Metabolic specialization of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells are endowed with four unusual properties. They are exceedingly small, exhibiting an intracellular volume two to three orders of magnitude smaller than that of normal mammalian cells. Their rate of cell division, wherein cell doubling takes place in only 4-5 h, is more rapid than even the fastest growing cancer cell lines. They do not senesce. Finally, mouse ES cells retain pluripotency adequate to give rise to all cell types present in either gender of adult mice. We have investigated whether some or all of these unusual features might relate to the possibility that mouse ES cells exist in a specialized metabolic state. By evaluating the abundance of common metabolites as a function of the conversion of mouse ES cells into differentiated embryoid bodies, it was observed that the most radical changes in metabolite abundance related to cellular building blocks associated with one carbon metabolism. These observations led to the discovery that mouse ES cells use the threonine dehydrogenase (TDH) enzyme to convert threonine into acetyl-coenzyme A and glycine, thereby facilitating consumption of threonine as a metabolic fuel. Here we describe the results of a combination of nutritional and pharmacological studies, providing evidence that mouse ES cells are critically dependent on both threonine and the TDH enzyme for growth and viability.

The yeast metabolic cycle: insights into the life of a eukaryotic cell.

The budding yeast Saccharomyces cerevisiae undergoes robust oscillations in oxygen consumption during continuous growth under nutrient-limited conditions. Comprehensive microarray studies reveal that more than half of the yeast genome is expressed periodically as a function of these respiratory oscillations, thereby specifying an extensively orchestrated program responsible for regulating numerous cellular outputs. Here, we summarize the logic of the yeast metabolic cycle (YMC) and highlight additional cellular processes that are predicted to be compartmentalized in time. Certain principles of temporal orchestration as seen during the YMC might be conserved across other biological cycles.

McBindall--a better name for CCAAT/enhancer binding proteins?

C/EBP-related transcription factors regulate the balance between cell proliferation and mitotic growth arrest during terminal differentiation. Three new studies give evidence that this regulation is mediated by protein:protein interactions completely distinct from the role of C/EBPs in gene expression.

A conserved family of prolyl-4-hydroxylases that modify HIF.

Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. We identified a conserved family of HIF prolyl hydoxylase (HPH) enzymes that appear to be responsible for this posttranslational modification. In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1alpha subunit under normoxic conditions was attenuated by coexpression of HPH. Suppression of HPH in cultured Drosophila melanogaster cells by RNA interference resulted in elevated expression of a hypoxia-inducible gene (LDH, encoding lactate dehydrogenase) under normoxic conditions. These findings indicate that HPH is an essential component of the pathway through which cells sense oxygen.

NPAS2: an analog of clock operative in the mammalian forebrain.

Neuronal PAS domain protein 2 (NPAS2) is a transcription factor expressed primarily in the mammalian forebrain. NPAS2 is highly related in primary amino acid sequence to Clock, a transcription factor expressed in the suprachiasmatic nucleus that heterodimerizes with BMAL1 and regulates circadian rhythm. To investigate the biological role of NPAS2, we prepared a neuroblastoma cell line capable of conditional induction of the NPAS2:BMAL1 heterodimer and identified putative target genes by representational difference analysis, DNA microarrays, and Northern blotting. Coinduction of NPAS2 and BMAL1 activated transcription of the endogenous Per1, Per2, and Cry1 genes, which encode negatively activating components of the circadian regulatory apparatus, and repressed transcription of the endogenous BMAL1 gene. Analysis of the frontal cortex of wild-type mice kept in a 24-hour light-dark cycle revealed that Per1, Per2, and Cry1 mRNA levels were elevated during darkness and reduced during light, whereas BMAL1 mRNA displayed the opposite pattern. In situ hybridization assays of mice kept in constant darkness revealed that Per2 mRNA abundance did not oscillate as a function of the circadian cycle in NPAS2-deficient mice. Thus, NPAS2 likely functions as part of a molecular clock operative in the mammalian forebrain.

Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors.

Clock:BMAL1 and NPAS2:BMAL1 are heterodimeric transcription factors that control gene expression as a function of the light-dark cycle. Although built to fluctuate at or near a 24-hour cycle, the clock can be entrained by light, activity, or food. Here we show that the DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers, whereas the oxidized forms inhibit. These observations raise the possibility that food, neuronal activity, or both may entrain the circadian clock by direct modulation of cellular redox state.

PAS kinase: an evolutionarily conserved PAS domain-regulated serine/threonine kinase.

PAS domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS domains followed by a canonical serine/threonine kinase domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS domain is a cis regulator of PASK catalytic activity. When the PAS domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS domains in a purified in vitro setting.

Impaired cued and contextual memory in NPAS2-deficient mice.

Neuronal PAS domain protein 2 (NPAS2) is a basic helix-loop-helix (bHLH) PAS domain transcription factor expressed in multiple regions of the vertebrate brain. Targeted insertion of a beta-galactosidase reporter gene (lacZ) resulted in the production of an NPAS2-lacZ fusion protein and an altered form of NPAS2 lacking the bHLH domain. The neuroanatomical expression pattern of NPAS2-lacZ was temporally and spatially coincident with formation of the mature frontal association/limbic forebrain pathway. NPAS2-deficient mice were subjected to a series of behavioral tests and were found to exhibit deficits in the long-term memory arm of the cued and contextual fear task. Thus, NPAS2 may serve a dedicated regulatory role in the acquisition of specific types of memory.

The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses intercellular signals to control the density-dependent expression of many virulence factors. The las and rhl quorum-sensing systems function, respectively, through the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone (C(4)-HSL), which are known to positively regulate the transcription of the elastase-encoding gene, lasB. Recently, we reported that a second type of intercellular signal is involved in lasB induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the Pseudomonas quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the las and rhl quorum-sensing systems, respectively. In order to define the role of PQS in the P. aeruginosa quorum-sensing cascade, lacZ gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes lasR, lasI, rhlR, and rhlI. We found that in P. aeruginosa, PQS caused a major induction of rhlI'-lacZ and had lesser effects on the transcription of lasR'-lacZ and rhlR'-lacZ. We also observed that the transcription of both rhlI'-lacZ and lasB'-lacZ was cooperatively effected by C(4)-HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the las and rhl quorum-sensing systems and that this signal is not involved in sensing cell density.

The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development.

Mice lacking the hypoxia-inducible transcription factor EPAS1 die at mid-gestation. Despite normal morphological development of the circulatory system, EPAS1-deficient mice display pronounced bradycardia. In addition to the vascular endothelium, EPAS1 is expressed intensively in the organ of Zuckerkandl (OZ), the principle source of catecholamine production in mammalian embryos. EPAS1-deficient embryos contained substantially reduced catecholamine levels. Mid-gestational lethality was rescued by administration of the catecholamine precursor DOPS to pregnant females. We hypothesize that EPAS1 expressed in the OZ senses hypoxia during mid-gestational development and translates this signal into an altered pattern of gene expression, leading to increases in circulating catecholamine levels and proper cardiac function.

The structure of GABPalpha/beta: an ETS domain- ankyrin repeat heterodimer bound to DNA.

GA-binding protein (GABP) is a transcriptional regulator composed of two structurally dissimilar subunits. The alpha subunit contains a DNA-binding domain that is a member of the ETS family, whereas the beta subunit contains a series of ankyrin repeats. The crystal structure of a ternary complex containing a GABPalpha/beta ETS domain-ankyrin repeat heterodimer bound to DNA was determined at 2. 15 angstrom resolution. The structure shows how an ETS domain protein can recruit a partner protein using both the ETS domain and a carboxyl-terminal extension and provides a view of an extensive protein-protein interface formed by a set of ankyrin repeats. The structure also reveals how the GABPalpha ETS domain binds to its core GGA DNA-recognition motif.

Molecular characterization of two mammalian bHLH-PAS domain proteins selectively expressed in the central nervous system.

Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS domain protein (NPAS) 1 and NPAS2, are members of the basic helix-loop-helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain. In situ hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human NPAS1 gene was mapped to chromosome 19q13.2-q13.3, and the mouse Npas1 gene to chromosome 7 at 2 centimorgans. Similarly, the human NPAS2 gene was assigned to chromosome 2p11.2-2q13, and the mouse Npas2 gene to chromosome 1 at 21-22 centimorgans. The chromosomal regions to which human NPAS1 and NPAS2 map are syntenic with those containing the mouse Npas1 and Npas2 genes, indicating that the mouse and human genes are true homologs.

Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.

Here we describe the cloning and characterization of a PAS domain transcription factor termed endothelial PAS-1 (EPAS1). This protein shares 48% sequence identity with hypoxia inducible factor (HIF-1alpha) and lesser similarity with other members of the basic helix-loop-helix/PAS domain family of transcription factors. Like HIF-1alpha, EPAS1 binds to and activates transcription from a DNA element originally isolated from the erythropoietin gene and containing the sequence 5'-GCCCTACGTGCTGTCTCA-3'. Activation by both HIF-1alpha and EPAS1 is stimulated by hypoxic conditions. EPAS1 forms a heterodimeric complex with the aryl hydrocarbon nuclear transporter prior to transcriptional activation of target genes. EPAS1 expression is limited to the endothelium of mouse embryos and, in agreement with its cell type-specific expression pattern, is capable of specifically activating the transcription of the endothelial tyrosine kinase gene Tie-2. These observations raise the possibility that EPAS1 may represent an important regulator of vascularization, perhaps involving the regulation of endothelial cell gene expression in response to hypoxia.

Differentiation of T-helper lymphocytes: selective regulation by members of the STAT family of transcription factors.

Interleukin-4 (IL-4) and interleukin-12 (IL-12) control the differentiation of T-helper cells. Here we summarize studies which investigate the mechanism by which these cytokines selectively reprogramme gene expression in T-lymphocytes. Cytokine stimulation leads to the phosphorylation of specific tyrosine residues within the intracellular domain of the corresponding cytokine receptor. These phosphotyrosines serve as docking sites for latent, cytoplasmic transcription factors known as signal transducers and activators of transcription (Stat) proteins. Receptor/Stat interaction is mediated by the src homology 2 (SH2) domain of the corresponding Stat protein. Although Stat binding to the intracellular domain of the cytokine receptor strongly depends on the phosphotyrosine residue, the recruitment of a specific Stat protein is dictated by amino acid residues C-terminal to the phosphotyrosine. Specific docking sites within individual cytokine receptors have been identified for almost all Stat proteins. The direct coupling between cytokine receptor and transcription factor helps to explain how different cytokines elicit distinct patterns of gene expression.

Regulation of adipose maturation and energy homeostasis.

The adipose tissue of mammals represents a dynamic organ disseminated throughout the body. It fluctuates in abundance according to the availability of metabolic energy supplies. Mature adipose tissue communicates with the central nervous system via a hormonal circuit that controls satiety. Adipogenesis can be recapitulated in cell culture, thus facilitating molecular biological studies of the regulatory proteins that control this process. Such studies have led to the identification of two families of transcription factors that regulate adipogenesis and mammalian energy homeostasis.