M. Celeste Simon

Targeted Disruption of the Mouse Stat1 Gene Results in Compromised Innate Immunity to Viral Disease

The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens.

The Combined Functions of Proapoptotic Bcl-2 Family Members Bak and Bax Are Essential for Normal Development of Multiple Tissues

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.

Mop3 Is an Essential Component of the Master Circadian Pacemaker in Mammals

Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.

PU.1 Functions in a Cell-Autonomous Manner to Control the Differentiation of Multipotential Lymphoid–Myeloid Progenitors

Transcription factor PU.1 is required for the development of lymphoid and myeloid progenitors during fetal hematopoiesis. By generating chimeric animals using PU.1-/- ES cells or PU.1(-/-) hematopoietic progenitors, we demonstrate that PU.1 functions in an exclusively cell-autonomous manner to regulate the development of the lymphoid-myeloid system. Multipotential lymphoid-myeloid progenitors (AA4.1+, Lin-) are significantly reduced in PU.1(-/-) embryos and fail to differentiate into B lymphoid or myeloid cells in vitro. These results suggest that the lymphoid and myeloid lineages develop in the fetal liver from a common hematopoietic progenitor not shared with erythrocytes and megakaryocytes. Finally, the Ikaros gene is expressed in PU.1 mutant embryos, suggesting that PU.1 and Ikaros are independently required for specification of embryonic lymphoid cell fates.

Cloning, Tissue Expression, and Chromosomal Localization of SUR2, the Putative Drug-Binding Subunit of Cardiac, Skeletal Muscle, and Vascular KATP Channels

ATP-sensitive inwardly rectifying potassium channels are expressed in a variety of tissues, including heart, skeletal, and smooth muscle, and pancreatic β-cells. Physiological and pharmacological studies suggest the presence of distinct KATP channels in these tissues. Recently, the KATP channel of β-cells has been reconstituted in functional form by coexpression of SUR, the sulfonylurea-binding protein, and the inwardly rectifying K+ channel subunit, KIR6.2. In this article, we describe the isolation of cDNAs encoding SUR-like proteins from mouse, SUR2A and SUR2B. Northern blotting showed that the highest expression of the SUR2 isoforms is in the heart and skeletal muscle, with lower levels in all other tissues. By reverse transcription-polymerase chain reaction, SUR2B is ubiquitously expressed, while the apparently alternatively spliced variant, SUR2A, is expressed exclusively in heart. In situ hybridization shows that the SUR2 isoforms are expressed in the parenchyma of the heart and skeletal muscle and in the vascular structures of other tissues. Human SUR2 was localized to chromosome 12, p12.1 by fluorescent in situ hybridization. The structure of the predicted protein and expression pattern of SUR2 suggests that it is the drug-binding channel-modulating subunit of the extrapancreatic KATP channel. Differences in sequence between SUR and between SUR2 isoforms may underlie the tissue-specific pharmacology of the KATP channel.

An abundant erythroid protein that stabilizes free α-haemoglobin

The development of red blood cells (erythrocytes) is distinguished by high-level production of the oxygen carrier, haemoglobin A (HbA), a heterotetramer of α- and β-haemoglobin subunits. HbA synthesis is coordinated to minimize the accumulation of free subunits that form cytotoxic precipitates. Molecular chaperones that regulate globin subunit stability, folding or assembly have been proposed to exist but have never been identified. Here we identify a protein stabilizing free α-haemoglobin by using a screen for genes induced by the essential erythroid transcription factor GATA-1 (refs 4, 5). Alpha Haemoglobin Stabilizing Protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free α-haemoglobin but not with β-haemoglobin or haemoglobin A (α2β2). Moreover, AHSP specifically protects free α-haemoglobin from precipitation in solution and in live cells. AHSP-gene-ablated mice exhibit reticulocytosis and abnormal erythrocyte morphology with intracellular inclusion bodies that stain positively for denatured haemoglobins. Hence, AHSP is required for normal erythropoiesis, probably acting to block the deleterious effects of free α-haemoglobin precipitation. Accordingly, AHSP gene dosage is predicted to modulate pathological states of α-haemoglobin excess, such as β-thalassaemia.

PU.1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation

We have previously shown using gene targeting that PU.1 is essential for the development of lymphoid and myeloid lineages during fetal liver hematopoiesis. We now show that PU.1 is required for the maturation of yolk sac-derived myeloid progenitors and for the differentiation of ES cells into macrophages. The role of PU.1 in regulating target genes, thought to be critical in the development of monocytes and granulocytes, has been analyzed. Early genes such as GM-CSFR, G-CSFR, and myeloperoxidase are expressed in PU.1-/- embryos and differentiated PU.1-/- ES cells. However, the expression of genes associated with terminal myeloid differentiation (CD11b, CD64, and M-CSFR) is eliminated in differentiated PU.1-/- ES cells. Development of macrophages is restored with the introduction of a PU.1 cDNA regulated by its own promoter. The PU.1-/- ES cells represent an important model for analyzing myeloid cell development.

Definition of multiple, functionally distinct TATA elements, one of which is a target in the hsp70 promoter for E1A regulation

We have dissected the human hsp70 promoter to define sequence elements allowing response to E1A. Alterations of sequence upstream of the TATA element, either with Bal 31 nuclease or by site-directed mutagenesis, had little or no effect on the response of the promoter to E1A. In general, the basal level was reduced, indicating that these sites interact with factors important for transcription, but regulation persisted. Although a CAT gene driven by just the hsp70 TATA (void of upstream sequences) could be stimulated by E1A, a similar construct containing the early SV40 TATA element was not. Analysis of several additional such constructions indicated that the specific sequence TATAA was crucial. Substitution of the TATAA sequence with the SV40 TATTTAT element in the context of the wild-type hsp70 promoter resulted in loss of E1A inducibility, but maintenance of heat inducibility. Replacement of this element with sequences not related to any TATA element resulted in loss of activity and inducibility. Thus, the SV40 TATA equivalent is functional in the context of the hsp70 promoter but cannot be induced by E1A. We conclude that the target for E1A induction of the hsp70 promoter is TATAA, and that multiple functionally distinct TATA elements, and presumably cognate transcription factors, can be distinguished in eukaryotic cells.

PU.1 and Spi-B Are Required for Normal B Cell Receptor–Mediated Signal Transduction

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.