Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation.

In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Here we demonstrate in the mouse that a single factor, the forkhead transcriptional regulator FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. Inducible deletion of Foxl2 in adult ovarian follicles leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Our results show that maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.

Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

miR-322/-503 cluster is expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification.

Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.

Germ Cell Nuclear Factor (GCNF) Represses Oct4 Expression and Globally Modulates Gene Expression in Human Embryonic Stem (hES) Cells.

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.

Induced multipotency in adult keratinocytes through down-regulation of ΔNp63 or DGCR8.

The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.

Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells.

All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy.

GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells.

Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17ß-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.

Ggnbp2 Is Essential for Pregnancy Success via Regulation of Mouse Trophoblast Stem Cell Proliferation and Differentiation.

The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.

Genome-Wide Identification of MESP1 Targets Demonstrates Primary Regulation Over Mesendoderm Gene Activity.

MESP1 is considered the first sign of the nascent cardiac mesoderm and plays a critical role in the appearance of cardiac progenitors, while exhibiting a transient expression in the developing embryo. We profiled the transcriptome of a pure population of differentiating MESP1-marked cells and found that they chiefly contribute to the mesendoderm lineage. High-throughput sequencing of endogenous MESP1-bound DNA revealed that MESP1 preferentially binds to two variants of E-box sequences and activates critical mesendoderm modulators, including Eomes, Gata4, Wnt5a, Wnt5b, Mixl1, T, Gsc, and Wnt3. These mesendoderm markers were enriched in the MESP1 marked population before the appearance of cardiac progenitors and myocytes. Further, MESP1-binding is globally associated with H(3)K(27) acetylation, supporting a novel pivotal role of it in regulating target gene epigenetics. Therefore, MESP1, the pioneer cardiac factor, primarily directs the appearance of mesendoderm, the intermediary of the earliest progenitors of mesoderm and endoderm organogenesis.

Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells.

How tumor suppressor p53 selectively responds to specific signals, especially in normal cells, is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation, induced by retinoic acid, versus DNA damage, caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and, in pluripotent hESCs, are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases, UTX and JMJD3, to chromatin. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs.

Sox2 is the faithful marker for pluripotency in pig: evidence from embryonic studies.

BACKGROUND: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. RESULTS: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. CONCLUSIONS: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks.

Revisiting the role of GCNF in embryonic development.

GCNF (NR6A1) is essential for embryonic development. GCNF belongs to the nuclear receptor (NR) gene family, it is distantly related to other NRs and is the only member of subfamily 6. As the ligand for GCNF has not been identified, GCNF is designated an orphan nuclear receptor. GCNF has been found to be a transcriptional repressor, through specific binding to DR0 response elements, which is found in the Oct4 proximal promoter for example. GCNF is expressed widely in early mouse embryos, and later in the developing nervous system. GCNF knockout mouse embryos die around E10.5. GCNF is required for the restriction of Oct4 expression to primordial germ cells after gastrulation. GCNF is expressed in ES/EC cells and during their differentiation, and has been reported to be required for pluripotency gene repression during retinoic acid (RA)-induced mES cell differentiation. GCNF can interact with DNA methylation proteins, and is suggested to recruit DNA methylation complexes to repress and silence Oct4 expression. Nuclear receptor regulation in embryonic development is a complex process, as different nuclear receptors have overlapping and distinct functions. In-depth exploration of GCNF function and mechanism of action will help to comprehensively understand the nuclear receptor regulation in embryonic development.

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF(-) (/) (-) ) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3'UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF(-) (/) (-) ESCs restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF(-) (/) (-) ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a.

Epigenetic reprogramming of the germ cell nuclear factor gene is required for proper differentiation of induced pluripotent cells.

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that germ cell nuclear factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells recapitulated the phenotype of Gcnf knockout (Gcnf(-/-)) ES cells. Reintroduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf(-/-) ES cells rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells.

Brief report: Oct4 and canonical Wnt signaling regulate the cardiac lineage factor Mesp1 through a Tcf/Lef-Oct4 composite element.

Oct4 is the gatekeeper of stem cell pluripotency, but recent evidences also support Oct4 as a key regulator of germ layer formation and lineage commitment. How Oct4 contributes to lineage commitment is not well understood. We identified a Tcf/Lef-Oct4 composite site in the promoter of the cardiac mesoderm gene Mesp1, with a nucleotide sequence identical to the previously established Sox2-Oct4 composite site. This Tcf/Lef-Oct4 composite site mediated synergistic activation of the Mesp1 promoter by Oct4 and canonical Wnt signaling. Transcription ternary complexes were formed with Oct4 and Wnt terminal components, Lef1. Point mutations on the Tcf/Lef-Oct4 composite site impaired Oct4 and Lef1 binding and Mesp1-ß-gal transgene reporter expression during mouse embryogenesis. In ZHBTc4 murine embryonic stem cells, the loss of Oct4 during differentiation impaired Mesp1 expression and the development of the cardiac program. This Tcf/Lef-Oct4 composite site appears to be a unique nodal point regulatory element that may drive pluripotency via Sox2-Oct4 and switch on lineage-related genes through Oct4's recruitment of Tcf/Lef factors.

Role of LIN28A in mouse and human trophoblast cell differentiation.

Proper regulation of trophoblast proliferation, differentiation, and function are critical for placenta development and function. The RNA-binding protein, LIN28A, has been well characterized as a potent regulator of differentiation in embryonic stem cells; however, little is known about the function of LIN28A in the placenta. We assessed LIN28A in vitro using mouse trophoblast stem (mTS) cells and human trophoblast cells (ACH-3P). We observed that LIN28A decreased and let-7 miRNA increased when mTS cells were induced to differentiate into mouse trophoblast giant cells (mTGCs) upon the removal of FGF4, heparin and conditioned medium. Similarly, we observed that LIN28A decreased in ACH-3P cells induced to syncytialize with forskolin treatment. To assess LIN28A in vivo we examined Embryonic Day 11.5 mouse placenta and observed abundant LIN28A in the chorioallantoic interface and labyrinth layer, with little LIN28A staining in spongiotrophoblast or differentiated mTGCs. Additionally, shRNA-mediated LIN28A knockdown in ACH-3P cells resulted in increased spontaneous syncytialization, and increased levels of syncytiotrophoblast markers hCG, LGALS13, and ERVW-1 mRNA. Additionally, targeted degradation of LIN28A mRNA increased responsiveness to forskolin-induced differentiation. In contrast, targeted degradation of Lin28a mRNA in mTS cells did not alter cell phenotype when maintained under proliferative culture conditions. Together, these data establish that LIN28A has a functional role in regulating trophoblast differentiation and function, and that loss of LIN28A in human trophoblast is sufficient to induce differentiation, but does not induce differentiation in the mouse.

The role of nuclear receptors in embryonic stem cells.

Embryonic stem (ES) cells, isolated from pre-implantation embryos, can grow indefinitely invitro (self-renewal) and have potential to differentiate into all cell types in the body (pluripotency). The nuclear receptor gene family is very important for controlling development, differentiation and homeostasis. Here, we review the new progress in understanding the role of nuclear receptors in ES cells focusing on the structure, expression and function of several nuclear receptors. LRH1, DAX1, Esrrß and TR2 play critical roles in maintaining pluripotency, while, GCNF, COUP-TFs and sumoylated TR2 are critical in regulating the exit from pluripotency. Nuclear receptors hold great potential as targets of manipulation of ES and iPS cells for applications in regenerative medicine, because they are ligand-activated transcription factors that can be regulated by small molecule agonists and antagonists.

Transcriptomine, a web resource for nuclear receptor signaling transcriptomes.

The nuclear receptor (NR) superfamily of ligand-regulated transcription factors directs ligand- and tissue-specific transcriptomes in myriad developmental, metabolic, immunological, and reproductive processes. The NR signaling field has generated a wealth of genome-wide expression data points, but due to deficits in their accessibility, annotation, and integration, the full potential of these studies has not yet been realized. We searched public gene expression databases and MEDLINE for global transcriptomic datasets relevant to NRs, their ligands, and coregulators. We carried out extensive, deep reannotation of the datasets using controlled vocabularies for RNA Source and regulating molecule and resolved disparate gene identifiers to official gene symbols to facilitate comparison of fold changes and their significance across multiple datasets. We assembled these data points into a database, Transcriptomine (http://www.nursa.org/transcriptomine), that allows for multiple, menu-driven querying strategies of this transcriptomic "superdataset," including single and multiple genes, Gene Ontology terms, disease terms, and uploaded custom gene lists. Experimental variables such as regulating molecule, RNA Source, as well as fold-change and P value cutoff values can be modified, and full data records can be either browsed or downloaded for downstream analysis. We demonstrate the utility of Transcriptomine as a hypothesis generation and validation tool using in silico and experimental use cases. Our resource empowers users to instantly and routinely mine the collective biology of millions of previously disparate transcriptomic data points. By incorporating future transcriptome-wide datasets in the NR signaling field, we anticipate Transcriptomine developing into a powerful resource for the NR- and other signal transduction research communities.

Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles.

To maintain the female reproductive lifespan, the majority of ovarian primordial follicles are preserved in a quiescent state in order to provide ova for later reproductive life. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Here we provide genetic evidence to show that the tumor suppressor tuberous sclerosis complex 1 (Tsc1), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the quiescence of primordial follicles. In mutant mice lacking the Tsc1 gene in oocytes, the entire pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in the oocyte, ending up with follicular depletion in early adulthood and causing premature ovarian failure (POF). We further show that maintenance of the quiescence of primordial follicles requires synergistic, collaborative functioning of both Tsc and PTEN (phosphatase and tensin homolog deleted on chromosome 10) and that these two molecules suppress follicular activation through distinct ways. Our results suggest that Tsc/mTORC1 signaling and PTEN/PI3K (phosphatidylinositol 3 kinase) signaling synergistically regulate the dormancy and activation of primordial follicles, and together ensure the proper length of female reproductive life. Deregulation of these signaling pathways in oocytes results in pathological conditions of the ovary, including POF and infertility.