UTX can promote and suppress gene targets in hNSCs
We previously used a neural differentiation course of hESCs to hNSCs and then neurons to describe the requirement of a UTX–53BP1 partnership for human neural differentiation [21]. Here, we want to more comprehensively characterize the activities of UTX during neural differentiation (Additional file 1: Figure S1A). By comparing UTX ChIP-seq datasets in hESCs and hNSCs [21], we found that UTX bound 3950 new sites but was released from 8016 sites in hNSCs compared to hESCs (Fig. 1a). We defined UTX-bound genes as those whose promoters (2 kb from transcription start sites) overlapped UTX ChIP-seq peaks (“Materials and methods”). The differential localization may be in part due to the significant downregulation of UTX expression during neural differentiation (Fig. 1b). Gene ontology (GO) analysis revealed that the UTX-bound genes in hNSC include regulators of transcription, macromolecule biosynthesis, cell cycle, generation of neurons, chromatin modifications, ephrin signaling, VEGF signaling, WNT signaling, and TGFβ signaling (Fig. 1b). In contrast, GO analysis of UTX-bound genes in hESCs include RNA-binding proteins, focal adhesion, translational regulation, nonsense-mediated decay, and ribosome assembly (Additional file 1: Figure S1B). These results suggest that UTX target genes change during neural differentiation, from regulation of RNA-binding, focal adhesion, translation, and non-sense-mediated decay in hESCs to the regulation of transcription, cell cycle, cell differentiation, chromatin structure, and signaling pathways in hNSCs.
We used the H7 and H9 hESCs to more comprehensively characterize the role of UTX during neural differentiation. We used two female hESC lines and not male hESCs (such as H1) because UTX has a functional homolog, UTY, whose gene locus locates at the Y chromosome. UTX and UTY has strong functional overlaps and the use of male lines have confounded phenotypic analyses [2, 22, 23]. We used CRISPR–Cas9 to generate UTX knockout (KO) that generated early translational stops in UTX (Fig. 1d, e). The H9 UTX-KO was used for the comparison with 53BP1-KO [21]. For UTX wild-type (UTX-WT) controls, we generated H7 and H9 hESCs expressing Cas9 and sgRNAs that target the luciferase locus and have no specificity to the human genome. UTX protein was detectable in UTX-WT hESCs but not in UTX-KO hESCs (Fig. 1e). Whole-genome sequencing of UTX-WT and UTX-KO hESCs confirmed the absence of off-target mutations (Additional file 1: Figure S1C–F).
UTX-WT and UTX-KO hESCs were differentiated along the neural lineage, and at time point ‘NSC’ of neural differentiation, they expressed known neural markers SOX2, TUJ1, NESTIN, and SOX1 (Fig. 1f, Additional file 1: Figure S2A). We used RNA-seq to profile the transcriptomes of 3 UTX-WT (H7 UTX-WT and 2 replicate H9 UTX-WT) and 4 UTX-KO (H7 UTX-KO and H9 UTX-KO1–3; Additional file 1: Figure S2B, C) hNSCs. Replicate datasets were merged in data analyses to identify consistent differences between UTX-WT and UTX-KO. We next compared UTX binding to differentially expressed genes between UTX-KO and UTX-WT hNSCs. We found that 52% (888/1718) of genes that were downregulated in UTX-KO vs. UTX-WT hNSCs were bound by UTX (P = 1.0e−13 by hypergeometric test assuming normal data distribution; Fig. 1g), suggesting that UTX binding in hNSCs positively correlates with their expression. Although the enrichment is not above statistical significance, 37% (508/1375) of genes with increased expression in UTX-KO vs. UTX-WT hNSCs were bound by UTX in hNSCs. These data suggest that UTX binding affects the promotion and suppression of UTX-bound genes and prompted us to investigate the effect of UTX during neural differentiation.
UTX modulates the self-renewal property in hNSCs
We examined NSC self-renewal by quantifying S phase, mitosis, and neurosphere formation. Briefly, we used the BrdU assay to detect cells in the S-phase and phospho-histone H3 (serine 10; PH3) staining to detect cells in mitosis (Additional file 1: Figure S2D). A quantitative comparison showed that more H7 UTX-KO hNSCs were in S phase than H7 UTX-WT but did not differ in PH3 quantification, and H9 UTX-KO and UTX-WT were not significantly different (Fig. 2a). To assess neurosphere formation, we serially passaged hNSCs in suspension (Fig. 2b). 53BP1 did not influence neurosphere formation (data not shown). When plated at high cell densities (50,000/6-well), UTX-WT hNSCs formed neurospheres, but UTX-KO neurospheres were densely packed and fused together. We could not accurately quantify neurospheres formed at high cell densities. When plated at low cell densities (10,000–25,000/6-well), UTX-KO hNSCs consistently formed more neurospheres than did UTX-WT across 3 passages (Fig. 2c, d), suggesting higher self-renewal of UTX-KO hNSCs.
We examined differences in gene expression between 5 UTX-KO (replicate formed by H9 UTX-KO 1–2) and 8 replicate UTX-WT neurosphere samples, which revealed 1259 downregulated genes and 1745 upregulated genes in UTX-KO neurospheres (using criteria of FDR < 0.05 and fold change > 2). Downregulated genes were highly enriched in functions related to cellular macromolecule biosynthesis, DNA metabolic processes including replication and repair, SRP-dependent cotranslational protein targeting to membrane, and mitotic phase transition. In contrast, upregulated genes were highly enriched in functions related to extracellular matrix organization, cytokine-mediated signaling, glycosaminoglycan biosynthesis, exocytosis, endoderm formation, and regulation of cell proliferation, motility, and migration (Additional file 1: Figure S2E). Glycosaminoglycans can have roles in cell signaling related to controlling proliferation and cell–cell adhesion. Gene set enrichment analysis (GSEA) showed that genes upregulated in UTX-KO neurospheres were preferentially bound by UTX in WT hNSCs (Fig. 2e). UTX-bound genes that were upregulated in UTX-KO neurospheres were enriched in functions related to extracellular matrix organization, regulation of cell proliferation, glycosaminoglycan biosynthesis, and signaling by VEGF, PDGF, p53, EGFR, TGFβ, and NGF (Fig. 2f). These processes likely influence neurosphere formation and growth. Our results suggest that in hNSCs, UTX binding leads to the suppression of genes involved in extracellular matrix organization, cell proliferation, and multiple signaling pathways, which together can influence the self-renewal property.
UTX controls the neurogenesis vs. gliogenesis fate
To determine if UTX-KO hNSCs display a similar differentiation potential as UTX-WT hNSCs, we induced neuronal differentiation and maturation (Additional file 1: Figure S3A). UTX-KO (H7 UTX-KO and H9 UTX-KO1) and UTX-WT (H7 and H9) hNSCs cultured in neuronal differentiation media for 5 days appeared morphologically similar. However, after culture in neuronal maturation media for 6 days (the “neuron” time point in Figure S3A), UTX-WT cells condensed their nuclei and extended axons, whereas UTX-KO cells enlarged and exhibited a fibroblastic morphology (Additional file 1: Figure S3B). During neuronal differentiation, there were more H9 UTX-KO cells in the S phase than in UTX-WT, but fewer H7 UTX-KO cells in the S phase than in UTX-WT (Additional file 1: Figure S3C). Mitotic indices did not differ between the groups (Additional file 1: Figure S3C). This difference is likely caused by differences between H9 and H7 lines and not the UTX-KO mutation.
The fibroblastic morphology of UTX-KO-differentiating cells resembled that of glia and, therefore, we examined the expression of neuronal and glial/astrocytic markers. After neuronal maturation, UTX-WT cells showed high expression of neuronal markers but low expression of glial/astrocytic markers. In contrast, UTX-KO cells showed high expression of glial/astrocytic differentiation markers and low expression of neuronal markers (Fig. 3a–c, Additional file 1: Figure S3D, E). RNA-seq analysis revealed numerous downregulated genes in differentiating UTX-KO (H7 UTX-KO and H9 UTX-KO 1–3) vs. UTX-WT (3 H9 replicate) cells that were enriched in functions related to nervous system development, axonogenesis, axon guidance, and generation of neurons (Fig. 3d, e), whereas upregulated genes were enriched in functions related to extracellular matrix organization, type-I interferon signaling, cytokine signaling, and neutrophil immunity (Additional file 1: Figure S3F). GSEA confirmed that downregulated genes were enriched in axonogenesis and that upregulated genes were enriched in glial/astrocytic markers (Fig. 3e). These results suggest that UTX-KO cells have decreased neuronal differentiation and increased glial/astrocytic differentiation.
To examine whether UTX gene binding in hNSCs potentially predicts gene expression patterns upon hNSC differentiation to neurons, we compared UTX-bound genes in hNSCs with genes differentially expressed in UTX-KO vs. UTX-WT cells at the “neuron” time point of differentiation. We found that 47% of genes with reduced expression in UTX-KO vs. UTX-WT “neurons” were bound by UTX in hNSCs (P = 4.2e−9; Fig. 3f). On the other hand, 45% of genes that were upregulated in UTX-KO vs. UTX-WT “neurons” were bound by UTX in hNSCs (P = 1.6e−4; Fig. 3f). These data suggest that UTX binding is strongly correlated with gene expression changes during differentiation.
Of the UTX-bound genes in hNSCs, GO analysis revealed that 68 are involved in the generation of neurons and 103 in axonogenesis (Fig. 1c). Additionally, 76 of 156 expressed genes implicated in glial/astrocyte differentiation were bound by UTX in hNSCs (Fig. 1c). Of these 76 genes, 17 known regulators of glial/astrocytic lineage were either downregulated (CDK5R1, ID4, KMT2A, LMNB1, and MEGF10) or upregulated (ANXA7, APOE, APP, COL4A1, DRD1, EN1, FZD4, HGSNAT, HMOX1, PI4K2A, SNTA1, and VIM) in UTX-KO vs. UTX-WT cells at the “neuron” time point of differentiation (Additional file 2: Table S1 lists literature information concerning the 17 genes). However, we could not unequivocally pinpoint that any of the 17 genes were crucial for UTX-KO hNSCs preferring gliogenesis at the expense of neurogenesis. These data suggest that UTX binding correlates with the positive and negative regulation of 36% (1963/5440; Additional file 1: Figure S6G) of its target genes, some of which are involved in neurogenesis and gliogenesis.
Chromatin modification by UTX influences neurogenesis vs. gliogenesis programs
UTX is an H3K27 demethylase that physically binds to and influences the activities of H3K4 methyltransferases, H3K27 acetyltransferase P300, and the chromatin modifier SWI-SNF. We examined H3K27me3, H3K4me3, and H3K27ac by ChIP-seq and potential differential chromatin accessibility by ATAC-seq [24] at the “NSC” time point of neural differentiation, using criteria of FDR-corrected p < 0.05 and fold change > 2 (Additional file 1: Figure S4a–e; “Materials and methods”). For ATAC-seq analysis, we focused on nucleosome-free regions to assay significant differences between H9 UTX-KO and UTX-WT hNSCs (“Materials and methods”). UTX-bound regions showed extensive overlap with at least 1 of the 4 chromatin features (Additional file 1: Figure S4F): 7414 of 10,271 UTX-bound regions overlapped with at least one feature (P < 1e−15 by Fisher’s exact test), whereas only 2857 (28%) of UTX-bound regions lacked all of these features. Further analyses showed that most of these 28% of UTX-bound located within gene encoding small nucleolar RNAs or miRNAs. These findings suggest that UTX preferentially binds to chromatin regions with the assayed features in hNSCs.
To determine whether glial/astrocytic differentiation of UTX-KO is associated with changes in chromatin structure, we systematically analyzed chromatin features at promoters and distal elements 2–50 kb from the genes involved in glial/astrocytic differentiation, axonogenesis, or neuronal differentiation that are differentially expressed during neuronal differentiation. UTX-KO had little effect on H3K27me3 levels (Additional file 1: Figure S5A, B), a finding that is consistent with those of previous developmental studies showing that Utx had little to modest impact on H3K27me3 dynamics or its demethylase activity is dispensable for organismal development [1, 23, 25, 26]. Instead of H3K27me3, UTX-KO appeared to alter H3K27ac and chromatin accessibility at developmentally important genes in hNSCs. The levels of H3K27ac, H3K4me3, and chromatin accessibility were significantly altered at promoters of more than 20 developmentally important genes in UTX-KO compared with UTX-WT (Additional file 1: Figure S5C–E). UTX-KO cells also showed significantly reduced levels of H3K27ac and chromatin accessibility at distal elements of 30 developmentally important genes (Additional file 1: Figure S5C–E). Furthermore, 15 of 30 distal elements of gene loci of TENM4, PAX6, NRXN3, EPHB3, SHANK3, NFASC, EPHB2, NCAM1, EPHB1, NR2E1, RGMA, CNTN4, ARSA, SMPD3, and FGFR2 had significantly reduced levels of both H3K27ac and chromatin accessibility (Additional file 1: Figure S5D–E), suggesting that at least 15 enhancers require UTX for proper chromatin composition. Altogether, these data suggest that UTX influences promoters and enhancers involved in neurogenesis and gliogenesis programs.
Among the features assayed, H3K27ac distribution appeared to be most disrupted by UTX-KO. We performed further analyses by segregating UTX-bound regions into two categories of significantly higher and lower H3K27ac signals in UTX-KO hNSCs. Distribution of the 2 categories among genic features and the other 3 chromatin features did not differ (Additional file 1: Figure S6A, B). However, GO analysis of genes associated with (or closest to) these regions differed. Regions with higher H3K27ac signals were enriched in functions related to miRNA-mediated inhibition of translation, Rho-guanyl-nucleotide exchange factor activity, and regulation of transcription by RNA polymerase II (Additional file 1: Figure S6C). Regions with lower H3K27ac signals were enriched with axonogenesis, axon guidance, neuron projection morphogenesis, transmembrane receptor tyrosine kinase signaling, mitogen-activated kinase, ephrin-mediated repulsion of cells, Wnt-activated receptor activity, and VEGFA–VEGFR2 signaling pathway (Additional file 1: Figure S6C). These data suggest that UTX binds and promotes H3K27ac levels at neurodevelopmentally important genes including those involved in key signaling pathways.
We next performed motif analysis to determine whether additional factors might correlate with these H3K27ac changes. Results revealed the significant enrichment of the motifs of ATOH1, TCF4, NEUROD1, MYOD, MYF5, and RRF2 in regions with lower H3K27ac signals; however, ATOH1, MYOD, and MYF5 had nondetectable expression in RNA-seq datasets (Additional file 1: Figure S6D). TCF4 and NEUROD1 both have essential role in neurogenesis [27, 28], and RFX2 is required for ciliogenesis that affects neural development [29]. There was no significant enrichment of motifs in regions with higher H3K27ac signals. We, therefore, concluded that in hNSCs, UTX-KO leads to H3K27ac increases in regions of little neurodevelopmental importance. In contrast, TCF4, NEUROD1, and RFX2 likely cooperate with or influence UTX to promote H3K27ac signals at genes involved in axonogenesis, axon guidance, neuron projection, and signaling pathways.
UTX suppresses an AP-1-mediated program of gliogenesis
Next, we performed ATAC-seq to examine differential nucleosome-free regions in UTX-KO and UTX-WT cells at the “neuron” time point of neural differentiation, using criteria of FDR-corrected P < 0.05 and fold change > 2 (Fig. 4a and Additional file 1: S7A–B; “Materials and methods”). We found significantly higher ATAC-seq signals in nucleosome-free regions of H7 UTX-KO vs. WT and H9 UTX-KO vs. WT differentiating cells. These regions were enriched for consensus binding motifs of JUN and FOS proteins (Fig. 4b and Additional file 1: S7C, D), which comprise the AP-1 transcription factor complex [30]. We used published AP-1 target genes—which were downregulated by Fos shRNA and having a nearby FOS ChIP-seq peak in mouse cortical neurons [31]—for GSEA to show their significant enrichment in upregulated genes and the nucleosome-free regions of UTX-KO vs. UTX-WT differentiating cells (Fig. 4C, D). Further, promoters of multiple glial/astrocytic genes such as CAV1, FGFR1, GFAP, KCNQ3, LTN1, POMT2, TK2, and VAC14 contained AP-1 motifs. Distal elements of 41 glial/astrocytic genes having significantly higher ATAC-seq signals were also AP-1 targets (Fig. 4d). However, only 19 AP-1 targets overlapped with UTX-bound genes in hNSCs. These data suggest that UTX suppresses the expression of AP-1 during neural differentiation.
Intriguingly, we found that UTX bound to JUN and JUNB loci in hNSCs (Fig. 4e), but FOSL1 and FOSL2 loci were not bound. Moreover, JUN, JUNB, FOSL1, and FOSL2 were upregulated in UTX-KO vs. UTX-WT cells at the “neuron” time point of differentiation (Fig. 4f and Additional file 1: S7E). The expressions of FOS and FOSB were undetectable. Potential differential chromatin accessibility, determined by ATAC-seq, at JUN and JUNB loci did not significantly differ between UTX-WT- and UTX-KO-differentiating cells, suggesting that UTX did not influence chromatin structure to suppress JUN and JUNB. Therefore, we investigated the control of RNA polymerase II in pausing–elongation, which has been proposed by others [9, 14, 32] as an alternative mechanism by which UTX regulates gene expression. We performed CUT&RUN-seq [33] (with spike-in controls; “Materials and methods”) to profile the distribution of RNA polymerase II with phosphorylated serine 2 in CTD (pS2-RNApol2) in UTX-WT and UTX-KO differentiating neurons. Compared to UTX-WT, levels of pS2-RNApol2 were lower at the promoters of JUN and JUNB loci in UTX-KO (Fig. 4e), suggesting the release of transcriptional pausing at these loci. Genome wide, pS2-RNApol2 CUT&RUN-seq signals significantly increased at 120 promoters and decreased at 99 promoters in UTX-KO vs. UTX-WT (Fig. 4g), suggesting UTX positively and negatively regulates RNA polymerase II pausing–elongation at some genes. Although the reduction of pS2-RNApol2 signal at JUNB locus did not pass the statistical threshold, the distribution was markedly disrupted by UTX-KO (Fig. 4e), suggesting that UTX may affect distribution as well as the total level of pS2-RNApol2 at potentially more genes.
We performed motif analysis to determine whether additional factors might correlate with differential gene expression in UTX-KO hNSCs. This analysis found significant enrichment of motifs of AP-1 components, p73, and Bach2 in upregulated genes (Additional file 1: Figure S7F) and Sox2, REST-NRSF, Sox4, Sox17, and Sox15 in downregulated genes of UTX-KO (Figure S7G). JUN and JUNB loci contained the Jun-AP1 motif. Our data suggest a network of transcription factors including AP-1 cooperate with or influence UTX to positively or negatively regulate gene expression in hNSCs.
To test whether aberrant AP-1 activity influences gene expression in UTX-KO differentiating cells, we employed two specific inhibitors of AP-1, SR-11302 [34] and T-5224 [35] (Additional file 1: Figure S7H). Compared with DMSO control treatment, inhibitor treatments did not affect the expression of NSC and neuronal markers or cell morphology, but significantly reduced the aberrant upregulation of glial/astrocytic markers S100B and GFAP in UTX-KO differentiating cells (Fig. 4h, i and Additional file 1: FigureS7I). Thus, inhibiting AP-1 activity suppresses glial/astrocytic gene expression in UTX-KO cells. As the differentiation course and drug treatment took 15 days (Additional file 1: Figure S7H), the effects of AP-1 inhibitor on UTX-KO differentiation might not be direct. Overall, our data suggest that UTX is required for AP-1 suppression, that AP-1 influences glial/astrocytic lineage genes, and that loss of UTX triggers the upregulation of AP-1 and promotion of gliogenesis.