bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis.
Authors: Turgay K , Hamoen L W , Venema G , Dubnau D
Published: June 2007

Development of genetic competence in Bacillus subtilis is controlled by the competence-specific transcription factor ComK. ComK activates transcription of itself and several other genes required for competence. The activity of ComK is controlled by other genes including mecA, clpC, and comS. We have used purified ComK, MecA, ClpC, and synthetic ComS to study their interactions and have demonstrated the following mechanism for ComK regulation. ClpC, in the presence of ATP, forms a ternary complex with MecA and ComK, which prevents ComK from binding to its specific DNA target. This complex dissociates when ComS is added, liberating active ComK. ClpC and MecA function as a molecular switch, in which MecA confers molecular recognition, connecting ClpC to ComK and to ComS.
A human protein required for the second step of pre-mRNA splicing is functionally related to a yeast splicing factor.
Authors: Horowitz D S , Krainer A R
Published: June 2007

We have identified a human splicing factor required for the second step of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step. The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved. A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes. The homology identifies one region important for the function of both proteins and may define a new protein motif. In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.
Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites.
Authors: Hurlin P J , Quéva C , Eisenman R N
Published: June 2007

The small constitutively expressed bHLHZip protein Max is known to form sequence-specific DNA binding heterodimers with members of both the Myc and Mad families of bHLHZip proteins. Myc:Max complexes activate transcription, promote proliferation, and block terminal differentiation. In contrast, Mad:Max heterodimers act as transcriptional repressors, have an antiproliferative effect, and are induced upon differentiation in a wide variety of cell types. We have identified a novel bHLHZip Max-binding protein, Mnt, which belongs to neither the Myc nor the Mad families and which is coexpressed with Myc in a number of proliferating cell types. Mnt:Max heterodimers act as transcriptional repressors and efficiently suppress Myc-dependent activation from a promoter containing proximal CACGTG sites. Transcription repression by Mnt maps to a 13-amino-acid amino-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. We show that this region of Mnt mediates interaction with mSin3 corepressor proteins and that its deletion converts Mnt from a repressor to an activator. Furthermore, wild-type Mnt suppresses Myc+Ras cotransformation of primary cells, whereas Mnt containing a SID deletion cooperates with Ras in the absence of Myc to transform cells. This suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. When mnt is expressed as a transgene under control of the beta-actin promoter in mice the transgenic embryos exhibit a delay in development and die during mid-gestation, when c- and N-Myc functions are critical. We propose that Mnt:Max:Sin3 complexes normally function to restrict Myc:Max activities associated with cell proliferation.
Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos.
Authors: Ludwig T , Chapman D L , Papaioannou V E , Efstratiadis A
Published: June 2007

Mutations of the human BRCA1 and BRCA2 genes encoding tumor suppressors have been implicated in inherited predisposition to breast and other cancers. Disruption of the homologous mouse genes Brca1 and Brca2 by targeting showed that they both have indispensable roles during embryogenesis, because nullizygous embryos become developmentally retarded and disorganized, and die early in development. In Brca1 mutants, the onset of abnormalities is earlier by one day and their phenotypic features and time of death are highly variable, whereas the phenotype of Brca2 null embryos is more uniform, and they all survive for at least 8.5 embryonic days. Observations with Brca1/Brca2 double nullizygotes raise the possibility that the two developmental pathways could be linked. Interestingly, the impact of the Brca1 or Brca2 null mutation is less severe in a p53 null background.
The Nf2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation.
Authors: McClatchey A I , Saotome I , Ramesh V , Gusella J F , Jacks T
Published: June 2007

The neurofibromatosis type II (NF2) tumor suppressor encodes a putative cytoskeletal associated protein, the loss of which leads to the development of Schwann cell tumors associated with NF2 in humans. The NF2 protein merlin belongs to the band 4.1 family of proteins that link membrane proteins to the cytoskeleton and are thought to be involved in dynamic cytoskeletal reorganization. Beyond its membership in this family, however, the function of merlin remains poorly understood. In order to analyze the function of merlin during embryogenesis and to develop a system to study merlin function in detail, we have disrupted the mouse Nf2 gene by homologous recombination in embryonic stem cells. Most embryos homozygous for a mutation at the Nf2 locus fail between embryonic days 6.5 and 7.0, exhibiting a collapsed extraembryonic region and the absence of organized extraembryonic ectoderm. The embryo proper continues to develop, but fails to initiate gastrulation. These observations are supported by the expression patterns of markers of the extraembryonic lineage and the lack of expression of mesodermal markers in the mutant embryos. Mosaic studies demonstrate that merlin function is not required cell autonomously in mesoderm, and support the proposition that merlin function is essential for the development of extraembryonic structures during early mouse development.
roughex down-regulates G2 cyclins in G1.
Authors: Thomas B J , Zavitz K H , Dong X , Lane M E , Weigmann K , Finley R L , Brent R , Lehner C F , Zipursky S L
Published: June 2007

Cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. In roughex mutants, cells accumulate Cyclin A protein in early G1 and progress into S phase precociously. When Roughex is overexpressed in S/G2 cells, Cyclin A is mislocalized to the nucleus and degraded, preventing mitosis. Whereas Roughex inhibits Cyclin A accumulation, Cyclin E down-regulates Roughex protein in vivo. Roughex binds to Cyclin E and is a substrate for a Cyclin E-Cdk complex in vitro. These data argue that Roughex inhibits Cyclin A accumulation in early G1 by targeting Cyclin A for destruction. In late G1, Roughex is destabilized in a Cyclin E-dependent process, releasing Cyclin A for its role in S/G2.
Nuclear entry, oligomerization, and DNA binding of the Drosophila heat shock transcription factor are regulated by a unique nuclear localization sequence.
Authors: Zandi E , Tran T N , Chamberlain W , Parker C S
Published: June 2007

In normally growing Drosophila cultured cells the Drosophila heat shock transcription factor (dHSF) is localized in the cytosol and translocates into the nucleus after heat shock. In the cytosol of nonshocked cells, the dHSF is present as a monomer that cannot bind DNA. Upon stress, the dHSF enters the nucleus where it is observed to be a trimer. A novel nuclear localization sequence (NLS) in the dHSF was found to be responsible for stress-dependent nuclear entry. Deletion of the NLS prevents nuclear entry, as expected, yet surprisingly also allows constitutive oligomerization and DNA binding in the cytosol. Further analysis of the NLS by mutagenesis suggests that the two functions of nuclear entry and oligomerization are separable in that distinct residues present in the NLS are responsible for each. Mutations in certain basic residues completely block nuclear entry, as expected for a constitutive NLS. In addition, two residues were found in the NLS that, when altered, allowed constitutive nuclear entry of dHSF independent of stress. These residues may interact with a putative cellular component or possibly other domains of the HSF to prevent nuclear entry in normally growing cells. The NLS can also function autonomously to target a beta-galactosidase fusion protein into the nucleus in a heat shock-dependent fashion.
Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development.
Authors: Biben C , Harvey R P
Published: June 2007

One of the first morphological manifestations of left/right (L/R) asymmetry in mammalian embryos is a pronounced rightward looping of the linear heart tube. The direction of looping is thought to be controlled by signals from an embryonic L/R axial system. We report here that morphological L/R asymmetry in the murine heart first became apparent at the linear tube stage as a leftward displacement of its caudal aspect. Beginning at the same stage, the basic helix-loop-helix (bHLH) factor gene eHand was expressed in a strikingly left-dominant pattern in myocardium, reflecting an intrinsic molecular asymmetry. In hearts of embryos lacking the homeobox gene Nkx2-5, which do not loop, left-sided eHand expression was abolished. However, expression was unaffected in Sc1-/- hearts that loop poorly because of hematopoietic insufficiency, and was right-sided in hearts of inv/inv embryos that display situs inversus. The data predict that eHand expression is enhanced in descendants of the left heart progenitor pool as one response to inductive signaling from the L/R axial system, and that eHand controls intrinsic morphogenetic pathways essential for looping. One aspect of the intrinsic response to L/R information falls under Nkx2-5 homeobox control.
The Spg1p GTPase is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe.
Authors: Schmidt S , Sohrmann M , Hofmann K , Woollard A , Simanis V
Published: June 2007

The spg1 gene (septum-promoting GTPase) was cloned as a multicopy suppressor of a dominant-negative mutant of the Cdc7p kinase. It encodes a small GTPase of the Ras superfamily. spg1 is an essential gene. Null or heat-sensitive alleles do not make a division septum, but growth, S-phase, and mitosis continue in the absence of cell division, producing elongated, multinucleate cells. Increased expression of Spg1p induces septum formation in G2, S-phase, and pre-Start G1-arrested cells. This requires the activity of Cdc7p kinase, but not p34(cdc2). Increased expression of Cdc7p bypasses the requirement for Spg1p. Spg1p and Cdc7p can be coimmunoprecipitated from cell extracts, and interact in the two-hybrid system. These data indicate that Spg1p is a key element in controlling the onset of septum formation in Schizosaccharomyces pombe, and that it acts through the Cdc7p kinase.
ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway.
Authors: Levchenko I , Yamauchi M , Baker T A
Published: June 2007

Transposition of phage Mu is catalyzed by an extremely stable transposase-DNA complex. Once recombination is complete, the Escherichia coli ClpX protein, a member of the Clp/Hsp100 chaperone family, initiates disassembly of the complex for phage DNA replication to commence. To understand how the transition between recombination and replication is controlled, we investigated how transposase-DNA complexes are recognized by ClpX. We find that a 10-amino-acid peptide from the carboxy-terminal domain of transposase is required for its recognition by ClpX. This short, positively charged peptide is also sufficient to convert a heterologous protein into a ClpX substrate. The region of transposase that interacts with the transposition activator, MuB protein, is also defined further and found to overlap with that recognized by ClpX. As a consequence, MuB inhibits disassembly of several transposase-DNA complexes that are intermediates in recombination. This ability of MuB to block access to transposase suggests a mechanism for restricting ClpX-mediated remodeling to the proper stage during replicative transposition. We propose that overlap of sequences involved in subunit interactions and those that target a protein for remodeling or destruction may be a useful design for proteins that function in pathways where remodeling or degradation must be regulated.
ELT-1, a GATA-like transcription factor, is required for epidermal cell fates in Caenorhabditis elegans embryos.
Authors: Page B D , Zhang W , Steward K , Blumenthal T , Priess J R
Published: June 2007

Epidermal cells are generated during Caenorhabditis elegans embryogenesis by several distinct lineage patterns. These patterns are controlled by maternal genes that determine the identities of early embryonic blastomeres. We show that the embryonically expressed gene elt-1, which was shown previously to encode a GATA-like transcription factor, is required for the production of epidermal cells by each of these lineages. Depending on their lineage history, cells that become epidermal in wild-type embryos become either neurons or muscle cells in elt-1 mutant embryos. The ELT-1 protein is expressed in epidermal cells and in their precursors. We propose that elt-1 functions at an early step in the specification of epidermal cell fates.
p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene.
Authors: Smith R C , Branellec D , Gorski D H , Guo K , Perlman H , Dedieu J F , Pastore C , Mahfoudi A , Denèfle P , Isner J M , Walsh K
Published: June 2007

gax, a diverged homeobox gene expressed in vascular smooth muscle cells (VSMCs), is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular injury that leads to cellular proliferation. Recombinant Gax protein microinjected into VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when introduced either during quiescence or early stages of G1. Overexpression of gax with a replication-defective adenovirus vector resulted in G0/G1 cell cycle arrest of VSMCs and fibroblasts. The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity. Fibroblasts deficient in p21 were not susceptible to a reduction in cdk2 activity or growth inhibition by gax overexpression. Localized delivery of the virus to denuded rat carotid arteries significantly reduced neointima formation and luminal narrowing. These data indicate that gax overexpression can inhibit cell proliferation in a p21-dependent manner and can modulate injury-induced changes in vessel wall morphology that result from excessive cellular proliferation.
The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins.
Authors: Barabino S M , Hübner W , Jenny A , Minvielle-Sebastia L , Keller W
Published: June 2007

Cleavage and polyadenylation specificity factor (CPSF), a key component of the mammalian RNA 3'-end processing machinery, consists of four subunits of 160, 100, 73, and 30 kD. Here we report the isolation and characterization of a cDNA encoding the 30-kD polypeptide. Antibodies raised against this protein inhibit cleavage and polyadenylation and coimmunoprecipitate the other CPSF subunits. The protein sequence contains five C3H-zinc-finger repeats and a putative RNA-binding zinc knuckle motif at the carboxyl terminus. Consistent with this observation, the in vitro translated 30-kD protein binds RNA polymers with a distinct preference for poly(U). In addition, an essential S. cerevisiae gene, YTH1, was cloned which is 40% identical to CPSF 30K at the protein level. Extracts prepared from a conditional yth1 mutant have normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Efficient polyadenylation activity can be restored by the addition of purified polyadenylation factor I (PF I). We demonstrate that Yth1p is a component of PF I that interacts in vivo and in vitro with Fip1p, a known PF I subunit.
Jun in Drosophila development: redundant and nonredundant functions and regulation by two MAPK signal transduction pathways.
Authors: Kockel L , Zeitlinger J , Staszewski L M , Mlodzik M , Bohmann D
Published: June 2007

Drosophila Jun is shown to be involved in different signal transduction pathways and developmental decisions. Dorsal closure, a morphogenetic process occurring during Drosophila embryogenesis, is regulated by Hemipterous (Hep) and Basket (Bsk), homologs of JNKK and JNK, respectively. Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of bsk and hep mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway that is comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly.
Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference.
Authors: Chua P R , Roeder G S
Published: June 2007

The TAM1 gene of Saccharomyces cerevisiae is expressed specifically during meiosis and encodes a protein that localizes to the ends of meiotic chromosomes. In a tam1 null mutant, there is an increase in the frequency of chromosomes that fail to recombine and an associated increase in homolog nondisjunction at meiosis I. The tam1 mutant also displays an increased frequency of precocious separation of sister chromatids and a reduced efficiency of distributive disjunction. The defect in distributive disjunction may be attributable to overloading of the distributive system by the increased number of nonrecombinant chromosomes. Recombination is not impaired in the tam1 mutant, but crossover interference is reduced substantially. In addition, chromosome synapsis is delayed in tam1 strains. The combination of a defect in synapsis and a reduction in interference is consistent with previous studies suggesting a role for the synaptonemal complex in regulating crossover distribution. tam1 is the only known yeast mutant in which the control of crossover distribution is impaired, but the frequency of crossing over is unaffected. We discuss here possibilities for how a telomere-associated protein might function in chromosome synapsis and crossover interference.
The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse.
Authors: Oh S P , Li E
Published: June 2007

Vertebrate animals exhibit segmented axial skeletons and lateral asymmetry of the visceral organs. The segment identity of individual vertebrae is believed to be determined by a combination of functionally active Hox genes that have defined expression boundaries along the anteroposterior axis (known as the axial Hox code). Disturbance of the Hox code by ectopic expression or mutation of Hox genes often leads to homeotic transformation of the vertebrae. Largely unknown, however, are the signaling molecules that provide the positional cues for the precise establishment and maintenance of the Hox code. In this study we show that disruption of the type IIB activin receptor (ActRIIB) by gene targeting results in altered expression of multiple Hox genes and abnormal patterning of the vertebrae, similar to but severer than retinoic acid (RA)-induced anterior transformation. We further show that RA and ActRIIB mutation have synergistic effects on vertebral patterning. Activin, Vg-1 and, type II activin receptors have been implicated in regulation of lateral asymmetry during chick and Xenopus development. We show here that the ActRIIB-/- mice die after birth with complicated cardiac defects including randomized heart position, malposition of the great arteries, and ventricular and atrial septal defects. In addition, the heart anomalies are associated with right pulmonary isomerism and splenic abnormalities, recapitulating the clinical symptoms of the human asplenia syndrome. These findings provide genetic evidence that the ActRIIB-mediated signaling pathway plays a critical role in patterning both anteroposterior and left-right axes in vertebrate animals.
E2F1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction.
Authors: Hsieh J K , Fredersdorf S , Kouzarides T , Martin K , Lu X
Published: June 2007

E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, we have demonstrated that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2F1-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it was clear that the transactivation and the apoptotic function of E2F1 are uncoupled. The transactivation-defective E2F1 mutants E2F1(1-374), E2F1(390-1)DF(delta mdm2), and E2F1(406-415)(delta Rb) can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 was proven to be essential for its apoptotic function, as the DNA-binding-defective mutants E2F1(132) and E2F1(132)(1-374) failed to induce apoptosis. Therefore Rb may inhibit E2F1-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by our observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and a Rb-binding-competent E2F1 mutant E2F1(390-1)DF(delta mdm2), it failed to inhibit the apoptosis induced by mutants E2F1(1-374) and E2F1(delta 406-415)(delta Rb), which are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. The results presented here may provide us some physiological implication of the repression function of the Rb-E2F1 complex.
Induction of DNA synthesis and apoptosis are separable functions of E2F-1.
Authors: Phillips A C , Bates S , Ryan K M , Helin K , Vousden K H
Published: June 2007

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.
Functions of the POU domain genes Skn-1a/i and Tst-1/Oct-6/SCIP in epidermal differentiation.
Authors: Andersen B , Weinberg W C , Rennekampff O , McEvilly R J , Bermingham J R , Hooshmand F , Vasilyev V , Hansbrough J F , Pittelkow M R , Yuspa S H , Rosenfeld M G
Published: June 2007

Here we report on investigation of the role of the POU domain genes Skin-1a/i (Skn-1a/i/Epoc/Oct-11) and Testes-1 (Tst-1/Oct-6/SCIP) in epidermis where proliferating basal keratinocytes withdraw from the cell cycle, migrate suprabasally, and terminally differentiate to form a multilayered, stratified epithelium. The expression of the Skn-1a/i and Tst-1 genes is linked to keratinocyte differentiation in vivo and in vitro, whereas the ubiquitous POU domain factor Oct-1 is expressed highly in both proliferating and post-mitotic keratinocytes. Analysis of Skn-1a/i gene-deleted mice reveals that the Skn-1a/i gene modulates the pattern of expression of the terminal differentiation marker loricrin and inhibits expression of genes encoding markers of the epidermal keratinocyte wounding response. Although epidermis from Tst-1 gene-deleted mice develops normally, epidermis from mice deleted for both Skn-1a/i and Tst-1 is hyperplastic and fails to suppress expression of K14 and Spr-1 in suprabasal cells when transplanted onto athymic mice. This suggests that Skn-1a/i and Tst-1 serve redundant functions in epidermis. Therefore, at least two POU domain genes, Skn-1a/i and Tst-1, serve both distinct and overlapping functions to regulate differentiation of epidermal keratinocytes during normal development and wound healing.
Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.
Authors: Maconochie M K , Nonchev S , Studer M , Chan S K , Pöpperl H , Sham M H , Mann R S , Krumlauf R
Published: June 2007

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.
Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian polycomb complex.
Authors: Alkema M J , Bronk M , Verhoeven E , Otte A , van 't Veer L J , Berns A , van Lohuizen M
Published: June 2007

The Bmi1 gene has been identified as a mouse Polycomb group (Pc-G) gene implicated in the regulation of Hox gene expression. Here we describe the characterization of a Bmi binding protein Mph1, which shares similarity to Drosophila polyhomeotic. Coimmunoprecipitation experiments indicate that Bmi1 and Mph1, as well as the Mel18 and M33 proteins described previously, are constituents of a multimeric protein complex in mouse embryos and human cells. A central domain of Bmi1 interacts with the carboxyl terminus of Mph1, whereas a conserved alpha-helical domain in the Mph1 protein is required for its homodimerization. Transgenic mice overexpressing various mutant Bmi1 proteins demonstrate that the central domain of Bmil is required for the induction of anterior transformations of the axial skeleton. Bmi1, M33, and Mph1 show an overlapping speckled distribution in interphase nuclei. These data provide molecular evidence for the existence of a mammalian Polycomb complex.
Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.
Authors: Xiao S H , Manley J L
Published: June 2007

ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.
RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo.
Authors: Enomoto S , McCune-Zierath P D , Gerami-Nejad M , Sanders M A , Berman J
Published: June 2007

In the yeast Saccharomyces cerevisiae, telomere repeat DNA is assembled into a specialized heterochromatin-like complex that silences the transcription of adjacent genes. The general DNA-binding protein Rap1p binds telomere DNA repeats, contributes to telomere length control and to telomeric silencing, and is a major component of telomeric chromatin. We identified Rap1p localization factor 2 (RLF2) in a screen for genes that alleviate antagonism between telomere and centromere sequences on plasmids. In rlf2 mutants, telomeric chromatin is perturbed: Telomeric silencing is reduced and Rap1p localization is altered. In wild-type cells, Rap1p and telomeres localize to bright perinuclear foci. In rlf2 strains, the number of Rap1p foci is increased, Rap1p staining is more diffuse throughout the nucleus, Rap1p foci are distributed in a much broader perinuclear domain, and nuclear volume is 50% larger. Despite the altered distribution of Rap1p in rlf2 mutant cells, fluorescence in situ hybridization to subtelomeric repeats shows that the distribution of telomeric DNA is similar in wild-type and mutant cells. Thus in rlf2 mutant cells, the distribution of Rap1p does not reflect the distribution of telomeric DNA. RLF2 encodes a highly charged coiled-coil protein that has significant similarity to the p150 subunit of human chromatin assembly factor-I(hCAF-I), a complex that is required for the DNA replication-dependent assembly of nucleosomes from newly synthesized histones in vitro. Furthermore, RLF2 is identical to CAC1, a subunit of yeast chromatin assembly factor-I (yCAF-I) which assembles nucleosomes in vitro. In wild-type cells, epitope-tagged Rlf2p expressed from the GAL10 promoter localizes to the nucleus with a pattern distinct from that of Rap1p, suggesting that Rlf2p is not a component of telomeric chromatin. This study provides evidence that yCAF-I is required for the function and organization of telomeric chromatin in vivo. We propose that Rlf2p facilitates the efficient and timely assembly of histones into telomeric chromatin.
HBP1: a HMG box transcriptional repressor that is targeted by the retinoblastoma family.
Authors: Tevosian S G , Shih H H , Mendelson K G , Sheppard K A , Paulson K E , Yee A S
Published: June 2007

A prominent feature of cell differentiation is the initiation and maintenance of an irreversible cell cycle arrest with the complex involvement of the retinoblastoma (RB) family (RB, p130, p107). We have isolated the HBP1 transcriptional repressor as a potential target of the RB family in differentiated cells. By homology, HBP1 is a sequence-specific HMG transcription factor, of which LEF-1 is the best-characterized family member. Several features of HBP1 suggest an intriguing role as a transcriptional and cell cycle regulator in differentiated cells. First, inspection of the HBP1 protein sequence revealed two consensus RB interaction motifs (LXCXE and IXCXE). Second, HBP1 interaction was selective for RB and p130, but not p107. HBP1, RB, and p130 levels are all up-regulated with differentiation; in contrast, p107 levels decline. Third, HBP1 can function as a transcriptional repressor of the promoter for N-MYC, which is a critical cell cycle and developmental gene. Fourth, because the activation of the N-MYC promoter in cycling cells required the E2F transcription factor, we show that E2F-1 and HBP1 represent opposite transcriptional signals that can be integrated within the N-MYC promoter. Fifth, the expression of HBP1 lead to efficient cell cycle arrest. The arrest phenotype was manifested in the presence of optimal proliferation signals, suggesting that HBP1 exerted a dominant regulatory role. Taken together, the results suggest that HBP1 may represent a unique transcriptional repressor with a role in initiation and establishment of cell cycle arrest during differentiation.
Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription.
Authors: Shykind B M , Kim J , Stewart L , Champoux J J , Sharp P A
Published: June 2007

The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.
Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape.
Authors: Wissmann A , Ingles J , McGhee J D , Mains P E
Published: June 2007

We have identified two genes associated with the hypodermal cell shape changes that occur during elongation of the Caenorhabditis elegans embryo. The first gene, called let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation, and let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). We suggest that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex and the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase.
A role for bone morphogenetic proteins in the induction of cardiac myogenesis.
Authors: Schultheiss T M , Burch J B , Lassar A B
Published: June 2007

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.
Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus.
Authors: Nutt S L , Urbánek P , Rolink A , Busslinger M
Published: June 2007

The Pax5 gene coding for the transcription factor BSAP has an essential role in B lymphopoiesis and midbrain development. Here we present a detailed analysis of the B-cell phenotype of Pax5 mutant mice that revealed a differential dependency of fetal and adult B lymphopoiesis on this transcriptional regulator. B-cell development is arrested in the bone marrow at the early pro-B (pre-BI) cell stage, which is characterized by expression of the early markers c-kit, CD43, lambda5, VpreB, and HSA and the absence of the later markers CD25 and BP-1. These pre-BI cells fail to express the BSAP target gene CD19 and are capable of long-term proliferation in vitro in the presence of stromal cells and IL-7. B-lymphoid progenitors could not be detected in the fetal liver of Pax5 mutant embryos. However, Pax5-deficient fetal liver cells gave rise to the development of pre-BI cells in bone marrow on transplantation into lethally irradiated mice. These data indicate different functions of Pax5 in the distinctive microenvironments of fetal liver and adult bone marrow. As shown by PCR analyses, the pre-BI cells in Pax5-deficient bone marrow have undergone D(H)-to-J(H) rearrangement of the immunoglobulin heavy-chain locus at normal frequency. In contrast, V(H)-to-D(H)J(H) rearrangements were reduced approximately 50-fold in Pax5-deficient pre-BI cells, suggesting a role for Pax5 in the developmental pathway controlling V-to-DJ recombination.
Identification of redox/repair protein Ref-1 as a potent activator of p53.
Authors: Jayaraman L , Murthy K G , Zhu C , Curran T , Xanthoudakis S , Prives C
Published: June 2007

p53 can be isolated from cells in a form that is inert for binding to DNA but that can be stimulated dramatically by phosphorylation, antibody binding, or short single strands of DNA. This suggests that upon genotoxic stress, cells can convert latent p53 to one that is active for DNA binding. Surprisingly, we observed that latent p53 is as effective in activating transcription in vitro as is active p53. We found that HeLa nuclear extracts can stimulate DNA binding by latent p53 and have purified from them a p53-stimulating protein that we have determined to be the product of the Ref-1 gene. Interestingly, Ref-1 is a dual function protein that can both regulate the redox state of a number of proteins and function as a DNA repair (A/P) endonuclease. We observed that oxidized forms of full-length and carboxy-terminally truncated p53 (p53 delta30), which are inactive for DNA binding, are both stimulated by the Ref-1 protein. However, in the presence of reducing agent, Ref-1 is an extremely potent stimulator of full-length p53 but not p53 delta30. These and additional data indicate that Ref-1 protein stimulates p53 by both redox-dependent and -independent means and imply a key role for it in p53 regulation. Importantly, we have also determined that Ref-1 can stimulate p53 transactivation in vivo. This is the first example of a noncovalent protein modifier of p53 function identified in cells.
RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication.
Authors: Lee D Y , Clayton D A
Published: June 2007

The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers.
ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function.
Authors: Bruhn L , Munnerlyn A , Grosschedl R
Published: June 2007

LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex.
A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function.
Authors: Goto H , Motomura S , Wilson A C , Freiman R N , Nakabeppu Y , Fukushima K , Fujishima M , Herr W , Nishimoto T
Published: June 2007

The temperature-sensitive BHK21 hamster cell line tsBN67 ceases to proliferate at the nonpermissive temperature after a lag of one to a few cell divisions, and the arrested cells display a gene expression pattern similar to that of serum-starved cells. The temperature-sensitive phenotype is reversible and results from a single missense mutation--proline to serine at position 134--in HCF, a cellular protein that, together with the viral protein VP16, activates transcription of herpes simplex virus (HSV) immediate-early genes. The tsBN67 HCF mutation also prevents VP16 activation of transcription at the nonpermissive temperature. The finding that the same point mutation in HCF disrupts both VP16 function and the cell cycle suggests that HCF plays a role in cell-cycle progression in addition to VP16-dependent transcription.
Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors.
Authors: Nakajima T , Uchida C , Anderson S F , Parvin J D , Montminy M
Published: June 2007

We have examined the mechanism by which the cAMP-responsive factor CREB stimulates target gene expression following its phosphorylation at Ser-133. Using an in vitro transcription assay, we found that two signals were required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAF(II)130. The adenovirus E1A oncoprotein was found to inhibit phospho(Ser-133) CREB activity by binding to CBP and specifically blocking recruitment of RNA Pol II to the promoter. Our results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes.