Showing papers in "Genes & Development in 1997"

Wnt signaling: a common theme in animal development

Abstract: Wnt proteins are now recognized as one of the major families of developmentally important signaling molecules, with mutations in Wnt genes displaying remarkable phenotypes in the mouse, Caenorhabditis elegans, and Drosophila. Among functions provided by Wnt proteins are such intriguing processes as embryonic induction, the generation of cell polarity, and the specification of cell fate. Until recently, our knowledge of the molecular mechanism of Wnt signaling was very limited, but over the past year, several major gaps have been filled. These include the identification of cell-surface receptors and a novel mechanism of relaying the signal to the cell nucleus. In addition, several components of Wnt signaling are implicated in the genesis of human cancer. These insights have come from different corners of the animal kingdom and have converged on a common pathway. At this junction in this rapidly evolving field, we review our current understanding of Wnt function and signaling mechanisms, doing so in a comparative approach. We have put emphasis on the latest findings, highlighting novelty and underscoring questions that remain. For additional literature, we refer to several previous reviews (McMahon 1992; Nusse and Varmus 1992; Klingensmith and Nusse 1994; Miller and Moon 1996; Moon et al. 1997). We have limited the number of references, particularly in the tables. Fully referenced forms of these tables can be found on the Wnt homepage (http://wwwleland.stanford.edu/∼rnusse/wntwindow.html).

Rho GTPases and signaling networks

Abstract: The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.

New functional activities for the p21 family of CDK inhibitors.

Abstract: The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors pZl'^^'', p27^^^, and p57^^^^ all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K^, respectively, mostly because of a decrease in X^ff. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a GQ arrest. In agreement with our other data, cyclin Dl and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin Dl complexes. This complex assembly occurs in parallel to an increase in cyclin Dl-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin Dl-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin Dl to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.

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

Abstract: 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.

Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.

Abstract: The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.

Stat5a is mandatory for adult mammary gland development and lactogenesis.

Abstract: Prolactin (PRL) induces mammary gland development (defined as mammopoiesis) and lactogenesis. Binding of PRL to its receptor leads to the phosphorylation and activation of STAT (signal transducers and activators of transcription) proteins, which in turn promote the expression of specific genes. The activity pattern of two STAT proteins, Stat5a and Stat5b, in mammary tissue during pregnancy suggests an active role for these transcription factors in epithelial cell differentiation and milk protein gene expression. To investigate the function of Stat5a in mammopoiesis and lactogenesis we disrupted this gene in mice by gene targeting. Stat5a-deficient mice developed normally and were indistinguishable from hemizygous and wild-type littermates in size, weight, and fertility. However, mammary lobuloalveolar outgrowth during pregnancy was curtailed, and females failed to lactate after parturition because of a failure of terminal differentiation. Although Stat5b has a 96% similarity with Stat5a and a superimposable expression pattern during mammary gland development it failed to counterbalance for the absence of Stat5a. These results document that Stat5a is the principal and an obligate mediator of mammopoietic and lactogenic signaling.

Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

Abstract: The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).

The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal.

Abstract: Serum and certain growth factors have the ability to inhibit programmed cell death (apoptosis) and promote survival. The mechanism by which growth factors deliver an anti-apoptotic signal and the mechanism by which this survival signal is uncoupled from mitogenesis are not clear. We studied five downstream effectors of growth factor receptors--Ras, Raf, Src, phosphoinositide 3-kinase (PI 3-kinase), and Akt (PKB)--for their abilities to block apoptosis. Activated forms of Ras, Raf, and Src, although transforming, were not sufficient to deliver a survival signal upon serum withdrawal. In contrast, inhibition of PI 3-kinase accelerated apoptosis, and an activated form of the serine/threonine kinase Akt, a downstream effector of PI 3-kinase, blocked apoptosis. The ability of Akt to promote survival was dependent on and proportional to its kinase activity. In Rat1a fibroblasts, activated Akt did not alter Bcl-2 or Bcl-X(L) expression but inhibited Ced3/ICE-like activity. Thus, the PI 3-kinase/Akt (PKB) signaling pathway transduces a survival signal that ultimately blocks Ced3/ICE-like activity. These results suggest that uncoupling of survival and mitogenesis can be explained by differing abilities of distinct mitogens to efficiently induce the PI 3-kinase/Akt signaling pathway.

The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses

Abstract: Gibberellins (GAs) are tetracyclic diterpenoid growth factors that are essential regulators of stem elongation and other plant developmental processes (Hooley 1994; Swain and Olszewski 1996). GA-related mutants have been identified in several plant species, including Arabidopsis (Ross 1994). GA-deficient Arabidopsis mutants display characteristic phenotypes, including dark green leaves and a dwarf growth habit attributable to reduced stem elongation (Koornneef and van der Veen 1980; Talon et al. 1990a; Sun and Kamiya 1994; Peng and Harberd 1997). gai is a semidominant mutation of Arabidopsis, which also confers a dark green, dwarf phenotype (Koornneef et al. 1985; Peng and Harberd 1993, 1997; Wilson and Somerville 1995). The gai mutation affects GA reception or subsequent signal transduction, and does not result in GA deficiency (Koornneef et al. 1985; Talon et al. 1990b; Wilson et al. 1992; Peng and Harberd 1993; Wilson and Somerville 1995). Dominant mutations conferring visible phenotypes resembling those attributable to GA deficiency are also known in other plants, including maize (D8 allelic series; Harberd and Freeling 1989; Winkler and Freeling 1994) and wheat (Rht homeoallelic series; Gale et al. 1975). Previous genetic and physiological analyses of gai, D8, and Rht indicate that all are gain-of-function mutations (Gale et al. 1975; Harberd and Freeling 1989; Peng and Harberd 1993; Winkler and Freeling 1994; Wilson and Somerville 1995) conferring reduced GA responses and increased endogenous GA levels (Lenton et al. 1987; Fujioka et al. 1988; Talon et al. 1990b). The increased endogenous GA levels found in gai, D8, and Rht mutants are likely to arise through perturbation of the feedback control mechanisms by which GAs regulate in planta GA levels negatively (Croker et al. 1990; Chiang et al. 1995; Phillips et al. 1995; Xu et al. 1995). These dominant GA-response mutations are of considerable agricultural significance. The Rht mutations are especially important because they are the genetic basis of the high-yielding, semi-dwarf wheat varieties of the “green revolution” (Gale and Youssefian 1985). We cloned GAI to enhance our understanding of the mechanisms of GA signal transduction, and because of the potential use for gai in crop improvement. Previous experiments had identified a T-DNA insertion, genetically linked to GAI, which contained a Ds transposable element (Peng and Harberd 1993). This Ds was used to clone GAI through targeted insertional mutagenesis. Comparison of GAI and gai DNA sequences shows that the predicted mutant protein (gai) lacks a short (17-amino-acid) segment of the GAI protein sequence. We propose that this structural alteration is responsible for the dominant, gain-of-function properties of gai. In addition, presumed null alleles of GAI confer increased resistance to the growth-retarding effects of paclobutrazol (PAC), an inhibitor of GA biosynthesis. These observations suggest the following hypotheses to explain the role of GAI in GA signaling. First, GAI is proposed to be a negative regulator that represses GA responses but whose activity is opposed by GA. Second, gai is proposed to be a mutant repressor that is relatively resistant to the effects of GA and, therefore, maintains repression irrespective of the presence of GA. Several recent publications have described extragenic mutations that suppress the phenotype conferred by gai (Carol et al. 1995; Wilson and Somerville 1995; Jacobsen et al. 1996) or by GA deficiency mutations (Jacobsen and Olszewski 1993; Silverstone et al. 1997). Here we extend the analysis of the phenotypes conferred by two of these suppressors (spy-7 and gar2-1). First, we compare the effects of spy-7 and gar2-1 (alone and in combination) on the growth of and PAC resistance of plants containing gai. We have also investigated the effects of spy-7 and gar2-1 on the regulation of GA biosynthesis, by comparing the steady-state levels of gene transcripts encoding GA C-20 oxidase, the enzyme that catalyzes the penultimate step in the synthesis of biologically active GAs (Phillips et al. 1995; Xu et al. 1995). Finally, we have investigated the effects of spy-7 and gar2-1 on steady-state levels of gai transcripts. The results of the above experiments indicate that GAI, SPY, and GAR2 operate within, or modulate, a signal-transduction pathway that represses growth and whose activity is opposed by GA. Because of the existence of mutations having comparable effects to gai and spy in other plant species (Swain and Olszewski 1996), and because GA is an essential growth regulator in a wide variety of plant species (Hooley 1994), it seems likely that the Arabidopsis GAI, SPY, and GAR2 genes define a system for GA-mediated growth regulation that is common to all higher plants.

GATA4 transcription factor is required for ventral morphogenesis and heart tube formation.

Abstract: Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development In the studies described in this report we have used gene targeting to produce GATA4-deficient mice Homozygous GATA4-deficient (GATA4 -/-) mice died between 85 and 105 days post coitum (dpc) GATA4 -/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube Analysis of cardiac development in the GATA4 -/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins However, consistent with the observed defect in ventral morphogenesis, these GATA4 -/procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo The defect in ventral migration of the GATA4 -/- procardiomyocytes was not cell intrinsic because GATA4 -/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/--C57BL/6 chimeric mice Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis

Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice

Abstract: Candidate transcription factors involved in pancreatic endocrine development have been isolated using insulin gene regulation as a paradigm. The cell-type restricted basic helix‐loop‐helix (bHLH) gene, BETA2/NeuroD, expressed in pancreatic endocrine cells, the intestine, and the brain, activates insulin gene transcription and can induce neurons to differentiate. To understand the importance of BETA2 in pancreatic endocrine cell differentiation, mice lacking a functional BETA2 gene were generated by gene targeting experiments. Mice carrying a targeted disruption of the BETA2 gene developed severe diabetes and died perinatally. Homozygous BETA2 null mice had a striking reduction in the number of insulin-producing b cells and failed to develop mature islets. Islet morphogenesis appeared to be arrested between E14.5 and E17.5, a period characterized by major expansion of the b cell population. The presence of severe diabetes in these mice suggests that proper islet structure plays an important role in blood glucose homeostasis. In addition, secretin- and cholecystokinin-producing enteroendocrine cells failed to develop in the absence of BETA2. The absence of these two pancreatic secretagogs may explain the abnormal cellular polarity and inability to secrete zymogen granules in pancreatic acinar exocrine cells. The nervous system appeared to develop normally, despite abundant expression of BETA2 in differentiating neurons. Thus, BETA2 is critical for the normal development of several specialized cell types arising from the gut endoderm.

Requirement for NF-κB in osteoclast and B-cell development

Abstract: NF-κB is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-κB. We have generated mice deficient in both the p50 and p52 subunits of NF-κB to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-κB in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.

Cyclin E-CDK2 is a regulator of p27Kip1.

Abstract: CDK inhibitors are thought to prevent cell proliferation by negatively regulating cyclin-CDK complexes. We propose that the opposite is also true, that cyclin-CDK complexes in mammmalian cells can promote cell cycle progression by directly down-regulating CDK inhibitors. We show that expression of cyclin E-CDK2 in murine fibroblasts causes phosphorylation of the CDK inhibitor p27Kip1 on T187, and that cyclin E-CDK2 can directly phosphorylate p27 T187 in vitro. We further show that cyclin E-CDK2-dependent phosphorylation of p27 results in elimination of p27 from the cell, allowing cells to transit from G1 to S phase. Moreover, mutation of T187 in p27 to alanine creates a p27 protein that causes a G1 block resistant to cyclin E and whose level of expression is not modulated by cyclin E. A kinetic analysis of the interaction between p27 and cyclin E-CDK2 explains how p27 can be regulated by the same enzyme it targets for inhibition. We show that p27 interacts with cyclin E-CDK2 in at least two distinct ways: one resulting in p27 phosphorylation and release, the other in tight binding and cyclin E-CDK2 inhibition. The binding of ATP to the CDK governs which state predominates. At low ATP ( 1 mM) p27 is more likely to be a substrate. Thus, we have identified p27 as a biologically relevant cyclin E-CDK2 substrate, demonstrated the physiological consequences of p27 phosphorylation, and developed a kinetic model to explain how p27 can be both an inhibitor and a substrate of cyclin E-CDK2.

Dna damage induces phosphorylation of the amino terminus of p53

Abstract: Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.

Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse.

Abstract: Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnan cy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

Meiotic chromosomes: it takes two to tango

Abstract: Meiosis is a special type of cell division that produces haploid gametes from diploid parental cells. Chromosome number is reduced during meiosis because a single round of DNA replication is followed by two rounds of chromosome segregation (Fig. 1). Fusion of two gametes during sexual reproduction restores the diploid chromosome complement. The second division of meiosis (the equational division) resembles mitosis: Sister chromatids separate and segregate. The first division, however, is unique. Reductional chromosome segregation at meiosis I differs from mitosis and meiosis II in a number of respects. First, sister chromatids remain associated with each other. Second, the two copies of the same chromosome (called homologous chromosomes or homologs) behave in a coordinate fashion, such that one chromosome moves to one pole of the spindle apparatus and its homolog moves to the opposite pole. This coordination in chromosome behavior depends on complex processes and elaborate structures that bring homologs together during meiotic prophase and hold them together until the transition between metaphase I and anaphase I. In most organisms, the relevant processes include alignment of homologs, assembly of the synaptonemal complex (SC, described below), genetic recombination, and the formation of chiasmata (stable connections between homologs formed at the sites of crossovers). These events occur during a very lengthy prophase that is divided into a series of substages based on changes in chromosome morphology (Table 1). This article reviews our current knowledge of the processes and structures that promote and maintain interactions between homologs and thereby ensure proper reductional chromosome segregation. Emphasis is placed on observations made in recent years, particularly those that have enhanced our understanding of the molecular mechanisms underlying meiotic chromosome behavior.

Multilineage gene expression precedes commitment in the hemopoietic system

Abstract: We have tested the hypothesis that multipotential hemopoietic stem and progenitor cells prime several different lineage-affiliated programs of gene activity prior to unilineage commitment and differentiation. Using single cell RT-PCR we show that erythroid (beta-globin) and myeloid (myeloperoxidase) gene expression programs can be initiated by the same cell prior to exclusive commitment to the erythroid or granulocytic lineages. Furthermore, the multipotential state is characterized by the coexpression of several lineage-affiliated cytokine receptors. These data support a model of hemopoietic lineage specification in which unilineage commitment is prefaced by a "promiscuous" phase of multilineage locus activation.

Mechanism and regulation of mRNA polyadenylation

Abstract: A poly(A) tail is found at the 38 end of nearly every fully processed eukaryotic mRNA and has been suggested to influence virtually all aspects of mRNA metabolism. Its proposed functions include conferring mRNA stability, promoting an mRNA’s translational efficiency, and having a role in transport of processed mRNA from the nucleus to the cytoplasm (for recent reviews, see Lewis et al. 1995; Sachs et al. 1997; Wickens et al. 1997). The reaction that catalyzes the addition of the poly(A) tail, an endonucleolytic cleavage followed by poly(A) synthesis, has also been the focus of intense investigation but, until recently, may have been viewed as a process that follows a predictable, isolated, and invariant path. Yet, as more is learned about 38-end formation, it becomes clear that the function of the polyadenylation machinery extends beyond simply adding poly(A) tails to mRNAs. The first report of a component of the mammalian cleavage and polyadenylation machinery was nearly 40 years ago in a paper describing an activity found in thymus nuclei extracts that could synthesize poly(A) from ATP (Edmonds and Abrams 1960). Ten years passed before poly(A) tails were identified as a post-transcriptionally added modification of mRNA 38 termini and a possible function was assigned to poly(A) polymerase (Darnell et al. 1971; Edmonds et al. 1971; Lee et al. 1971). But nearly another decade elapsed before it was found that transcription proceeds past the polyadenylation site, revealing that a mechanism other than transcriptional termination generates mRNA 38 ends (Ford and Hsu 1978; Nevins and Darnell 1978; Manley et al. 1982). The pace of discovery quickened with the development of cell extracts that reproduce the reaction, and this allowed the subsequent and still ongoing biochemical characterization of mRNA 38-end formation (Manley 1983; Moore and Sharp 1984, 1985). The results from this work have shown that nuclear cleavage and poly(A) addition occurs in a coupled reaction and is carried out by a suprisingly large complex of multisubunit proteins (for recent reviews, see Keller 1995; Manley 1995). Several years were devoted to detailing the mechanism of 38-end formation, assigning relatively simple functions to each separable factor of the complex polyadenylation machinery. With the cloning of cDNAs encoding many of these factors, we have enjoyed an accelerated pace in understanding their precise functions, as well as the unexpected bonuses of finding that these basal factors link nuclear polyadenylation to a variety of cellular processes and that they can be important targets for regulating gene expression. Here we describe how information gained from studies done over the last few years has enhanced our understanding of the structure and function of the proteins catalyzing polyadenylation. We concentrate on mammalian systems but also highlight progress that points to both similarities and differences in yeast polyadenylation. From reviewing the latest events, we not only see how far we have come in 40 years but also become more aware of the rich path of discovery that lies ahead.

Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway

Abstract: The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination in an in vitro system or in vivo, and it is markedly stabilized (t1/2 approximately 3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells, and although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.

The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl

Abstract: Plant developmental processes are controlled by both endogenous programs and environmental stimuli. As a photomorphogenetic mutant, hy5 of Arabidopsis has been isolated and characterized. Our detailed characterization has revealed that the mutant is deficient in a variety of stimulus responses, including gravitropic response and waving growth of roots, as well as light-dependent hypocotyl elongation. In the roots and hypocotyl, the hy5 mutation also affects greening and specific cell proliferation such as lateral root formation and secondary thickening. Those phenotypes indicate that the HY5 gene is responsible for the regulation of fundamental developmental processes of the plant cell: cell elongation, cell proliferation, and chloroplast development. Molecular cloning of the HY5 gene using a T-DNA-tagged mutant has revealed that the gene encodes a protein with a bZIP motif, one of the motifs found in transcriptional regulators. Nuclear localization of the HY5 protein strongly suggests that the HY5 gene modulates the signal transduction pathways under the HY5-related development by controlling expression of genes downstream of these pathways.

Normal human chromosomes have long G-rich telomeric overhangs at one end

Abstract: Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 +/- 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3' overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.

A role for bone morphogenetic proteins in the induction of cardiac myogenesis.

Abstract: Little is known about the molecular mechanisms that govern heart specification in vertebrates. Here we demonstrate that bone morphogenetic protein (BMP) signaling plays a central role in the induction of cardiac myogenesis in the chick embryo. At the time when chick precardiac cells become committed to the cardiac muscle lineage, they are in contact with tissues expressing BMP-2, BMP-4, and BMP-7. Application of BMP-2-soaked beads in vivo elicits ectopic expression of the cardiac transcription factors CNkx-2.5 and GATA-4. Furthermore, administration of soluble BMP-2 or BMP-4 to explant cultures induces full cardiac differentiation in stage 5 to 7 anterior medial mesoderm, a tissue that is normally not cardiogenic. The competence to undergo cardiogenesis in response to BMPs is restricted to mesoderm located in the anterior regions of gastrula- to neurula-stage embryos. The secreted protein noggin, which binds to BMPs and antagonizes BMP activity, completely inhibits differentiation of the precardiac mesoderm, indicating that BMP activity is required for myocardial differentiation in this tissue. Together, these data imply that a cardiogenic field exists in the anterior mesoderm and that localized expression of BMPs selects which cells within this field enter the cardiac myocyte lineage.

SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.

Abstract: Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.

Transcription elongation factor P-TEFb is required for HIV-1 Tat transactivation in vitro

Abstract: P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

Xist-deficient mice are defective in dosage compensation but not spermatogenesis.

Abstract: The X-linked Xist gene encodes a large untranslated RNA that has been implicated in mammalian dosage compensation and in spermatogenesis. To investigate the function of the Xist gene product, we have generated male and female mice that carry a deletion in the structural gene but maintain a functional Xist promoter. Mutant males were healthy and fertile. Females that inherited the mutation from their mothers were also normal and had the wild-type paternal X chromosome inactive in every cell. In contrast to maternal transmission, females that carry the mutation on the paternal X chromosome were severely growth-retarded and died early in embryogenesis. The wild-type maternal X chromosome was inactive in every cell of the growth-retarded embryo proper, whereas both X chromosomes were expressed in the mutant female trophoblast where X inactivation is imprinted. However, an XO mouse with a paternally inherited Xist mutation was healthy and appeared normal. The imprinted lethal phenotype of the mutant females is therefore due to the inability of extraembryonic tissue with two active X chromosomes to sustain the embryo. Our results indicate that the Xist RNA is required for female dosage compensation but plays no role in spermatogenesis.

Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice.

Abstract: We have isolated cDNA clones that encode a novel human gene related to agouti. Sequence analysis of this gene, named ART, for agouti-related transcript, predicts a 132-amino-acid protein that is 25% identical to human agouti. The highest degree of identity is within the carboxyl terminus of both proteins. Like agouti, ART contains a putative signal sequence and a cysteine rich carboxyl terminus, but lacks the region of basic residues and polyproline residues found in the middle of the agouti protein. Both agouti and ART contain 11 cysteines, and 9 of these are conserved spatially. ART is expressed primarily in the adrenal gland, subthalamic nucleus, and hypothalamus, with a lower level of expression occurring in testis, lung, and kidney. The murine homolog of ART was also isolated and is predicted to encode a 131-amino-acid protein that shares 81% amino acid identity to humans. The mouse was found to have the same expression pattern as human when assessed by RT-PCR. Examination by in situ hybridization using mouse tissues showed localized expression in the arcuate nucleus of the hypothalamus, the median eminence, and the adrenal medulla. In addition, the hypothalamic expression of ART was elevated approximately 10-fold in ob/ob and db/db mice. ART was mapped to human chromosome 16q22 and to mouse chromosome 8D1-D2. The expression pattern and transcriptional regulation of ART, coupled with the known actions of agouti, suggests a role for ART in the regulation of melanocortin receptors within the hypothalamus and adrenal gland, and implicates this novel gene in the central control of feeding.

Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.

Abstract: The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-/os serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor KB (NF-KB) by a mechanism that involves phosphorylation of iKBa and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-KB by TNFa depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-KB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-KB-dependent transcription.

An unusual form of transcriptional silencing in yeast ribosomal DNA.

Abstract: Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcription al silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Tyl retrotransposons marked with a weakened URA3 gene (Tyl-mURAS) efficiently integrated into rDNA. The tnURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. METIS and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and METIS silencing, but deletion of SIRl or SIRS did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.

The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase.

Abstract: Bone morphogenetic proteins (BMPs) are members of the TGF-beta family that regulate cell proliferation, apoptosis, and differentiation, and participate in the development of most tissues and organs in vertebrates. Smad proteins function downstream of TGF-beta receptor serine/threonine kinases and undergo serine phosphorylation in response to receptor activation. Smad1 is regulated in this fashion by BMP receptors, and Smad2 and Smad3 by TGF-beta and activin receptors. Here, we report that BMP receptors phosphorylate and activate Smad1 directly. Phosphorylation of Smad1 in vivo involves serines in the carboxy-terminal motif SSXS. These residues are phosphorylated directly by a BMP type I receptor in vitro. Mutation of these carboxy-terminal serines prevents several Smad1 activation events, namely, Smad1 association with the related protein DPC4, accumulation in the nucleus, and gain of transcriptional activity. Similar carboxy-terminal serines in Smad2 are required for its phosphorylation and association with DPC4 in response to TGF-beta, indicating the generality of this process of Smad activation. As a direct physiological substrate of BMP receptors, Smad1 provides a link between receptor serine/threonine kinases and the nucleus.