deactivate expression of pluripotency regulators, such as Pou5f1, Sox2, and Nanog. These results are in alignment with a previous study which showed that pluripotency genes were not completely silenced during differentiation of Kdm5b knockdown ES cells (24). Also, Kdm5b knockdown ES cells were unable to fully escape self-renewal, as demonstrated by the persistence of self-renewing cells following prolonged differentiation in the absence of Kdm5b. Temporal transcriptome analysis revealed that developmental genes are misexpressed during differentiation in the absence of Kdm5b, further suggesting that Kdm5b is critical for promoting differentiation. Lineage-specific genes were underexpressed following EB differentiation of shKdm5b ES cells and Kdm5b^−/− ES cells relative to shLuc ES cells (Fig. 3D, E, and G; also see Fig. S1, S2, S4, and S6 in the supplemental material). Moreover, we observed delayed EB differentiation (Fig. 2A to C; also see Fig. S1C and S2C) and inhibited teratoma differentiation (see Fig. S4) of shKdm5b ES cells and Kdm5b^−/− ES cells, demonstrating the importance of Kdm5b for differentiation. It is possible that Kdm5b-depleted ES cells fail to properly differentiate due to a combination of inefficient deactivation of self-renewal genes and activation of differentiation genes. While a combined depletion of Oct4 and Kdm5b results in downregulation of Oct4 target genes, a loss of Oct4 is insufficient to induce normal differentiation of Kdm5b-depleted ES cells (Fig. 4C to E), suggesting that Kdm5b plays an important role during differentiation. Because KDM5B has been shown to bind promoters of active genes marked by H3K4me3 in human ES cells (55) and developmental genes marked with H3K4me3 (24) in mouse ES cells, it is plausible that KDM5B is required to deactivate self-renewal genes during differentiation to reset the underlying chromatin and to repress developmental regulators in ES cells. In the latter case, KDM5B may prevent activation of developmental genes in ES cells by demethylating promoters and enhancers. This is in alignment with ChIP binding profiles of KDM5B at promoters of active genes and developmental regulators in ES cells (24, 55).

KDM5B is a barrier to the reprogramming of differentiated cells.

Results presented in this study demonstrate that Kdm5b is a barrier to the reprogramming process, where Kdm5b knockdown 4TF-MEFs exhibited accelerated reprogramming relative to control 4TF-MEFs. These findings reveal that Kdm5b is dispensable for reprogramming. Moreover, depletion of Kdm5b in MEFs prior to induction of OSKM and in the early stages of reprogramming facilitates rapid deactivation of fibroblast-specific genes, as determined by decreased expression of mesenchymal master regulators in shKdm5b 4TF-MEFs relative to shLuc 4TF-MEFs, and decreased H3K4 methylation levels at fibroblast-specific enhancers. Thus, our findings suggest that Kdm5b is a barrier to the reprogramming process. Depletion of Kdm5b in MEFs leads to deactivation of pro-EMT genes prior to induction of OSKM, and during OSKM overexpression depletion of Kdm5b facilitates a rapid resetting of the underlying epigenetic state in a manner that is conducive for reprogramming.

Our results also demonstrate that shKdm5b iPS cells are functionally similar to shKdm5b ES cells and Kdm5b^−/− ES cells; they are both unable to fully differentiate and deactivate self-renewal genes. shKdm5b iPS cells also exhibited delayed differentiation (Fig. 6E and F), persistent expression of self-renewal genes (Fig. 6G), and underexpression of differentiation genes (Fig. 6G), which is consistent with the phenotype of shKdm5b ES cells and Kdm5b^−/− ES cells. These results suggest that while a loss of Kdm5b accelerates the early stages of reprogramming and acquisition of a primitive cell state, Kdm5b is important for full pluripotency of resulting iPS cells. In other words, while Kdm5b is a barrier to the reprogramming process, Kdm5b is important for ES cell and iPS cell differentiation. Moreover, our results demonstrating that KDM5B is important for ES cell differentiation are in agreement with our finding that depletion of Kdm5b leads to accelerated reprogramming. In both cases, depletion of Kdm5b favors a cellular state that is resistant to terminal differentiation. Kdm5b knockdown ES cells are unable to efficiently undergo lineage commitment, and Kdm5b knockdown MEFs continue to proliferate while control MEFs undergo terminal differentiation. After culture for an extended time prior to initiation of reprogramming (Fig. 8A), proliferation of shLuc 4TF-MEFs decreased relative to shKdm5b 4TF-MEFs. Escape from terminal differentiation in the absence of Kdm5b may in part explain the increased reprogramming efficiency. Additionally, if KDM5B is required to actively regulate H3K4 methylation at enhancer regions of MEF-specific genes, the absence of KDM5B may explain the rapid deactivation of these enhancers during early reprogramming. Alternatively, if KDM5B participates in maintaining silencing of pluripotency gene enhancers in differentiated cells, depletion of KDM5B may allow for rapid activation of pluripotency genes during reprogramming. Application of these results may be useful to reprogram terminally differentiated cells that are otherwise refractory to reprogramming using small-molecule inhibitors of Kdm5b. Altogether, these findings demonstrate that KDM5B is critical for regulating epigenetic states of differentiated cells.

In summary, data presented here demonstrate critical roles of the KDM5B demethylase in ES cells and provide novel insight into mechanisms whereby KDM5B functions to regulate ESC self-renewal, differentiation, and reprogramming.

Supplementary Material

Supplemental material

supp_33_24_4793__index.html^{(1.2KB, html)}

ACKNOWLEDGMENTS

The DNA Sequencing Core, Light Microscopy, and Transgenic Core facilities of NHLBI assisted with this work. We thank Kristian Helin for generously providing the Kdm5b conditional knockout ES cells.

Footnotes

Published ahead of print 7 October 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00692-13.

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[B1] 1. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947–956 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B3] 3. Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH. 2008. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133:1106–1117 [DOI] [PubMed] [Google Scholar]

[B4] 4. Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka IR. 2006. Dissecting self-renewal in stem cells with RNA interference. Nature 442:533–538 [DOI] [PubMed] [Google Scholar]

[B5] 5. Ang YS, Tsai SY, Lee DF, Monk J, Su J, Ratnakumar K, Ding J, Ge Y, Darr H, Chang B, Wang J, Rendl M, Bernstein E, Schaniel C, Lemischka IR. 2011. Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network. Cell 145:183–197 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Pasini D, Cloos PA, Walfridsson J, Olsson L, Bukowski JP, Johansen JV, Bak M, Tommerup N, Rappsilber J, Helin K. 2010. JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells. Nature 464:306–310 [DOI] [PubMed] [Google Scholar]

[B7] 7. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK, Bell GW, Otte AP, Vidal M, Gifford DK, Young RA, Jaenisch R. 2006. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441:349–353 [DOI] [PubMed] [Google Scholar]

[B8] 8. Ringrose L, Paro R. 2004. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu. Rev. Genet. 38:413–443 [DOI] [PubMed] [Google Scholar]

[B9] 9. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K. 2007. High-resolution profiling of histone methylations in the human genome. Cell 129:823–837 [DOI] [PubMed] [Google Scholar]

[B10] 10. Schubeler D, MacAlpine DM, Scalzo D, Wirbelauer C, Kooperberg C, van Leeuwen F, Gottschling DE, O'Neill LP, Turner BM, Delrow J, Bell SP, Groudine M. 2004. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18:1263–1271 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA. 2005. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122:517–527 [DOI] [PubMed] [Google Scholar]

[B12] 12. Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, Richmond TA, Wu Y, Green RD, Ren B. 2005. A high-resolution map of active promoters in the human genome. Nature 436:876–880 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Sims RJ, III, Nishioka K, Reinberg D. 2003. Histone lysine methylation: a signature for chromatin function. Trends Genet. 19:629–639 [DOI] [PubMed] [Google Scholar]

[B14] 14. Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J, Bernstein BE, Emre NC, Schreiber SL, Mellor J, Kouzarides T. 2002. Active genes are tri-methylated at K4 of histone H3. Nature 419:407–411 [DOI] [PubMed] [Google Scholar]

[B15] 15. Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T. 2004. Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol. 6:73–77 [DOI] [PubMed] [Google Scholar]

[B16] 16. Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X, Burlingame AL, Roeder RG, Brivanlou AH, Allis CD. 2005. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 121:859–872 [DOI] [PubMed] [Google Scholar]

[B17] 17. Mosammaparast N, Shi Y. 2010. Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases. Annu. Rev. Biochem. 79:155–179 [DOI] [PubMed] [Google Scholar]

[B18] 18. Lee MG, Wynder C, Bochar DA, Hakimi MA, Cooch N, Shiekhattar R. 2006. Functional interplay between histone demethylase and deacetylase enzymes. Mol. Cell. Biol. 26:6395–6402 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Klose RJ, Zhang Y. 2007. Regulation of histone methylation by demethylimination and demethylation. Nat. Rev. Mol. Cell Biol. 8:307–318 [DOI] [PubMed] [Google Scholar]

[B20] 20. Christensen J, Agger K, Cloos PA, Pasini D, Rose S, Sennels L, Rappsilber J, Hansen KH, Salcini AE, Helin K. 2007. RBP2 belongs to a family of demethylases, specific for tri- and dimethylated lysine 4 on histone 3. Cell 128:1063–1076 [DOI] [PubMed] [Google Scholar]

[B21] 21. Frankenberg S, Smith L, Greenfield A, Zernicka-Goetz M. 2007. Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation. BMC Dev. Biol. 7:8. 10.1186/1471-213X-7-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Madsen B, Spencer-Dene B, Poulsom R, Hall D, Lu PJ, Scott K, Shaw AT, Burchell JM, Freemont P, Taylor-Papadimitriou J. 2002. Characterisation and developmental expression of mouse Plu-1, a homologue of a human nuclear protein (PLU-1) which is specifically up-regulated in breast cancer. Gene Expr. Patterns 2:275–282 [DOI] [PubMed] [Google Scholar]

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PERMALINK

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

Benjamin L Kidder

Gangqing Hu

Zu-Xi Yu

Chengyu Liu

Keji Zhao

Abstract

INTRODUCTION

MATERIALS AND METHODS

ES cell culture.

Lentiviral infection.

Generation of iPS cells and mouse chimeras.

Teratoma formation.

qRT-PCR expression analysis.

RNA-Seq analysis.

ChIP-Seq.

Microarray data accession number.

RESULTS

Kdm5b-depleted ES cells exhibit reduced self-renewal.

Fig 1.

KDM5B is important for ES cell differentiation.

Fig 2.

KDM5B is important for silencing of pluripotency genes during ES cell differentiation.

Fig 3.

Depletion of Oct4 is insufficient to rescue the differentiation defect of Kdm5b-depleted ES cells.

Fig 4.

Depletion of Kdm5b leads to accelerated reprogramming.

Fig 5.

Fig 6.

Kdm5b is important for iPS cell differentiation.

KDM5B regulates H3K4 methylation at MEF-specific promoters and enhancers during early reprogramming.

Fig 7.

Sustained knockdown of Kdm5b prior to OSKM overexpression allows for rapid reprogramming.

Fig 8.

DISCUSSION

KDM5B regulates ES cell differentiation.

KDM5B is a barrier to the reprogramming of differentiated cells.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

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