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GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression.

During early mammalian development, genesis of the first two cell lineages, inner cell mass (ICM) and trophectoderm (TE), is dependent upon functions of key transcription factors that are expressed in a regulated and spatially restricted fashion. In this study, we demonstrate that during early mouse development, mRNA expression of transcription factor GATA3 is induced at the 4-cell stage and is consistently present during pre-implantation embryonic development. Interestingly, at the blastocyst stage, Gata3 mRNA is selectively up-regulated within the TE lineage, and GATA3 protein is abundantly present only in the TE but not in the ICM. Using mouse trophoblast stem cells (TS cells) as a model, we found that, knockdown of GATA3 by RNA interference (RNAi) down-regulates expression of caudal-type homeobox 2 (CDX2), a key regulator of the TE lineage. Chromatin immunoprecipitation (ChIP) analyses revealed that, in TS cells, GATA3 directly regulates Cdx2 transcription from a conserved GATA motif at the intron 1 region of the Cdx2 locus. ChIP analyses with mouse blastocysts also detected GATA3 occupancy at intron 1 of the Cdx2 locus. In addition, down-regulation of GATA3 in pre-implantation mouse embryos reduces Cdx2 expression and inhibits morula to blastocyst transformation. Our results indicate a novel function of GATA3, in which it is selectively expressed in TE, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation.

Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.

Self‐Renewal Versus Lineage Commitment of Embryonic Stem Cells: Protein Kinase C Signaling Shifts the Balance

The intricate molecular mechanisms that regulate ESC pluripotency are incompletely understood. Prior research indicated that activation of the Janus kinase–signal transducer and activator of transcription (STAT3) pathway or inhibition of extracellular signal‐regulated kinase/glycogen synthase kinase 3 (ERK/GSK3) signaling maintains mouse ESC (mESC) pluripotency. Here, we demonstrate that inhibition of protein kinase C (PKC) isoforms maintains mESC pluripotency without the activation of STAT3 or inhibition of ERK/GSK3 signaling pathways. Our analyses revealed that the atypical PKC isoform, PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ–nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling axis. Furthermore, inhibition of PKC isoforms permits derivation of germline‐competent ESCs from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts toward induced pluripotent stem cells. Our results indicate that PKC signaling is critical to balancing ESC self‐renewal and lineage commitment. STEM Cells 2011;29:618–628

Regulation of angiogenesis by histone chaperone HIRA-mediated incorporation of lysine 56-acetylated histone H3.3 at chromatin domains of endothelial genes.

Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.

EED and KDM6B coordinate the first mammalian cell lineage commitment to ensure embryo implantation.

ABSTRACT The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.

Inhibition of Protein Kinase C Signaling Maintains Rat Embryonic Stem Cell Pluripotency

Background: Signaling mechanisms regulating rat embryonic stem cell (rESC) pluripotency are understood poorly. Results: Inhibition of PKC signaling promotes rESC self-renewal without compromising developmental potency. Conclusion: PKC signaling contributes to the balance of self-renewal versus differentiation of rESCs. Significance: PKC signaling could be targeted to derive rat pluripotent stem cells to establish transgenic models and for regenerative studies. Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria.

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype.

Genetic redundancy of GATA factors in the extraembryonic trophoblast lineage ensures the progression of preimplantation and postimplantation mammalian development

GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure. Summary: During trophoblast development in mice, GATA2 and GATA3 act synergistically by directly regulating a large number of common genes, and together are important to ensure trophoblast lineage progression.

Regulation of mitochondrial function and cellular energy metabolism by protein kinase C-λ/ι: a novel mode of balancing pluripotency.

Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self‐renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι‐hypoxia‐inducible factor 1α‐PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts. Stem Cells 2014;32:2880–2892