Summary
THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression.
Keywords: Thanatos-associated protein domain-containing apoptosis-associated protein 1, THAP1, dystonia, differentiation, survival, embryonic stem cells, transcriptomics, neuroectodermal differentiation, apoptosis, zinc finger transcription factor
Highlights
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Wild-type THAP1 regulates ESC potential, survival, and proliferation
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THAP1 is essential for ESC differentiation, particularly neuroectodermal
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Thap1C54Y or ΔExon2 ESCs prolong expression of pluripotent genes upon differentiation
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Thap1C54Y or ΔExon2 EBs show increased cell death and abnormal differentiation
Ehrlich and colleagues identified THAP1, mutations of which cause dystonia (DYT6), as an essential regulator of ESC survival, proliferation, and differentiation. As THAP1 mutations result in a neurologic disease, this work examined the effects of a causative mutation, C54Y, and an essentially null allele, ΔExon2, on neuronal differentiation.
Introduction
THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a member of a large family of proteins which are primarily transcription factors (Gervais et al., 2013, Roussigne et al., 2003). The THAP domain, an atypical zinc finger (CysX2-4CysX35-53CysX2His), is highly conserved and is part of the DNA-binding domain (DBD) with homology to P-transposable elements (Majumdar and Rio, 2015). THAP1 mutations cause DYT6 dystonia (Fuchs et al., 2009), and mutations are located throughout the protein, with about 50% in the DNA-binding domain. Importantly, recessive mutations have been identified (Houlden et al., 2010, Schneider et al., 2011, Xiromerisiou et al., 2012). THAP1 functions, targets, and the mechanisms by which its mutations lead to dystonia are largely unknown, including the effects of mutations on DNA binding (Campagne et al., 2012).
Functional studies of THAP1 in human umbilical vein endothelial cells (HUVECs) show a role in the S phase of mitosis via modulation of pRb-E2F cell-cycle target genes, including RRM1 (Clouaire et al., 2005). In vitro, a coiled-coil domain is required for dimerization (Sengel et al., 2011). Other interactors include prostate apoptosis response-4 protein (Par-4), an effector of cell death linked to prostate cancer and neurodegenerative diseases (Roussigne et al., 2003); HCF-1, a transcriptional co-activator involved in cell-cycle regulation; and O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc and thereby also participates in epigenetic regulation of gene expression with an essential function in dividing cells (Mazars et al., 2010).
In mouse models of DYT6 which harbor either a disease-causing C54Y mutation in the DBD or a null allele (ΔExon2) (Ruiz et al., 2015), rare homozygous embryos survived to day 14. They were small with defects in peripheral organs and brain, which showed deficits in the number and morphology of neurons. To study the impact of the mutant alleles on stem cell maintenance and differentiation, we generated mouse embryonic stem cells (mESCs) homozygous for either the C54Y (Thap1C54Y) or ΔExon2 (Thap1ΔExon2) alleles. Herein, we characterize both ESCs, which are viable with intact stem cell characteristics, but with abnormalities in cell death, cell cycle, and proliferation rate, that are more severe in the ΔExon2 than in the C54Y homozygote. Furthermore, we show that during differentiation of embryoid bodies (EBs), wild-type THAP1 is required for repression of a cohort of core pluripotency-associated genes and survival in ΔExon2 cells, and apparently for terminal neuronal differentiation in C54Y homozygote surviving cells.
Results
Generation of Thap1-Recombinant Embryonic Stem Cells
To explore the function of THAP1 in ESCs, we derived ESCs from two mouse alleles, (1) Thap1C54Y, a constitutive knockin (KI) of the C54Y causative mutation in the DBD of THAP1, and (2) Thap1ΔExon2, a constitutive knockout (KO), allele lacking exon 2 (Ruiz et al., 2015; Figure 1A). Genotypes of wild-type (WT), Thap1C54Y/C54Y, and Thap1 ΔExon2/ΔExon2 ESCs were analyzed by PCR and compared with WT, Thap1C54Y/+, and Thap1 ΔExon2/+ heterozygote mice (Figure 1B). Consistent with THAP1 autorepression (Erogullari et al., 2014) and failure of THAP1C54Y to bind at the Tor1a promoter (Gavarini et al., 2010), Thap1C54Y cells exhibited higher levels of Thap1 mRNA than WT ESCs, whereas full-length Thap1 mRNA was undetectable in Thap1ΔExon2 ESCs (Figure 1C). THAP1 antibodies recognize several THAP1-like immunoreactive (THAP1-LIR) species (Ortiz-Virumbrales et al., 2014). Subcellular fractionation followed by western blot analysis revealed the presence of three distinct THAP1-LIR species in the nuclear fraction at ∼29 kDa, ∼50 kDa, and ∼75 kDa (Figure 1D, upper panel). Only the ∼75-kDa species was induced or drastically reduced in Thap1C54Y or Thap1ΔExon2 ESCs, respectively, following the same pattern of the corresponding mRNA as assessed by qRT-PCR (Figure 1C). Thus, the ∼29- and ∼50-kDa species appear to be largely composed of cross-reacting proteins in ESCs. In murine brain, the ∼29-kDa species was also non-specific, whereas the 50- and 75-kDa THAP1-LIR species were nuclear and neuron specific, and virtually undetectable in ΔExon2 embryos (Ortiz-Virumbrales et al., 2014). Primers spanning exon 1 and exon 3 of Thap1 mRNA amplified a de-repressed transcript, i.e., its expression was induced, in Thap1C54Y/C54Y and Thap1ΔExon2 ESCs (Figure 1E). This naturally occurring THAP1ΔExon2 represents less than 1% relative to the major isoform (containing exon 2) (Figure 1F) and in vivo does not substitute for the loss of the full-length isoform.
Loss of THAP1 Alters ESC Viability
To investigate THAP1 function in self-renewal, we analyzed the expression of stage-specific embryonic antigen 1 (SSEA1) and POU5F1 (Palmqvist et al., 2005) in all three genotypes (Figure 2A, top and middle panels). There were no detectable differences. The level of alkaline phosphatase (AP) staining (Figure 2A, bottom panels), characteristic of undifferentiated ESCs, was also genotype independent. These data suggest that THAP1 is not required to maintain the pluripotency state of ESCs.
To assess the proliferation rates of WT, Thap1C54Y, and Thap1ΔExon2 ESCs, we counted the cells at predetermined times after plating. Thap1C54Y ESC proliferation rate was slightly slower than in WT cells, whereas deletion of THAP1 Exon2 severely compromised proliferation (Figure 2B). Viability was not affected in Thap1C54Y ESCs as indicated by annexin V, whereas the number of viable cells was reduced ∼25% in Thap1ΔExon2 ESCs compared with WT (Figure 2C). Propidium iodide (PI) staining followed by DNA flow cytometry revealed a significant reduction in G2/M-phase cell populations and a corresponding increase in the number of cells in S phase (Figure 2D) in the Thap1ΔExon2 ESCs. Again, Thap1C54Y showed no significant differences compared with WT ESCs. Thus, loss of full-length THAP1, but not the C54Y mutation, affects cell viability by increasing the rate of cell death and arresting the cell cycle at the S phase.
Global Expression Profile of Thap1C54Y and Thap1ΔExon2 ESCs
We studied the global transcriptional response in both ESC lines via high-throughput RNA sequencing (RNA-Seq), and identified a larger number of differentially regulated genes in Thap1ΔExon2 than in Thap1C54Y ESCs (Figure 3A and Table S1). Specifically, comparing Thap1C54Y with WT ESCs, there were 391 upregulated and 163 downregulated genes, whereas comparing Thap1ΔExon2 with WT we identified 1,245 upregulated and 1,237 downregulated genes (Figure 3A). Notably, 93% of the upregulated and 89.5% of the downregulated Thap1C54Y genes were common to both genotypes (Figure 3B), but a heatmap illustrated that Thap1C54Y and Thap1ΔExon2 alleles have distinct effects on the transcriptional ESC profile (Figure 3C). These differences were also reflected by gene ontology (GO) analyses of biological processes of uniquely differentially expressed genes (Figure S1), and could not be attributed to metabolic or apoptotic gene expression dysregulation. Interestingly, GO analyses of biological processes of upregulated genes (log2 fold change = +1 of the Thap1ΔExon2 ESC dataset) revealed categories related to embryonic pattern specification, synapsis, chromosome organization, meiosis, and negative regulation of cell differentiation (Figure 3D, upper panel). Analysis of downregulated genes (log2 fold change = −1 of the Thap1ΔExon2 ESC dataset) demonstrated enrichment for processes involved in neuron development, including neuron differentiation, axon guidance, axonogenesis, and cell projection organization (Figure 3D, lower panel). Known pluripotency genes, including Rex1, Prdm14, and Nanog were increased in both Thap1C54Y and Thap1ΔExon2 ESCs, whereas Klf4, Esrrb, and Pou5F1 were increased only in Thap1ΔExon2 ESCs, all compared with WT ESCs (Figure 3E). In contrast, ectodermal markers, e.g., Gbx2 and Sox11, were decreased in Thap1C54Y ESCs, and Pax6, Sox11, Olig2, and Fgf5 were decreased in Thap1ΔExon2 ESCs, all compared with WT. Endoderm specification genes, including Gata4, Gata6, Sox17, and Foxa2, were also significantly decreased in both genotypes compared with WT ESCs (Figure 3E), whereas no differences in mesoderm markers were detected (data not shown). The RNA-Seq results were confirmed by qRT-PCR for selected genes (Figure 3F). Altogether, these results provide initial evidence that pluripotency factors and developmental regulators are differentially, and oppositely, regulated after C54Y mutation of Thap1, and that such changes in the ESC transcriptome are even more apparent after loss of full-length THAP1.
THAP1 Genomic Binding Analysis
To gain an overview of the global role of THAP1 in ESCs, we performed chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq). We identified 1,731 high-confidence peaks bound by THAP1, corresponding to 1,052 target genes (Table S2), 65 of which were upregulated and 43 were downregulated in Thap1ΔExon2 ESCs (Figure 4A). THAP1 binding was enriched at the transcription start site (TSS), extending over a 2-kb interval (Figure 4B). THAP1 bound only slightly more at promoters (27.04%) than in intergenic regions (24.10%) (Figure 4C). We used public ESC ChIP-Seq datasets for p300, Med1, and monomethylated histone H3 Lys4 (H3K4me1), acetyl histone H3 Lys27 (H3K27ac), and trimethylated histone H3 Lys27 (H3K27me3) (Arnold et al., 2013, Heintzman et al., 2007, Heintzman et al., 2009, Rada-Iglesias et al., 2011, Visel et al., 2009) to determine that THAP1 occupation was detected at activated enhancers but not with the H3K27me3 silent mark (Figure 4D). By clustering the ChIP-Seq enrichment profiles of RNA polymerase II Ser-5 phosphorylated (Pol II Ser-5P) and the histone modification trimethylated histone H3 Lys4 (H3K4me3) with high-confidence THAP1 peaks, we found that THAP1 occupancy occurred at promoter regions of activated genes (Figure 4E) associated with GO categories related to metabolic processes, including nucleobase-containing compound and RNA metabolic, and neurologic system process (Figures 4F and S2A). THAP1 also occupied bivalent or poised genes that are associated with developmental processes, system development, cell communication, and nervous system and ectoderm development, among others (Figures 4F and S2B). Occupancy of THAP1 at silent genes was related to diverse functions, including developmental and immune system process (Figures 4F and S2C). We identified the top DNA motifs enriched at the THAP-domain-binding sequence (THABS) and de novo motifs using the top 1,000 THAP1 peaks and the algorithm MEME (Figure 4G, left panel). Motifs #1 and #3 overlapped with the THABS identified by ENCODE in the human K562 cell line (Kheradpour and Kellis, 2014). Motif #3 contained the core GGCA sequence, essential for recognition by the THAP domain (Clouaire et al., 2005). Secondary motifs overrepresented in THABS overlapped with the binding sequence for RE-silencing transcription factor (REST), which represses the expression of neuronal genes in differentiated non-neuronal cells (Ballas et al., 2005), and Yin Yang 1 (YY1), which plays multiple roles in the development of the central and peripheral nervous systems (He and Casaccia-Bonnefil, 2008; Figure 4G, right panel). Consistent with previous reports, inspection of individual gene tracks showed THAP1 binding at the core promoter of Thap1 and Tor1A (Erogullari et al., 2014, Gavarini et al., 2010, Kaiser et al., 2010), thus validating our approach (Figures 5A and 5B, left panel). ChIP-qPCR using anti-THAP1 antibody confirmed THAP1 occupancy at these sites in WT but not in Thap1C54Y and Thap1ΔExon2 ESCs (Figures 5A and 5B, right panel), and at promoters of the cell-cycle genes Aurora A kinase (Aurka) and tumor protein p53 (Trp53), at the apoptosis genes Bcl-2-associated death promoter (Bad) and DNA fragmentation factor subunit beta (Dffb), and at the splicing factor 3a subunit 2 (Sf3a2). Despite this occupancy, the expression of these genes is not altered in Thap1C54Y and Thap1ΔExon2 ESCs compared with WT (Table S1), suggesting that THAP is present on specific promoters as part of an inactive complex poised to respond to defined developmental stimuli yet to be elucidated. Binding of THAP1 at the non-specific region of chromosome 8 was not detected (Figure 5C).
Thap1C54Y and Thap1ΔExon2 EBs Show Increased Cell Death and Fail to Properly Differentiate
To study lineage specification, we assayed EB formation and differentiation (Höpfl et al., 2004). The absence of leukemia inhibitory factor (LIF), replacement of fetal calf serum (FCS) with KnockOut serum replacement, and growth in suspension triggered cell differentiation. WT, Thap1C54Y, and Thap1ΔExon2 ESCs formed EB aggregates (Figure S3A). It was immediately apparent that Thap1ΔExon2 EBs were smaller and all died between days 12 and 16. We assayed mRNA levels of markers that either promote or inhibit cell death (Youle and Strasser, 2008). Compared with WT, the expression of Bax was higher in Thap1C54Y and Thap1ΔExon2 EBs, and Puma also increased in the latter, with a peak at day 8 observed in both genotypes (Figure S3B, upper panel). The expression of the anti-apoptotic markers Xiap and Bcl-2 was also higher in Thap1ΔExon2 EBs compared with Thap1C54Y and WT EBs, with a striking 50- to 75-fold upregulation of Bcl-2 in Thap1ΔExon2 EBs (Figure S3B, lower panel). Cell viability in EBs was actually decreased in both Thap1C54Y and Thap1ΔExon2 ESCs compared with WT ESCs as indicated by annexin V (Figure 6A) and TUNEL staining (Figure 6B), and cleaved caspase-3 staining (Figure S4A), more so in the ΔExon2 EBs. Cell death was not diminished in either genotype when EBs were grown in 40 μM Z-VAD-FMK, a pan-caspase inhibitor (Figure S4B). Thap1ΔExon2 EBs demonstrated the highest percentage of cell death whereas an intermediate level was observed in Thap1C54Y EBs, compared with WT, over the 4-day period (Figure S4B). Failure of Z-VAD-FMK to preserve survival is consistent with literature showing that cellular commitment to apoptosis occurs at the level of BCL-2 family-regulated mitochondrial outer membrane permeabilization (i.e., cytochrome c release), and that subsequent death phenotypes occur whether or not caspases activate (Chipuk and Green, 2005).
We also used qRT-PCR to assay the expression of differentiation markers in EBs, representing the three germ layers, many of which were already dysregulated in the ESCs. Thap1 mRNA levels were constant in WT EBs up to day 16, the last day examined. In Thap1C54Y ESCs, Thap1 mRNA was higher than in WT, and increased steadily up to day 16. Thap1 mRNA was undetectable in Thap1ΔExon2 EBs (Figure 6C, top left panel). The expression of ESC genes Prdm14 and Rex1 increased progressively between day 1 and day 12 in Thap1ΔExon EBs, and the latter also progressively increased in Thap1C54Y EBs (Figure 6C, top right panel), suggesting that failure to silence pluripotency genes during differentiation may contribute to restriction of developmental potential. Thap1C54Y and Thap1ΔExon2 EBs expressed markers of endodermal lineage (Figure S3C, upper panel) following a pattern similar to that of WT EBs. However, the expression of the mesodermal markers T (i.e., Brachyury) and Flk1 was abnormal in the Thap1C54Y and Thap1ΔExon2 EBs (Figure S3C, lower right panel). Consistent with the GO analysis, the expression of the ectodermal markers Sox11, Nestin, and Tuj1 were decreased in Thap1C54Y and Thap1ΔExon2 EBs, whereas the marker of primitive ectoderm Fgf5 was normally expressed in Thap1C54Y/C54Y and Thap1ΔExon2 EBs compared with WT EBs (Figure S3C, lower panel). Failure of Thap1C54Y to increase expression of Tuj1 suggests a stall at early ectodermal differentiation.
THAP1 Is Essential for Neural Differentiation of Mouse ESCs
To further investigate the role of THAP1 during neural differentiation of mESCs from EBs, we used one protocol based on retinoic acid (RA)-dependent neural differentiation (Figure 7A, protocol #1; Okada et al., 2004), and a second protocol without RA but with the addition of N2 supplemented with epidermal growth factor β and insulin (Figure S5A, protocol #2; Okabe et al., 1996). Survival of mutant cells, particularly ΔExon2, was improved in protocol #2 relative to #1. Using protocol #1, undifferentiated, neural progenitor Nestin-positive cells were detected in Thap1C54Y EBs, but at a lower expression level than in protocol #2, which has a longer culture period. Using protocol #2, Nestin was detected in both WT and Thap1C54Y differentiated cells, but TuJ1-positive neuronal projections, indicative of more mature neurons, were detected almost exclusively in WT ESCs and were abnormally restricted to the internal portion of cell mass in Thap1C54Y ESCs, again consistent with a stall at early ectodermal differentiation (Figure S5B). Thap1ΔExon2 EBs failed to develop Nestin- or TuJ1-immunopositive neuronal projections with either protocol (Figures 7B and S5B). The quantification of neuronal projections in the three genotypes is illustrated in the bar graphs (Figures 7C and S5C). Expression of the ESC genes Prdm14, Nanog, and Rex1 in samples from protocol #1 (Figure 6) was abnormally enhanced in Thap1C54Y and Thap1ΔExon2 EBs, even at the later stages of neuronal differentiation (Figure 7D, upper panel). We also monitored progression of ESC differentiation toward neural fates by analysis of Pax6, Sox11, Nestin, and Tuj1 expression with qRT-PCR. Thap1C54Y and Thap1ΔExon2 failed to activate the expression of these ectodermal markers, suggesting a role for THAP1 in multiple stages of neuronal differentiation (Figure 7D, lower panel). Similar results were obtained by expression analysis in protocol #2 (Figure S5D). Collectively, our results show that THAP1 is required to silence the expression of the pluripotency factors and to activate the expression of the neural factors (Figure 7E).
Discussion
We have identified THAP1 as a major regulator of ESC survival and, to a lesser degree, proliferation and differentiation. As THAP1 mutations result in a neurologic disease, we were particularly interested in the effects of a causative mutation, C54Y, on neuronal differentiation. We had previously posited, based on observations of homozygote mutant embryos, that THAP1 is required in multiple stages and layers during embryogenesis, organogenesis, and maturation of the nervous system, including neurogenesis and neuritogenesis, and that the C54Y variant is frequently unable to substitute for the WT (Ruiz et al., 2015).
In all assays, the homozygous ΔExon2 phenotype was more severe than that of the homozygous C54Y phenotype, confirming that C54Y does not confer a complete loss of function. Both genotypes showed a decreased rate of proliferation, more so in the ΔExon2, consistent with regulation of the cell cycle by THAP1 in HUVECs and lymphoblasts (Cayrol et al., 2007, Vemula et al., 2014). Only the ΔExon2 homozygote showed a decrease in viability in the ESC stage. Thus, a loss of WT THAP1 promotes cell death even though THAP1 is reportedly a pro-apoptotic factor (Roussigne et al., 2003). The lack of a requirement for THAP1 for pluripotency is in marked contrast to RONIN/THAP11, another member of the THAP family to have been characterized in this manner (Dejosez et al., 2008).
We compared this study of the global transcriptome in ESCs harboring a mutation of THAP1 with previous microarray assays of HUVECs with up- or downregulation of THAP1 and of human lymphoblasts harboring a disease-causing intronic variant of THAP1, both of which revealed that the main pathway regulated by THAP1 is the cell cycle. The cell-cycle abnormalities and cell death described herein bear a strong resemblance to these HUVECs and lymphoblasts. Similar to the Thap1ΔExon2 ESCs, the mutant human lymphoblastoid cells had decreased viability and showed a reduced number of cells in the G2 phase (Vemula et al., 2014). In HUVECs, knockdown of THAP1 resulted in an impaired G1/S phase (Cayrol et al., 2007). Despite the presence of cell-cycle abnormalities in the THAP1 null cells, cell-cycle genes were not a highly ranked GO category, but genes pertaining to meiosis and X chromosome inactivation categories were also differentially expressed in Thap1C54Y/C54Y and Thap1ΔExon2 compared with WT ESCs.
We examined the role of THAP1 in ESC differentiation by performing EB formation assays. EB formation and growth were highly compromised in THAP1ΔExon2 cells. Indeed, we showed that cell viability was compromised in both Thap1C54Y and Thap1ΔExon2 EBs, with a greater cell death phenotype after loss of full-length THAP1. The extent of cell death in the ΔExon2 EBs precludes a conclusion regarding an effect on differentiation. Our investigations into the mechanism of cell death implicate the mitochondrial pathway of apoptosis as responsible for cell death commitment and caspase activation. Cell death execution, however, apparently can proceed independently of caspase activation. Delineation of a definitive sequence of events leading to cell death requires further characterization. Although there is increased cell death in the Thap1C54Y EBs, their survival is sufficient to enable some conclusions regarding differentiation. Specifically, mesoderm specification may be delayed, but, more strikingly for a mutation that leads to a neurologic disease, neuronal differentiation appears to stall at an early stage.
The mechanism(s) via which the Thap1 mutations prevent repression of pluripotency markers is not entirely obvious, but there are leads, particularly in the categories of dysregulated genes. An in silico search for THABS motifs in mouse Nanog, Prdm14, Pou5f1, Esrrb, and Klf4 was negative, and ChIP-Seq did not uncover binding sites in these genes. Regulation of the pluripotency network requires cooperation between multiple transcription factors, including the pluripotency factors themselves, signal transduction pathways, and epigenetic modifications. Notably, of the DEGs detected by RNA-Seq, many genes in these categories are altered, e.g., Bmp4, Dnmt3a and Dnmt3b, Satb1, Klf10, Sirt6, and Sox17, with a greater degree of dysregulation in the Thap1ΔExon2 relative to the Thap1C54Y ESCs. Interestingly, Dnmt3a and 3b, Satb1, and Sox17 KO ESCs display phenotypes that are similar in many ways to those of the Thap1 mESCs. Specifically, upon exposure to differentiation factors, they simultaneously fail to downregulate pluripotency genes and upregulate lineage-specific genes, some of which are already decreased prior to differentiation (Bergsland et al., 2011, Chen et al., 2003, Savarese et al., 2009). Thus, the mechanism via which THAP1 regulates these genes requires further examination but may represent a pathway for inverse regulation of pluripotency and differentiation genes.
The low percentage (10%) of overlap between RNA-Seq and ChIP-Seq datasets suggests an alternative role of THAP1 in regulating gene expression other than direct binding at DNA, as exists with other zinc-finger factors (Aguilo et al., 2015). This conclusion may also be supported by the relatively low log2 fold changes observed in the Thap1C54Y ESCs, as much greater changes are seen in Thap1ΔExon2 ESCs, where the full-length transcription factor is eliminated. It is possible that the mutant THAP1 proteins are able to bind to some, but not all, targets of WT THAP1, and in addition, may bind to sequences that are normally not bound by WT THAP1 (Campagne et al., 2012). Also, these mutant proteins may participate in THAP1 protein interactions. For example, THAP1 interacts with HCF-1 via a motif that does not include amino acid 54 (Mazars et al., 2010). Therefore, the C54Y form may still be able to interact with HCF-1, Par-4, and partners as yet to be identified, thereby recruiting such transcription factors to the target DNA. This hypothesis would be consistent with the fact that there are individuals with dystonia with homozygote THAP1 mutations. In the case where THAP1 binding is decreased or eliminated, there may be increased binding of transcription factors with an affinity for similar motifs. As noted, these include YY1 and REST, thereby altering levels of their direct and indirect targets, which include many of the RNA-Seq DEGs. In fact, there is a very strong super-shift with anti-YY1 in electrophoretic mobility shift assays using the Tor1a THABS and embryonic brain nuclear extract (data not shown).
In summary, we have identified THAP1 as an essential regulator of mESC potential, including viability and likely neuronal differentiation, the latter particularly for the naturally occurring C54Y mutation. These activities are likely due in part to direct regulation of gene expression but to a large extent to indirect regulation, the mechanisms of which are important to identify. Notably, dysregulated pathways in ESCs with mutations of THAP1 overlap with those seen in mouse models of DYT6, and the ESC system may potentially be exploited to discover how following differentiation, mutations of the ubiquitously expressed THAP1 protein can lead to a strictly neurologic disease.
Experimental Procedures
Preparation of nuclear extracts and qRT-PCR gene expression analysis were performed as previously described (Ruiz et al., 2015).
Further experimental details and lists of antibodies, public datasets, and primer sets utilized in this study may be found in Supplemental Experimental Procedures and other Supplemental Information.
Cell Culture and EB Formation
Wild-type (WT), Thap1C54Y, and Thap1ΔExon2 ESCs were maintained on 0.1% gelatin-coated tissue culture plates on irradiated mouse embryonic fibroblasts (MEFs) in DMEM containing 15% FCS, 0.1 mM 2-mercaptoethanol, L-glutamine, non-essential amino acids, and LIF (1,000 units/mL) and penicillin-streptomycin (Thermo Fisher). Cells were passaged once onto gelatin-coated dishes and then aggregated to form EBs in 10-cm Petri dishes at 5 × 106/plate in the absence of LIF. Cells were harvested for extraction of total RNA at the indicated time points. All cell cultures were maintained at 37°C with 5% CO2.
Differentiation Assays
Induction of neural differentiation was performed based on generation of EBs, using two different approaches (Okabe et al., 1996, Okada et al., 2004), with and without addition of RA as indicated in Supplemental Experimental Procedures.
Cellular Proliferation, Apoptosis Assay, and Cell-Cycle Analyses
Cellular proliferation, apoptosis assay, and cell-cycle analysis were performed using a Muse Cell Analyzer (Millipore) following the manufacturer's instructions.
Alkaline Phosphatase Activity
AP activity was measured using the Stemgent Alkaline Phosphatase Staining kit (Stemgent) following the manufacturer's recommendations.
Chromatin Immunoprecipitation with High-Throughput Sequencing
THAP1 ChIP-Seq was performed as described in Supplemental Experimental Procedures.
RNA-Seq and Analysis
RNA-Seq library preparation was performed at the Weill-Cornell Medical College Genomic Core Facility using the TrueSeq RNA sample preparation kit (Illumina RS-122-2001) and sequenced by the Illumina HiSeq 2,500 platform as 100-bp pair-ended reads.
Gene Ontology Analysis
For functional profiling of THAP1 binding regions identified by ChIP-Seq, GO analysis was performed using Panther software (http://pantherdb.org) (Mi et al., 2016). GO analysis from RNA-Seq datasets was performed using the web tool The Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/) (Huang et al., 2009a, Huang et al., 2009b).
Statistical Analysis
All values are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA followed by Bonferroni's multiple testing corrections using GraphPad Prism 5 Software. A probability value of p < 0.05 was considered statistically significant.
Author Contributions
Conceptualization, F.A., Z.Z., M.J.W., L.J.O., P.G.-A., T.P.Z., and M.E.E.; Methodology, K.K.; Formal analysis, F.A., Z.Z., R.S., C.W, and W.Z.; Investigation, F.A., Z.Z., K.N., R.W., M.H., and Y.S.; Writing – Original Draft, F.A. and M.E.E.; Writing – Review & Editing, Z.Z., P.G.-A., L.J.O., M.J.W., T.P.Z., and M.E.E.; Funding Acquisition, M.E.E.; Supervision and Project Administration, F.A. and M.E.E.
Acknowledgments
We thank Dr. A. Alonso of the Weill-Cornell College of Medicine's Epigenomic Sequencing Core for expert advice for sequencing and library preparation. This work was funded by NIH R01NS081282 (to M.E.E. and L.J.O.) New York State Stem Cell Science (NYSTEM C029553 to M.E.E.), and the Huffington Foundation (T.P.Z.).
Published: June 1, 2017
Footnotes
Supplemental Information includes Supplemental Experimental Procedures, five figures, and four tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2017.04.032.
Contributor Information
Francesca Aguilo, Email: francesca.aguilo@umu.se.
Michelle E. Ehrlich, Email: michelle.ehrlich@mssm.edu.
Accession Numbers
All next-generation sequencing data are deposited in NCBI GEO database under accession numbers GEO: GSE86947 and GSE86911.
Supplemental Information
References
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