bluuebunny/crossref_metadata_2025 huggingface dataset:
Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation.
Authors: Wright R , Stephens C , Zweiger G , Shapiro L , Alley M R
Published: June 2007

CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional. cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.
Regulation of mRNA export in response to stress in Saccharomyces cerevisiae.
Authors: Saavedra C , Tung K S , Amberg D C , Hopper A K , Cole C N
Published: June 2007

The response of eukaryotic cells to heat shock and other forms of stress occurs at both transcriptional and post-transcriptional levels. We used in situ hybridization to determine whether stress affected the subcellular distribution of poly(A)+ RNA in Saccharomyces cerevisiae. Following induction of stress by either heat shock (42 degrees C) or addition of a high concentration of ethanol (10%), the nucleocytoplasmic export of most poly(A)+ RNA was blocked. In situ hybridization indicated that heat-inducible SSA4 and SSA1 mRNAs were exported from nuclei under these same conditions. On the other hand, both GAL1 and URA3 transcripts expressed from the SSA4 promoter accumulated in nuclei following heat shock. Sequences within either the 5' 1600 or the 3' 500 nucleotides of SSA4 mRNA were sufficient to direct GAL1 mRNA to the cytoplasm during stress. The export of SSA4 mRNA following stress required functional nuclear pore complexes, as SSA4 mRNA accumulated in nuclei following heat shock of cells containing temperature-sensitive nucleoporins. However, the selective export of SSA4 mRNA was maintained in heat-shocked cells carrying temperature-sensitive alleles of RNA1, PRP20, or an inducible dominant-negative allele of GSP1, the S. cerevisiae homolog of RAN/TC4. The results reported here suggest that there is selective export of mRNA in yeast.
Targeted disruption of p107: functional overlap between p107 and Rb.
Authors: Lee M H , Williams B O , Mulligan G , Mukai S , Bronson R T , Dyson N , Harlow E , Jacks T
Published: June 2007

To explore the physiological role of p107, a member of retinoblastoma gene (Rb) family, we disrupted the mouse gene by homologous recombination in embryonic stem cells. p107 homozygous mutant mice were viable, fertile, and displayed no obvious abnormalities. To investigate possible functional overlap between p107 and Rb, mice with mutations at both loci were generated. Rb+/-;p107-/- mice have a pronounced growth retardation and increased mortality rate during the first 3 weeks after birth. The Rb+/-;p107-/- pups that survive to adulthood did not show any altered tumor predisposition when compared with Rb+/- mice but developed multiple dysplastic lesions of the retina. Embryos homozygous for both Rb and p107 died at approximately 11.5 days of gestation, 2 days earlier than embryos homozygous for Rb alone. Histological examination revealed accelerated apoptosis in the liver and the central nervous system of Rb-/-;p107-/- embryos relative to Rb-/- embryos. These results provide the first in vivo evidence that p107 and Rb have overlapping functions in some tissues of the developing and adult mouse.
Shared role of the pRB-related p130 and p107 proteins in limb development.
Authors: Cobrinik D , Lee M H , Hannon G , Mulligan G , Bronson R T , Dyson N , Harlow E , Beach D , Weinberg R A , Jacks T
Published: June 2007

The p130 protein shares extensive sequence similarity with pRB, the product of the retinoblastoma gene, and is a major E2F-associated protein in quiescent cells. To investigate its biological function, we have mutated p130 via gene targeting in the mouse. Homozygous mutation of p130 had little discernible effect on development or on the growth of mouse embryo fibroblasts in culture. Much of the E2F activity that normally associates with p130 in serum-starved mouse embryo fibroblasts associated instead with the highly related p107 protein. To determine whether p130 and p107 have overlapping biological roles, we produced mice having simultaneous inactivation of the p130 and p107 genes. Such mice exhibited deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal lethality. These findings indicate that p130 and p107 play an important role in limb development through their abilities to control chondrocyte proliferation. Thus, in certain settings p107 and p130 perform growth-regulatory functions that are not fulfilled by pRB.
The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse.
Authors: Zhao G Q , Deng K , Labosky P A , Liaw L , Hogan B L
Published: June 2007

Bone morphogenetic protein 8B (BMP8B) is a member of the TGFbeta superfamily of growth factors. In the mouse, Bmp8b is expressed in male germ cells of the testis and trophoblast cells of the placenta, suggesting that it has a role in spermatogenesis and reproduction. To investigate these possibilities, we have generated mice with a targeted mutation in Bmp8b. Here, we show that homozygous Bmp8b(tm1blh) mutant males exhibit variable degrees of germ-cell deficiency and infertility. Detailed analysis reveals two separable defects in the homozygous mutant testes. First, during early puberty (2 weeks old or younger) the germ cells of all homozygous mutants either fail to proliferate or show a marked reduction in proliferation and a delayed differentiation. Second, in adults, there is a significant increase in programmed cell death (apoptosis) of spermatocytes, leading to germ-cell depletion and sterility. Sertoli cells and Leydig cells appear relatively unaffected in mutants. This study therefore provides the first genetic evidence that a murine germ cell-produced factor, BMP8B, is required for the resumption of male germ-cell proliferation in early puberty, and for germ-cell survival and fertility in the adult.
A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site.
Authors: Lewis J D , Izaurralde E , Jarmolowski A , McGuigan C , Mattaj I W
Published: June 2007

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing. Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons. The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. Data in Colot et al. (this issue) indicate that this function of CBC is conserved in humans and yeast.
The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.
Authors: Colot H V , Stutz F , Rosbash M
Published: June 2007

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.
Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.
Authors: McLean W H , Pulkkinen L , Smith F J , Rugg E L , Lane E B , Bullrich F , Burgeson R E , Amano S , Hudson D L , Owaribe K , McGrath J A , McMillan J R , Eady R A , Leigh I M , Christiano A M , Uitto J
Published: June 2007

Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLEC1), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.
Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice.
Authors: Telling G C , Haga T , Torchia M , Tremblay P , DeArmond S J , Prusiner S B
Published: June 2007

Transgenic mice overexpressing approximately eightfold the mouse (Mo) prion protein (PrP) gene carrying the P102L mutation of GSS developed neurodegeneration between 150 and 300 days of age, while controls expressing the wild-type MoPrP-A transgene at the same level remained healthy. Mice overexpressing the wild-type MoPrP-A transgene were highly susceptible to inoculated mouse prions, exhibiting abbreviated scrapie incubation times of 45 days. After crossing the mutant transgene onto a null (Prnp 0/0) background, the resulting Tg(MoPrP-P101L)Prnp 0/0 mice displayed a highly synchronous onset of illness at 145 days of age, which was shortened to 85 days upon breeding to homozygosity for the transgene array. Besides occasional PrP plaques and modest spongiform degeneration, Tg(MoPrP-P101L) mice suffered from a myopathy and a peripheral neuropathy. Disruption of the wild-type MoPrP gene increased the number of PrP plaques and the severity of spongiform degeneration. Brain extracts prepared from spontaneously ill transgenic mice transmitted disease to Tg196/Prnp 0/0 mice, expressing low levels of the mutant transgene. Our results demonstrate that the presence of wild-type PrP genes, the level of PrP transgene expression, and the sequence of the transgene can profoundly modify experimental prion disease.
Tst-1/Oct-6/SCIP regulates a unique step in peripheral myelination and is required for normal respiration.
Authors: Bermingham J R , Scherer S S , O'Connell S , Arroyo E , Kalla K A , Powell F L , Rosenfeld M G
Published: June 2007

The terminal differentiation of myelinating glia involves complex interactions that culminate in the formation of myelin. The POU domain transcription factor Tst-1/Oct-6/SCIP is expressed transiently during myelination, and we report here that it has a critical role in this developmental process. Deletion of the Tst-1/Oct-6/SCIP gene produces a severe defect in peripheral myelination by arresting Schwann cell maturation before axonal wrapping. Unexpectedly, the activation of major myelin-specific genes appears to be unaffected by the Tst-1/Oct-6/SCIP mutation, demonstrating that multiple, independently regulated events are required for terminal differentiation of Schwann cells. In addition, aberrant differentiation and migration of specific neurons in Tst-1/Oct-6/SCIP mutant homozygotes is associated with a fatal breathing defect, providing a model for investigating the regulation of pulmonary homeostasis.
grim, a novel cell death gene in Drosophila.
Authors: Chen P , Nordstrom W , Gish B , Abrams J M
Published: June 2007

A genomic interval at 75C1,2 is required for programmed cell death in Drosophila. We identified a new activator of apoptosis, grim, which maps between two previously identified cell death genes in this region reaper (rpr) and head involution defective (hid). Expression of grim RNA coincided with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggered extensive apoptosis in both transgenic animals and in cell culture. Cell killing by grim was blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and did not require the functions of rpr or hid. The predicted grim protein shares an amino-terminal motif in common with rpr. However, grim was sufficient to elicit apoptosis in at least one context, where rpr was not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors.
Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development.
Authors: Liu C Y , Flesken-Nikitin A , Li S , Zeng Y , Lee W H
Published: June 2007

BRCA1 is proposed to be a tumor suppressor gene. To explore the biological function of BRCA1, a partial deletion (amino acids 300-361) of mouse Brca1 exon 11 was introduced into the genome of embryonic stem (ES) cells by homologous recombination. Mice carrying one mutated allele of Brca1 appear normal and are fertile up to 10 months of age without any sign of illness. However, no viable progeny homozygous for the Brca1 mutant allele were obtained. Detailed analysis of large numbers of embryos at different stages of development indicated that the homozygous mutant concepti are severely retarded in growth as early as embryonic day 4.5 (E4.5) and are resorbed completely by E8.5. Although the homozygotes at E5.5-E6.5 are able to synthesize DNA and display distinguishable embryonic and extraembryonic structures, they fail to differentiate and form egg cylinders. Consequently, they were unable to form primitive streaks and undergo gastrulation. Consistent with these in vivo results, blastocysts homozygous for mutated Brca1 alleles are at a considerable disadvantage when grown in vitro. These observations suggest that Brca1 has an important role in the early development of mouse embryos.
The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb.
Authors: Dash A B , Orrico F C , Ness S A
Published: June 2007

The c-Myb transcription factor is a proto-oncoprotein whose latent transforming activity can be unmasked by truncation of either terminus. Because both ends of Myb are involved in negative regulation, we tested whether they could associate in a two-hybrid assay and identified a carboxy-terminal motif that interacts with the amino-terminal DNA-binding domain. The EVES motif is highly conserved in vertebrate c-Myb proteins and contains a known site of phosphorylation previously implicated in the negative regulation of c-Myb. Interestingly, a related EVES motif is present in p100, a ubiquitously expressed transcriptional coactivator found in diverse species. We show that p100 interacts with and influences the activity of c-Myb, implicating it in the regulation of c-Myb, differentiation, and cell growth. Our results suggest that Myb is regulated by a novel mechanism in which intramolecular interactions and conformational changes control the intermolecular associations among Myb, p100, and the transcriptional apparatus.
A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway.
Authors: Baker J C , Harland R M
Published: June 2007

A functional assay to clone mouse mesoderm inducers has identified the mouse gene Mad related 2 (Madr2). Madr2 induces dorsal mesoderm from Xenopus ectoderm and can mimic the organizer in recruiting neighboring cells into a second axis. By analyzing the expression of a lacZ/Madr2 fusion protein, we find Madr2 confined to the nucleus in the deep, anterior cells of the second axis, whereas in epidermal and more posterior cells the protein is cytoplasmically localized. This context-dependent nuclear localization suggests that in certain regions of the embryo, Madr2 responds to a localized signal and amplifies this signal to form the second axis. Furthermore, although Madr2 remains unlocalized in ectodermal explants, addition of activin enhances the concentration of Madr2 in the nucleus. Significantly, a functional lacZ fusion to a carboxy-terminal portion of Madr2 is nuclear localized even in the absence of activin. This indicates that Madr2 contains a domain that can activate downstream components and a repressive domain that anchors the protein in the cytoplasm. Nuclear localization of Madr2 in response to activin, and the activin-like phenotypes induced by overexpression of Madr2, indicate that Madr2 is a signal transduction component that mediates the activity of activin.
Frequent provirus insertional mutagenesis of Notch1 in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notch1 for oncogenesis.
Authors: Girard L , Hanna Z , Beaulieu N , Hoemann C D , Simard C , Kozak C A , Jolicoeur P
Published: June 2007

The MMTVD/myc transgenic mice spontaneously develop oligoclonal CD4+CD8+ T-cell tumors. We used provirus insertional mutagenesis in these mice to identify putative collaborators of c-myc. We found that Notch1 was mutated in a high proportion (52%) of these tumors. Proviruses were inserted upstream of the exon coding for the transmembrane domain and in both transcriptional orientations. These mutations led to high expression of truncated Notch1 RNAs and proteins (86-110 kD). In addition, many Notch1-rearranged tumors showed elevated levels of full-length Notch1 transcripts, whereas nearly all showed increased levels of full-length (330-kD) or close to full-length (280-kD) Notch1 proteins. The 5' end of the truncated RNAs were determined for some tumors by use of RT-PCR and 5' RACE techniques. Depending on the orientation of the proviruses, viral LTR or cryptic promoters appeared to be utilized, and coding potential began in most cases in the transmembrane domain. Pulse-chase experiments revealed that the 330-kD Notch1 proteins were processed into 110- and 280-kD cleavage products. These results suggest that Notch1 can be a frequent collaborator of c-myc for oncogenesis. Furthermore, our data indicate that Notch1 alleles mutated by provirus insertion can lead to increased expression of truncated and full-length (330/280-kD) Notch1 proteins, both being produced in a cleaved and uncleaved form.
KAP-1, a novel corepressor for the highly conserved KRAB repression domain.
Authors: Friedman J R , Fredericks W J , Jensen D E , Speicher D W , Huang X P , Neilson E G , Rauscher F J
Published: June 2007

The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING-B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors.
Diversity and specialization of mammalian SWI/SNF complexes.
Authors: Wang W , Xue Y , Zhou S , Kuo A , Cairns B R , Crabtree G R
Published: June 2007

The SWI/SNF complex in yeast facilitates the function of transcriptional activators by opposing chromatin-dependent repression of transcription. We demonstrate that in mammals SWI/SNF complexes are present in multiple forms made up of 9-12 proteins that we refer to as BRG1-associated factors (BAFs) ranging from 47 to 250 kD. We have isolated cDNAs for human BAF155, BAF170, and BAF60. BAF155 and BAF170 are encoded by separate genes that are both homologs of yeast SWI3. Both contain a region of similarity to the DNA binding domain of myb, but lack the basic residues known to be necessary for interaction with DNA. The two SWI3 homologs copurify on antibody columns specific for either BAF155 or BAF170, indicating that they are in the same complex. BAF60 is encoded by a novel gene family. An open reading frame from yeast, which is highly homologous, encodes the previously uncharacterized 73-kD subunit of the yeast SWI/SNF complex required for transcriptional activation by the glucocorticoid receptor (Cairns et al., this issue). BAF60a is expressed in all tissues examined, whereas BAF60b and BAF60c are expressed preferentially in muscle and pancreas, respectively. BAF60a is present within the 2000-kD BRG1 complex, whereas BAF60b is in a distinct complex that shares some but not all subunits with the BRG1 complex. The observed similarity between mammalian BAF190, BAF170, BAF155, BAF60, and BAF47 and yeast SNF2/SWI2, SWI3, SWI3, SWP73, and SNF5, respectively, underscores the similarity of the mammalian and yeast complexes. However, the complexes in mammals are more diverse than the SWI/SNF complex in yeast and are likely dedicated to developmentally distinct functions.
Ras1 signaling and transcriptional competence in the R7 cell of Drosophila.
Authors: Kauffmann R C , Li S , Gallagher P A , Zhang J , Carthew R W
Published: June 2007

The receptor tyrosine kinase Sevenless determines R7 cell fate by activation of the Ras1 pathway in a subset of equivalent cells competent to respond in the Drosophila eye. We show that the prospero gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-responsive ETS transcription factors. Restriction of high-level prospero expression to the R7 cell appears as a subsequent event, which requires Sevenless activation of the Ras1/MAP kinase pathway. We show that Phyllopod, a nuclear factor whose expression is induced by Sevenless, interacts with another nuclear factor, Sina, to form a complex, and that both factors are involved in upregulating transcription of the prospero gene in the eye. Ultimately, prospero expression is required for proper connectivity of R7 photoreceptor axons to their synaptic targets. Our results suggest that specific transcriptional responses are linked to the mode of activation of the Ras1/MAP kinase signal transduction pathway.
The Caenorhabditis elegans gene lin-17, which is required for certain asymmetric cell divisions, encodes a putative seven-transmembrane protein similar to the Drosophila frizzled protein.
Authors: Sawa H , Lobel L , Horvitz H R
Published: June 2007

Mutations in the gene lin-17 result in the disruption of a variety of asymmetric cell divisions in Caenorhabditis elegans. We have found that lin-17 encodes a protein with seven putative transmembrane domains. The LIN-17 protein is most similar to the Drosophila Frizzled protein and its vertebrate homologs. Studies using a lin-17-green fluorescent protein translational fusion indicate that lin-17 is expressed in mother cells before asymmetric cell divisions and in both daughter cells after the divisions. Our results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions and raise the possibility that the LIN-17 protein acts as a receptor for the Wnt protein LIN-44, which also controls asymmetric cell divisions.
The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation.
Authors: Brissette J L , Li J , Kamimura J , Lee D , Dotto G P
Published: June 2007

Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype which affects the hair, skin, and thymus suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. We show here that in skin whn is specifically expressed in epithelial cells and not the mesenchymal cells, and using a hair reconstitution assay, we demonstrate that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture revealed that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes displayed a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, we directly show that whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model will be discussed.
Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1.
Authors: Brass A L , Kehrli E , Eisenbeis C F , Storb U , Singh H
Published: June 2007

Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.1. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses alpha/beta-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of alpha/beta-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.1-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.
Dual roles of ATM in the cellular response to radiation and in cell growth control.
Authors: Xu Y , Baltimore D
Published: June 2007

The gene mutated in ataxia-telangiectasia (AT) patients, denoted ATM, encodes a putative protein or lipid kinase. To elucidate the functions of ATM, we disrupted the mouse ATM gene through homologous recombination in mice. Consistent with cellular defects of AT patients, the ATM-/- cells are hypersensitive to gamma-irradiation and defective in cell-cycle arrest following radiation, correlating with a defective up-regulation of p53. In addition, ATM-/- mouse thymocytes are more resistant to apoptosis induced by gamma-irradiation than normal thymocytes. ATM-/- fibroblasts are inefficient in G1 to S-phase progression following serum stimulation and senesce after only a few passages in culture. They have an increased constitutive level of p21CP1/WAF1. The ATM protein is therefore critical both for cellular responses to ionizing radiation and for normal cell-cycle progression. ATM+/- fibroblasts and thymocytes showed intermediately defective responses to irradiation but no growth defect, suggesting that the increased cancer risk of AT heterozygotes could be attributable to poor checkpoint function.
Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo.
Authors: Bryant G O , Martel L S , Burley S K , Berk A J
Published: June 2007

Regions on the surface of human TATA-box binding protein (TBP) required for activated transcription in vivo were defined by construction of a library of 89 surface residue mutants with radical substitutions that were assayed for their ability to support activated transcription in vivo, basal transcription in vitro, and TFIIA and TFIIB binding in vitro. Four epitopes were identified in which substitutions in two to four neighboring surface residues greatly inhibited activated transcription in vivo. One epitope in which substitutions inhibited both basal and activated transcription (E284, L287) is the interface between TBP and TFIIB. Another (A184, N189, E191, R205) is the recently determined interface between TBP and TFIIA. Mutations in residues in this TFIIA interface greatly inhibit activated, but not basal transcription, demonstrating a requirement for the TFIIA-TBP interaction for activated transcription in vivo in mammalian cells. The remaining two activation epitopes (TBP helix 2 residues R231, R235, R239, plus F250; and G175, C176, P247) are probably interfaces with other proteins required for activated transcription. The library of mutants responded virtually identically to two different types of activators, GL4-E1A and GAL4-VP16, indicating that transcriptional activation by different classes of activators requires common interactions with TBP.
The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion.
Authors: Lilly M A , Spradling A C
Published: June 2007

Early during Drosophila oogenesis the 16 interconnected cells of each germ-line cyst choose between two alternative fates. The single future oocyte enters meiosis, arrests, and becomes transcriptionally quiescent. The remaining 15 cells initiate a series of polyploid cell cycles to prepare for their role as nurse cells. Like many other polyploid and polytene cells, during nurse cell growth the major satellite DNAs become highly under-represented by a mechanism that has remained obscure. We implicate the cell-cycle regulator cyclin E in DNA under-representation by identifying a hypomorphic, female sterile cycE mutation, cycE01672, that increases the amount of satellite DNA propagated in nurse cells. In mutant but not wild-type endomitotic nurse cells, "late S" patterns of bromodeoxyuridine incorporation are observed similar to those in mitotic cells. CycE protein still cycles in cycE01672 germ-line cysts but at reduced levels, and it is found throughout a longer fraction of the cell cycle. Our experiments support the view that oscillating levels of CycE control the polyploid S phase. Moreover, they indicate that a checkpoint linking the presence of unreplicated DNA to the CycE oscillator is lacking, leading to incomplete replication of late-replicating sequences such as satellite DNAs. Unexpectedly, two to three of the 16 cells in cycE01672 cysts frequently differentiate as oocytes, implicating cell-cycle programming in oocyte determination.
Genetic analysis of the Müllerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation.
Authors: Mishina Y , Rey R , Finegold M J , Matzuk M M , Josso N , Cate R L , Behringer R R
Published: June 2007

Müllerian-inhibiting substance (MIS) is a member of the transforming growth factor-beta (TGF-beta) gene family. MIS expression in males causes the regression of the Müllerian ducts, an essential process in male sexual differentiation. Recently, an MIS type II receptor gene has been isolated that is expressed during embryogenesis in mesenchymal cells adjacent to the Müllerian duct epithelium and in Sertoli and granulosa cells of the fetal and adult, male and female gonads, respectively. MIS receptor mutant males develop as internal pseudohermaphrodites, possessing a complete male reproductive tract and also a uterus and oviducts, a phenocopy of MIS ligand-deficient male mice. They express both MIS mRNA and protein, showing that ligand was present, but target organs were hormone-insensitive. All produce sperm, but the majority were infertile because the presence of their female reproductive organs blocks sperm transfer into females. Focal seminiferous tubule atrophy accompanied by Leydig cell hyperplasia was observed and began as early as 2 months of age. The phenotype of MIS ligand/MIS receptor double mutant males was indistinguishable from those of each single mutant. MIS receptor/alpha-inhibin double mutant males developed testicular stromal tumors and large fluid-filled uteri that were identical in phenotype to MIS ligand/alpha-inhibin double mutant males. These studies provide in vivo evidence that MIS is the only ligand of the MIS type II receptor, in contrast to the complexity of other TGF-beta gene family signaling pathways.
RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.
Authors: Navas T A , Sanchez Y , Elledge S J
Published: June 2007

In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair. Examination of the inducibility of RNR3 in response to UV damage has revealed that the various checkpoint genes can be arranged in a pathway consistent with their requirement to arrest cells at different stages of the cell cycle. While RAD9, RAD24, and MEC3 are required to activate the DNA damage checkpoint when cells are in G1 or G2, POL2 is required to sense UV damage and replication blocks when cells are in S phase. The phosphorylation of the essential central transducer, Rad53p, is dependent on POL2 and RAD9 in response to UV damage, indicating that RAD53 functions downstream of both these genes. Mutants defective for both pathways are severely deficient in Rad53p phosphorylation and RNR3 induction and are significantly more sensitive to DNA damage and replication blocks than single mutants alone. These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal. Each of these pathways subsequently activates the central transducers Mec1p/Esr1p/Sad3p and Rad53p/Mec2p/Sad1p, which are required for both cell-cycle arrest and transcriptional responses.
MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea.
Authors: Xu J R , Hamer J E
Published: June 2007

Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cerevisiae MAP kinases FUS3/KSS1, and a GST-Pmk1 fusion protein has kinase activity in vitro. pmk1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants. pmk1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmk1 acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GAL1 promoter in yeast, PMK1 can rescue the mating defect in a fus3 kss1 double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.
Repression of human heat shock factor 1 activity at control temperature by phosphorylation.
Authors: Knauf U , Newton E M , Kyriakis J , Kingston R E
Published: June 2007

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.
TrkB and TrkC neurotrophin receptors cooperate in promoting survival of hippocampal and cerebellar granule neurons.
Authors: Minichiello L , Klein R
Published: June 2007

The Trk family of protein tyrosine kinases (TrkA/B/C) are receptors for neurotrophins, a family of closely related proteins that are important physiological regulators of the survival of specific neurons within the peripheral nervous system (PNS) of vertebrates. In contrast to the PNS, brains of mutant mice deficient in a single neurotrophin or Trk receptor species do not show signs of major cell loss. However, in double mutant mice, we now show that reducing the expression of both TrkB and TrkC causes massive cell death of postnatal hippocampal and cerebellar granule neurons. Kinetic analysis of neuronal death in the hippocampus showed that dentate gyrus granule neurons become dependent on TrkB and TrkC after the first postnatal week, shortly after the period of naturally occurring cell death, indicating a role of these receptors in supporting postmitotic neurons. Correlating with the loss of granule cells, the number of mossy fibers projecting to CA3 pyramidal neurons was markedly reduced in mice carrying mutant trkB/trkC alleles, demonstrating impairment of excitatory pathways in the hippocampus. In the cerebellum, TrkB and TrkC receptors were specifically required for premigratory granule neurons located in the external granule layer. In contrast, cerebellar Purkinje cells were found to be poorly differentiated, but showed no signs of increased cell death. These results provide in vivo evidence that neurotrophins are essential physiological survival factors for specific central neurons. Moreover, they suggest that central, in contrast to peripheral, neurons are capable of using more than one neurotrophin/Trk receptor signaling pathway to stay alive.
The role of EKLF in human beta-globin gene competition.
Authors: Wijgerde M , Gribnau J , Trimborn T , Nuez B , Philipsen S , Grosveld F , Fraser P
Published: June 2007

We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.
Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration and branching in the Drosophila tracheal system.
Authors: Lee T , Hacohen N , Krasnow M , Montell D J
Published: June 2007

Receptor tyrosine kinases (RTKs) are members of a diverse class of signaling molecules well known for their roles in cell fate specification, cell differentiation, and oncogenic transformation. Recently several RTKs have been implicated in cell and axon motility, and RTKs are known to mediate chemotactic guidance of tissue culture cells. We have investigated whether the Drosophila FGF receptor homolog, Breathless (BTL), whose activity is necessary for each phase of branching morphogenesis in the embryonic tracheal system, might play a role in guiding the directed migration of tracheal cells. We found that expression of a constitutively active receptor during tracheal development interfered with directed tracheal cell migration and led to extra secondary and terminal branch-forming cells. Reduction in endogenous BTL signaling enhanced the cell migration defects while suppressing the ectopic branching defects. These results are consistent with a model for tracheal development in which spatially regulated BTL activity guides tracheal cell migration and quantitatively regulated BTL activity determines the patterns of secondary and terminal branching cell fates.
The Drosophila short gastrulation gene prevents Dpp from autoactivating and suppressing neurogenesis in the neuroectoderm.
Authors: Biehs B , François V , Bier E
Published: June 2007

The short gastrulation (sog) gene is expressed in broad lateral stripes comprising the neuroectoderm of the Drosophila blastoderm embryo. sog encodes a predicted secreted protein that functions nonautonomously to antagonize the activity of the TGF-beta-like Decapentaplegic (Dpp) signaling pathway in the dorsal region of the embryo. Recently, it has been shown that sog and dpp are functionally equivalent to their respective Xenopus homologs chordin and BMP-4. In this report we provide the first direct evidence that sog plays a local role in the lateral region of the blastoderm embryo to oppose Dpp activity in the neuroectoderm. In the dorsal region, Dpp signaling both suppresses neurogenesis and maintains expression of genes that promote dorsal cell fates (dorsalization). We show that Dpp also can perform both of these functions in the neuroectoderm. In wild-type embryos, the ability of Dpp to induce expression of dorsal markers including itself (autoactivation) in the neuroectoderm is blocked by sog. We propose that Sog protects the neuroectoderm from an invasive positive feedback loop created by Dpp diffusion and autoactivation. We show that the two functions of Dpp signaling, neural suppression and dorsalization, are triggered by distinct thresholds of Dpp activity. Epistasis experiments reveal that all observed sog activity can be accounted for by Sog functioning as a dedicated Dpp antagonist. Finally, we provide evidence that Sog functions as a diffusible morphogen in the blastoderm embryo. These data strongly support the view that the primary phylogenetically conserved function of the Drosophila sog and dpp genes and the homologous Xenopus chordin and BMP-4 genes is to subdivide the primitive embryonic ectoderm into neural versus non-neural domains.
Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins.
Authors: Hateboer G , Kerkhoven R M , Shvarts A , Bernards R , Beijersbergen R L
Published: June 2007

E2F transcription factors are key regulators of transcription during the cell cycle. E2F activity is regulated at the level of transcription and DNA binding and by complex formation with the retinoblastoma pocket protein family. We show here that free E2F-1 and E2F-4 transcription factors are unstable and that their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. We show that binding of E2F-1 to pRb or E2F-4 to p107 or p130 protects E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins.