Lats2 is critical for the pluripotency and proper differentiation of stem cells.

Differentiation is a highly controlled process essential for embryonic and adult development. Moreover, disruption of proper differentiation is often associated with human diseases, including cancer. We analyzed the involvement of the tumor-suppressor Lats2 in mouse embryonic stem cell (mESC) pluripotency and differentiation, and report that mESCs lacking Lats2 are unable to sustain stemness and are not able to initiate and coordinate developmental transcriptional programs. Lats2-/- mESCs retain bivalent 'poised' chromatin marks on developmental genes and exhibit germ layer ambiguity both in vitro and in vivo. Importantly, in coordinating proper germ layer specification, Lats2 engages in a feedback loop with another tumor suppressor, p53.

Regulatory mechanisms involved in activator-protein-1 (AP-1)-mediated activation of glutathione-S-transferase gene expression by chemical agents.

Induction of murine glutathione-S-transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element, EpRE, composed of an Ets and two adjacent activator protein-1 (AP-1)-like sites and activated by the Fos/Jun heterodimeric complex (AP-1). The mechanism of this induction was examined in the present study. We find that the regulation of EpRE-mediated GST Ya gene expression by 3-methylcholanthrene, tert-butylhydroquinone and beta-naphthoflavone is associated with an induction of AP-1 DNA-binding activity and that the AP-1 complex induced in hepatoma cells by these chemicals contains members of the Fos and Jun protein families. We show that tert-butylhydroquinone induces c-fos gene expression and indicate the formation of a transcriptionally active AP-1 complex that contains Fos/Jun heterodimer. In F9 cells, which are considered to lack AP-1 complex, a careful examination reveals that tert-butylhydroquinone induces a low level of an AP-1-related activity responsible for the enhanced expression of EpRE as well as of AP-1 reporter constructs. We find that protein phosphorylations mediate the activation of the GST Ya gene by chemical agents since okadaic acid, an inhibitor of protein phosphatases, can mimic this activation while protein kinase inhibitors abolish it. Evidence is presented that 3-methylcholanthrene, tert-butylhydroquinone and beta-naphthoflavone use a signal transduction pathway to Fos/Jun-dependent GST Ya gene expression via Ras and protein-tyrosine kinase activity. Furthermore, we find that activation by phorbol 12-myristate 13-acetate, which uses both protein kinase C and protein-tyrosine kinase activities, may share a common pathway with these chemicals downstream of Ras.

Signaling pathways in the induction of c-fos and c-jun proto-oncogenes by 3-methylcholanthrene.

3-methylcholanthrene (MC), a potent promutagen and procarcinogen, is also an inducer of mammalian CYPIAI (cytochrome P1-450) gene. The CYPIAI enzyme is responsible for the detoxification of MC and its oxidation into reactive epoxide intermediates. Through its epoxide metabolites, MC functions also as an inducer of drug-metabolizing enzyme glutathione S-transferase (GST) gene expression. Induction of murine GST Ya gene by MC and a variety of other chemical agents is mediated by a regulatory element composed of two adjacent AP-1-like sites, and activated by the Fos/Jun heterodimeric complex (AP-1). In cultured cells, MC causes the induction of AP-1 activity, which is the result of an increased expression of c-Fos and c-Jun proteins. The mechanisms involved in MC activation of c-fos and c-jun gene expression were examined in the present study. Evidence is presented that stimulation of c-fos transcription by MC involves a signal transduction pathway, which includes activation of the small G protein Ras, Raf-1 kinase, and the mitogen-activated protein (MAP) kinases, ERK1 and ERK2. Furthermore, we find that phorbol 12-myristate 13-acetate, which uses both protein kinase C and protein-tyrosine kinase activities to induce c-fos promoter, may share a common pathway with MC downstream of Ras. The signal transduction pathway induced by MC to stimulate c-jun promoter involves Ras activation and the JNK group of MAP-kinases.

[Modification of DNA with dihydrazide of succinic acid for preparation of hybridization probes]. / Modifikatsiia DNK digidrazidom iantarnoi kisloty dlia polucheniia gibridizatsionnykh zondov.

A two-step chemical method of introduction of nonradioactive labels in DNA was proposed. At first step DNA is modified by succinic dihydrazide at pH 5.0 and 95 degrees C, or at pH 4.5 and 37 degrees C in presence of sodium bisulfite. Then FITC or biotin are joined to the hydrazide groups. DNA modified in this way were shown to be effective hybridisation probes.

[Multiple hybridization in genome analysis. 1. Reaction of diamine and bisulfite with cytosine for the introduction of nonradioactive markers into DNA]. / Mnozhestvennaia gibridizatsiia v analize genmov. 1. Reaktsiia diaminov i bisul'fita s tsitozinom dlia vvedeniia v DNK neradioaktivnykh metok.

A two-step chemical method has been developed for the introduction of biotin and other low-molecular derivatives into DNA. The method is based on the interaction of aliphatic diamines with cytosine residues of the polynucleotide chain in the presence of sodium bisulfite with subsequent addition of biotin. DNA modified this way may be used as an effective probe for non-isotopic hybridization which can be used simultaneously with 32P-labelled probes.