POU Transcription Factor Research Articles

The Nuclear NF-κB Regulator IκBζ: Updates on Its Molecular Functions and Pathophysiological Roles.

More than a decade after the discovery of the classical cytoplasmic IκB proteins, IκBζ was identified as an additional member of the IκB family. Unlike cytoplasmic IκB proteins, IκBζ has distinct features, including its nuclear localization, preferential binding to NF-κB subunits, unique expression properties, and specialized role in NF-κB regulation. While the activation of NF-κB is primarily controlled by cytoplasmic IκB members at the level of nuclear entry, IκBζ provides an additional layer of NF-κB regulation in the nucleus, enabling selective gene activation. Human genome-wide association studies (GWAS) and gene knockout experiments in mice have elucidated the physiological and pathological roles of IκBζ. Despite the initial focus to its role in activated macrophages, IκBζ has since been recognized as a key player in the IL-17-triggered production of immune molecules in epithelial cells, which has garnered significant clinical interest. Recent research has also unveiled a novel molecular function of IκBζ, linking NF-κB and the POU transcription factors through its N-terminal region, whose role had remained elusive for many years.

IκBζ is a dual-use coactivator of NF-κB and POU transcription factors

OCA-B, OCA-T1, and OCA-T2 belong to a family of coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IκBζ (encoded by the NFKBIZ gene) as an additional coactivator of POU TFs. Although originally discovered as an inducible regulator of NF-κB, we show here that IκBζ shares a microhomology with OCA proteins and uses this segment to bind to POU TFs and octamer-motif-containing DNA. Our functional experiments suggest that IκBζ requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by epigenomic analysis of MYD88L265P-mutant lymphoma cells, which revealed colocalization of IκBζ with the POU TF OCT2 and NF-κB:p50 at hundreds of DNA elements harboring octamer and κB motifs. These results suggest that IκBζ is a transcriptional coactivator that can amplify and integrate the output of NF-κB and POU TFs at inducible genes in immune cells.

STEM-24. WDR5 REPRESENTS A THERAPEUTICALLY EXPLOITABLE TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA

Abstract Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

OCT4 protein and gene expression analysis in the differentiation of spermatogonia stem cells into neurons by immunohistochemistry, immunocytochemistry, and bioinformatics analysis.

Spermatogonia Stem Cells (SSCs) are potential candidates for reprogramming and regeneration. Recent studies have revealed that differentiated cells can be reverted to pluripotent by overexpressing a set of pluripotent transcription factors. OCT4 (encoded by pou5f1), a POU transcription factor family member, is essential to the potential that controls pluripotency, and it is widely expressed in pluripotent stem cells, although it decreased or suppressed after differentiation. In this investigated research, we examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein-protein interaction and enrichment analysis. We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming. Our findings can help us better understand the process of differentiation of spermatogonia stem cells into neurons, and it can be effective in finding new and more efficient treatments for neurogenesis and repair of neurons. We examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein-protein interaction and enrichment analysis. We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming.

361 WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma

OBJECTIVES/GOALS: Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. METHODS/STUDY POPULATION: Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. RESULTS/ANTICIPATED RESULTS: In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. DISCUSSION/SIGNIFICANCE: Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

The functional diversity of the POUV-class proteins across vertebrates.

POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo. The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins-zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage.

Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3(refs.2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNAoctamermotif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.

Regulation of Oct4 in stem cells and neural crest cells.

During embryonic development, cells gradually restrict their developmental potential as they exit pluripotency and differentiate into various cell types. The POU transcription factor Oct4 (encoded by Pou5f1) lies at the center of the pluripotency machinery that regulates stemness and differentiation in stem cells, and is required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Several studies have revealed that Oct4 and other stemness genes are also expressed in multipotent cell populations such as neural crest cells (NCCs), and are required to expand the NCC developmental potential. Transcriptional regulation of Oct4 has been studied extensively in stem cells during early embryonic development and reprogramming, but not in NCCs. Here, we review how Oct4 is regulated in pluripotent stem cells, and address some of the gaps in knowledge about regulation of the pluripotency network in NCCs.

EP183: Functional assessment of a novel POU4F3 missense variant

The POU-domain family of transcription factors have a wide variety of functions and are critical regulators of many developmental processes. They are characterized by a common bipartite DNA-binding structure with POU-specific and POU-homeodomain subdomains, joined by an unstructured linkage region. Both subdomains are required for high affinity DNA binding of POU transcription factors. The POU4F3 gene has been found to be essential for inner-ear hair cell maintenance and is associated with autosomal-dominant nonsyndromic hearing loss with variable age of onset. Here we describe an individual with a novel heterozygous missense variant in POU4F3 exhibiting bilateral childhood onset sensorineural hearing loss. The proband’s mother also exhibited hearing loss. Functional assessment of this variant was performed to determine the impact on protein localization, and familial segregation studies were performed to better understand the pathogenicity of this variant. The proband underwent exome sequencing (ES) which revealed a maternally inherited missense variant in POU4F3 (NM_002700.2, c.856A>G, p.(Lys286Glu)). Family members were subsequently given audiograms and tested for this variant. To functionally assess the variant HA-tagged wild-type and mutant POU4F3 were expressed by using the mammalian expression vector pcDNA3.1 and transfected in Hek293 cells. To determine the subcellular localization of the HA-tagged wild-type and mutant POU4F3, transiently transfected cells were fixed with 4% paraformaldehyde, incubated with primary anti-HA and secondary goat-anti-mouse Alexa488-labelled antibodies. Images were taken with a fluorescence microscope and processed using ImageJ. To determine the number of cells expressing POU4F3 outside the nucleus, slides were blinded and 100 cells of each transfected mutant were scored; each construct was transfected in duplicate. Audiograms revealed that one of the proband’s brothers had mild hearing loss, while the other was unaffected. Familial variant testing then showed that the affected brother is heterozygous for the POU4F3 variant while the unaffected brother does not harbor the variant. The mother was previously shown to have hearing loss, and the variant was maternally inherited. Functional assessment of the variant demonstrated that it results in significant mis-localization from the nucleus to the cytoplasm compared to wild type controls (p value: 0.009). This affect was more significant than positive controls included in the analysis. The proband’s variant is not reported in population databases and is predicted to be deleterious by REVEL (0.89) and CADD (27.7). This variant was clinically classified as a VUS, with the following criteria applied: PM2+PP3. This variant falls within the homeodomain of the protein, which is required for DNA binding of POU transcription factors. Pathogenic missense variants in POU4F3 have previously been shown to disrupt the nuclear localization of the protein, so this was assessed in cells expressing the p.(Lys286Glu) variant. The proband’s variant was found to result in significant mis-localization of POU4F3 to the cytoplasm, suggesting an impact on the protein function. Finally, the variant was found to segregate with hearing loss in the proband’s mother and affected brother, allowing us to apply PP1 criteria based on the ClinGen hearing loss evaluation guidelines. Taken together these data are suggestive that this variant is causative for the proband, however this remains a VUS.

POU4F3 Acts as a Tumor Suppressor in Lung Adenocarcinoma via the Endoplasmic Reticulum Stress Signaling Pathway

Lung cancer is the most common malignancy worldwide, and LUAD is the primary type of lung cancer. Recently, the POU transcription factor family has been associated with the development of multicancer, especially lung cancer. However, the relationship between POU domain class Ⅳ transcription factor Ⅲ (POU4F3) and lung cancer remains unknown. We detected the expression of POU4F3 in human LUAD and adjacent tissues with immunohistochemical staining, and we found that POU4F3 was expressed less in LUAD tissues compared with adjacent tissues. Patients with higher POU4F3 expression have more prolonged overall survival. We then constructed SPCA1 and A549 cells with stable overexpression or inhibition of POU4F3. We found that overexpressed POU4F3 suppressed LUAD cell proliferation in vitro and in vivo, according to CCK-8, colony formation, and xenograft assays. LUAD cell apoptosis was suppressed by POU4F3 overexpression based on Flow cytometry. The downregulation of POU4F3 yielded the opposite patterns. Next, we explored the possible mechanisms through which POU4F3 promoted the apoptosis of LUAD cells. Western blotting suggested that overexpression of POU4F3 significantly increased protein expression levels of the PERK/eIF2α/ATF4/CHOP and IRE1α/XBP1s pathways of ERS, while POU4F3 absence reversed the expressions of the above essential proteins in ERS pathways in SPCA1 and A549 cells. However, we found that PERK inhibitor but not IRE1 inhibitor can reverse the effect of POU4F3 overexpression on apoptosis. This study indicated that POU4F3 may work as a tumor suppressor in LUAD via regulating the PERK/eIF2α/ATF4/CHOP pathway. We made it possible to develop POU4F3 as a diagnostic, therapeutic, and prognostic target of LUAD.

Cnidarian hair cell development illuminates an ancient role for the class IV POU transcription factor in defining mechanoreceptor identity.

Although specialized mechanosensory cells are found across animal phylogeny, early evolutionary histories of mechanoreceptor development remain enigmatic. Cnidaria (e.g. sea anemones and jellyfishes) is the sister group to well-studied Bilateria (e.g. flies and vertebrates), and has two mechanosensory cell types - a lineage-specific sensory effector known as the cnidocyte, and a classical mechanosensory neuron referred to as the hair cell. While developmental genetics of cnidocytes is increasingly understood, genes essential for cnidarian hair cell development are unknown. Here, we show that the class IV POU homeodomain transcription factor (POU-IV) - an indispensable regulator of mechanosensory cell differentiation in Bilateria and cnidocyte differentiation in Cnidaria - controls hair cell development in the sea anemone cnidarian Nematostella vectensis. N. vectensis POU-IV is postmitotically expressed in tentacular hair cells, and is necessary for development of the apical mechanosensory apparatus, but not of neurites, in hair cells. Moreover, it binds to deeply conserved DNA recognition elements, and turns on a unique set of effector genes - including the transmembrane receptor-encoding gene polycystin 1 - specifically in hair cells. Our results suggest that POU-IV directs differentiation of cnidarian hair cells and cnidocytes via distinct gene regulatory mechanisms, and support an evolutionarily ancient role for POU-IV in defining the mature state of mechanosensory neurons.

Key features of the POU transcription factor Oct4 from an evolutionary perspective.

Oct4, a class V POU-domain protein that is encoded by the Pou5f1 gene, is thought to be a key transcription factor in the early development of mammals. This transcription factor plays indispensable roles in pluripotent stem cells as well as in the acquisition of pluripotency during somatic cell reprogramming. Oct4 has also been shown to play a role as a pioneer transcription factor during zygotic genome activation (ZGA) from zebrafish to human. However, during the past decade, several studies have brought these conclusions into question. It was clearly shown that the first steps in mouse development are not affected by the loss of Oct4. Subsequently, the role of Oct4 as a genome activator was brought into doubt. It was also found that the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) could proceed without Oct4. In this review, we summarize recent findings, reassess the role of Oct4 in reprogramming and ZGA, and point to structural features that may underlie this role. We speculate that pluripotent stem cells resemble neural stem cells more closely than previously thought. Oct4 orthologs within the POUV class hold key roles in genome activation during early development of species with late ZGA. However, in Placentalia, eutherian-specific proteins such as Dux overtake Oct4 in ZGA and endow them with the formation of an evolutionary new tissue-the placenta.

POU2F2 regulates glycolytic reprogramming and glioblastoma progression via PDPK1-dependent activation of PI3K/AKT/mTOR pathway

The POU Class Homeobox 2 (POU2F2) is a member of POU transcription factors family, which involves in cell immune response by regulating B cell proliferation and differentiation genes. Recent studies have shown that POU2F2 acts as tumor-promoting roles in some cancers, but the underlying mechanism remains little known. Here, we identified that the highly expressed POU2F2 significantly correlated with poor prognosis of glioblastoma (GBM) patients. POU2F2 promoted cell proliferation and regulated glycolytic reprogramming. Mechanistically, the AKT/mTOR signaling pathway played important roles in the regulation of POU2F2-mediated aerobic glycolysis and cell growth. Furthermore, we demonstrated that POU2F2 activated the transcription of PDPK1 by directly binding to its promoter. Reconstituted the expression of PDPK1 in POU2F2-knockdown GBM cells reactivated AKT/mTOR pathway and recovered cell glycolysis and proliferation, whereas this effect was abolished by the PDPK1/AKT interaction inhibitor. In addition, we showed that POU2F2-PDPK1 axis promoted tumorigenesis by regulating glycolysis in vivo. In conclusion, our findings indicate that POU2F2 leads a metabolic shift towards aerobic glycolysis and promotes GBM progression in PDPK1-dependent activation of PI3K/AKT/mTOR pathway.

Expression of Eya1 in mouse taste buds.

Taste substances are detected by taste receptor cells in the taste buds in the oral epithelium. Individual taste receptor cells contribute to evoking one of the five taste qualities: sweet, umami, bitter, sour, and salty (sodium). They are continuously replaced every few weeks by new ones generated from local epithelial stem cells. A POU transcription factor, Pou2f3 (also known as Skn-1a), regulates the generation and differentiation of sweet, umami, and bitter cells. However, the molecular mechanisms underlying terminal differentiation into these Pou2f3-dependent taste receptor cells remain unknown. To identify the candidate molecules that regulate the differentiation of these taste receptor cells, we searched for taste receptor type-specific transcription factors using RNA-sequence data of sweet and bitter cells. No transcription factor gene showing higher expression in sweet cells than in bitter cells was found. Eyes absent 1 (Eya1) was identified as the only transcription factor gene showing higher expression in bitter cells than in sweet cells. In situ hybridization revealed that Eya1 was predominantly expressed in bitter cells and also in the putative immature/differentiating taste bud cells in circumvallate and fungiform papillae and soft palate. Eya1 is a candidate molecule that regulates the generation and differentiation of bitter cells.

The POU Transcription Factor POU-M2 Regulates Vitellogenin Receptor Gene Expression in the Silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) play critical roles in egg formation by transporting vitellogenin (Vg) into oocytes in insects. Although the function of VgR in insects is well studied, the transcriptional regulation of this gene is still unclear. Here, we cloned the promoter of the VgR gene from Bombyx mori (BmVgR), and predicted many POU cis-response elements (CREs) in its promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that the POU transcription factor POU-M2 bound directly to the CREs of the promoter. Overexpression of POU-M2 in an ovarian cell line (BmNs) enhanced BmVgR transcription and promoter activity detected by quantitative reverse transcription PCR and luciferase reporter assays. Analyses of expression patterns indicated that POU-M2 was expressed in ovary at day two of wandering stage initially, followed by BmVgR. RNA interference of POU-M2 significantly reduced the transcription of BmVgR in ovary and egg-laying rate. Our results suggest a novel function for the POU factor in silkworm oogenesis by its involvement in BmVgR regulation and expands the understanding of POU factors in insect VgR expression.

Reconstruction of protein domain evolution using single-cell amplified genomes of uncultured choanoflagellates sheds light on the origin of animals.

Understanding the origins of animal multicellularity is a fundamental biological question. Recent genome data have unravelled the role that co-option of pre-existing genes played in the origin of animals. However, there were also some important genetic novelties at the onset of Metazoa. To have a clear understanding of the specific genetic innovations and how they appeared, we need the broadest taxon sampling possible, especially among early-branching animals and their unicellular relatives. Here, we take advantage of single-cell genomics to expand our understanding of the genomic diversity of choanoflagellates, the sister-group to animals. With these genomes, we have performed an updated and taxon-rich reconstruction of protein evolution from the Last Eukaryotic Common Ancestor (LECA) to animals. Our novel data re-defines the origin of some genes previously thought to be metazoan-specific, like the POU transcription factor, which we show appeared earlier in evolution. Moreover, our data indicate that the acquisition of new genes at the stem of Metazoa was mainly driven by duplications and protein domain rearrangement processes at the stem of Metazoa. Furthermore, our analysis allowed us to reveal protein domains that are essential to the maintenance of animal multicellularity. Our analyses also demonstrate the utility of single-cell genomics from uncultured taxa to address evolutionary questions.This article is part of a discussion meeting issue ‘Single cell ecology’.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Genome-Wide Analysis and Hormone Regulation of Chitin Deacetylases in Silkworm.

Chitin deacetylases (CDAs) are a group of enzymes involved in chitin metabolism in insects; they play a critical role in molting, pupation, and the modification of chitin. In this study, we identified several CDAs in the silkworm, Bombyx mori (BmCDA), and investigated the effect of various hormones on their expression in B. mori larvae and embryo cell lines (BmE). Eight genes encoding BmCDAs were identified in the silkworm genome. They showed different expression patterns in different tissues, and were classified into three types based on where they were expressed: the exoskeleton, digestive organs, and genital organs. Moreover, we found that some BmCDAs showed upregulated expression during the molting period, especially during the fourth molting period in larvae. We also verified that the expression of BmCDA1–6 was upregulated by treatment with 20-hydroxyecdysone not only in larvae, but also in BmE cells. Interestingly, juvenile hormone analog treatment also upregulated the expression of some BmCDAs. The overexpression of several transcription factors revealed that the POU transcription factor POUM2 may play a major role in the regulation of BmCDA expression. Finally, the silencing of BmCDA1 and BmCDA2 did not lead to abnormal phenotypes or death, but may have led to delays in silkworm pupation. These results provide important information about lepidopteran insects in terms of chitin deacetylases and the regulation of their expression.

Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells.

SummaryThe POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.