Jeffrey J. Kim

Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells

Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOHs effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOHs effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis.

Cdk2ap1 Is Required for Epigenetic Silencing of Oct4 during Murine Embryonic Stem Cell Differentiation

Oct4 is a known master regulator of stem cell renewal and differentiation. Expression of Oct4 during differentiation is regulated by promoter methylation by the nucleosome remodeling and histone deacetylation (NuRD) complex. Here, we show that Cdk2ap1, a negative regulator of Cdk2 function and cell cycle, promotes Oct4 promoter methylation during murine embryonic stem cell differentiation to down-regulate Oct4 expression. We further show that this repressor function of Cdk2ap1 is dependent on its physical interaction with the methyl DNA-binding protein, Mbd3. Our data support a potential molecular link between the known differentiation promoters, including bone morphogenetic proteins and transforming growth factor signaling, and embryonic stem cell differentiation.

Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation

Mouse embryonic stem cells (mESCs) maintain pluripotency and indefinite self-renewal through yet to be defined molecular mechanisms. Leukemia inhibitory factor has been utilized to maintain the symmetrical self-renewal and pluripotency of mESCs in culture. It has been suggested that molecules with significant cellular effects on retinoblastoma protein (pRb) or its related pathways should have functional impact on mESC proliferation and differentiation. However, the involvement of pRb in pluripotent differentiation of mESCs has not been extensively elaborated. In this paper, we present novel experimental data indicating that Cdk2ap1 (cyclin-dependent kinase 2-associating protein 1), an inhibitor of G1/S transition through down-regulation of CDK2 and an essential gene for early embryonic development, confers competency for mESC differentiation. Targeted disruption of Cdk2ap1 in mESCs resulted in abrogation of leukemia inhibitory factor withdrawal-induced differentiation, along with altered pRb phosphorylation. The differentiation competency of the Cdk2ap1−/− mESCs was restored upon the ectopic expression of Cdk2ap1 or a nonphosphorylatable pRb mutant (mouse Ser788 → Ala), suggesting that the CDK2AP1-mediated differentiation of mESCs was elicited through the regulation of pRb. Further analysis on mESC maintenance or differentiation-related gene expression supports the phosphorylation at serine 788 in pRb plays a significant role for the CDK2AP1-mediated differentiation of mESCs. These data clearly demonstrate that CDK2AP1 is a competency factor in the proper differentiation of mESCs by modulating the phosphorylation level of pRb. This sheds light on the role of the establishment of the proper somatic cell type cell cycle regulation for mESCs to enter into the differentiation process.

Double Edge: CDK2AP1 in Cell-cycle Regulation and Epigenetic Regulation

Cancer research has been devoted toward an understanding of the molecular regulation and functional significance of cell-cycle regulators in the pathogenesis and development of cancers. Cyclin-dependent Kinase 2-associated Protein 1 (CDK2AP1) is one such cell-cycle regulator, originally identified as a growth suppressor and a prognostic marker for human oral/head and neck cancers. Functional importance and the molecular mechanism of CDK2AP1-mediated cell-cycle regulation have been documented over the years. Recent progress has shown that CDK2AP1 is a competency factor in embryonic stem cell differentiation. Deletion of CDK2AP1 leads to early embryonic lethality, potentially through altered differentiation capability of embryonic stem cells. More intriguingly, CDK2AP1 exerts its effect on stem cell maintenance/differentiation through epigenetic regulation. Cancer cells and stem cells share common cellular characteristics, most prominently in maintaining high proliferative potential through an unconventional cell-cycle regulatory mechanism. Cross-talk between cellular processes and molecular signaling pathways is frequent in any biological system. Currently, it remains largely elusive how cell-cycle regulation is mechanistically linked to epigenetic control. Understanding the molecular mechanism underlying CDK2AP1-mediated cell-cycle regulation and epigenetic control will set an example for establishing a novel and effective molecular link between these two important regulatory mechanisms.

Discovery of consensus gene signature and intermodular connectivity defining self-renewal of human embryonic stem cells

Molecular markers defining self‐renewing pluripotent embryonic stem cells (ESCs) have been identified by relative comparisons between undifferentiated and differentiated cells. Most of analysis has been done under a specific differentiation condition that may present significantly different molecular changes over others. Therefore, it is currently unclear if there are true consensus markers defining undifferentiated human ESCs (hESCs). To identify a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions, we have performed microarray analysis on undifferentiated hESCs (H1 and H9) and differentiated EBs and validated our results using publicly available expression array datasets. We constructed consensus modules by Weighted Gene Coexpression Network Analysis and discovered novel markers that are consistently present in undifferentiated hESCs under various differentiation conditions. We have validated top markers (downregulated: LCK, KLKB1, and SLC7A3; upregulated: RhoJ, Zeb2, and Adam12) upon differentiation. Functional validation analysis of LCK in self‐renewal of hESCs using LCK inhibitor or gene silencing with siLCK resulted in a loss of undifferentiation characteristics—morphological change, reduced alkaline phosphatase activity, and pluripotency gene expression, demonstrating a potential functional role of LCK in self‐renewal of hESCs. We have designated hESC markers to interactive networks in the genome, identifying possible interacting partners and showing how new markers relate to each other. Furthermore, comparison of these datasets with available datasets from induced pluripotent stem cells (iPSCs) revealed that the level of these newly identified markers was correlated to the establishment of iPSCs, which may imply a potential role of these markers in gaining of cellular potency. Stem Cells 2014;32:1468–1479

A Novel Regulatory Factor Recruits the Nucleosome Remodeling Complex to Wingless Integrated (Wnt) Signaling Gene Promoters in Mouse Embryonic Stem Cells

Background: Nucleosome remodeling is integral in transcriptional control, yet the exact mechanisms involved remain to be elucidated. Results: CDK2AP1 is a novel regulatory factor that guides the NuRD complex. Conclusion: CDK2AP1 guides NuRD onto promoters involved in Wnt signaling of mouse embryonic stem cells. Significance: Understanding NuRD regulation is critical in understanding transcriptional regulation. The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Alcohol-induced suppression of KDM6B dysregulates the mineralization potential in dental pulp stem cells

Epigenetic changes, such as alteration of DNA methylation patterns, have been proposed as a molecular mechanism underlying the effect of alcohol on the maintenance of adult stem cells. We have performed genome-wide gene expression microarray and DNA methylome analysis to identify molecular alterations via DNA methylation changes associated with exposure of human dental pulp stem cells (DPSCs) to ethanol (EtOH). By combined analysis of the gene expression and DNA methylation, we have found a significant number of genes that are potentially regulated by EtOH-induced DNA methylation. As a focused approach, we have also performed a pathway-focused RT-PCR array analysis to examine potential molecular effects of EtOH on genes involved in epigenetic chromatin modification enzymes, fibroblastic markers, and stress and toxicity pathways in DPSCs. We have identified and verified that lysine specific demethylase 6B (KDM6B) was significantly dysregulated in DPSCs upon EtOH exposure. EtOH treatment during odontogenic/osteogenic differentiation of DPSCs suppressed the induction of KDM6B with alterations in the expression of differentiation markers. Knockdown of KDM6B resulted in a marked decrease in mineralization from implanted DPSCs in vivo. Furthermore, an ectopic expression of KDM6B in EtOH-treated DPSCs restored the expression of differentiation-related genes. Our study has demonstrated that EtOH-induced inhibition of KDM6B plays a role in the dysregulation of odontogenic/osteogenic differentiation in the DPSC model. This suggests a potential molecular mechanism for cellular insults of heavy alcohol consumption that can lead to decreased mineral deposition potentially associated with abnormalities in dental development and also osteopenia/osteoporosis, hallmark features of fetal alcohol spectrum disorders.

Genome-wide transcriptomic alterations induced by ethanol treatment in human dental pulp stem cells (DPSCs)

Human dental pulp stem cells (DPSCs) isolated from adult dental pulp are multipotent mesenchymal stem cells that can be directed to differentiate into osteogenic/odontogenic cells and also trans-differentiate into neuronal cells. The utility of DPSC has been explored in odontogenic differentiation for tooth regeneration. Alcohol abuse appears to lead to periodontal disease, tooth decay and mouth sores that are potentially precancerous. Persons who abuse alcohol are at high risk of having seriously deteriorated teeth, gums and compromised oral health in general. It is currently unknown if alcohol exposure has any impact on adult stem cell maintenance, stem cell fate determination and plasticity, and stem cell niche environment. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE57255. Our data provide transcriptomic changes that are occurring by EtOH treatment of DPSCs at 24-hour and 48-hour time point.

Cdk2ap2 Is a Novel Regulator for Self-Renewal of Murine Embryonic Stem Cells

In this study we present data to support the role for Cdk2ap2 in regulating self-renewal of mouse embryonic stem cells (mESCs) under permissive conditions, and cell survival during differentiation of the mESCs into terminally differentiated cell types. To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2(tr/tr)). The Cdk2ap2(tr/tr) mESCs, when grown in a complete growth medium containing leukemia inhibitory factor (LIF), showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation, including Brachyury (T), Afp, and S100a, when compared to Wt mESCs. Cdk2ap2(tr/tr) mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Furthermore, Cdk2ap2(tr/tr) mESCs were unable to form teratomas in severe combined immunodeficiency (SCID) mice. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis.

Molecular effect of ethanol during neural differentiation of human embryonic stem cells in vitro

Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. To better understand the molecular effect of alcohol on the process of neural differentiation, we have performed gene expression microarray analysis on human embryonic stem cells being directed to neural rosettes and neural precursor cells in the presence of ethanol treatment. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE56906. Our data provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development.