bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo.
Authors: Zhou J , Barolo S , Szymanski P , Levine M
Published: June 2007

Enhancers integrate positive and negative regulatory information to direct localized patterns of gene expression in the Drosophila embryo. Here we present evidence for the occurrence of cis regulatory elements that control the levels of gene expression by modulating enhancer-promoter interactions. For this purpose we have investigated the Drosophila bithorax complex (BX-C) because genetic studies suggest that the BX-C contains as much as 300 kb of cis regulatory information. A specialized DNA element, Fab-7, has been proposed to function as a boundary element that separates the iab-6 and iab-7 cis regulatory regions within the Abd-B domain of the BX-C. A 1.2-kb Fab-7 DNA fragment was placed between divergently transcribed white and lacZ test promoters and challenged with several defined enhancers expressed in the early embryo. These studies suggest that Fab-7 functions as an attenuator, which weakens gene expression by reducing enhancer-promoter interactions. Fab-7 selectively blocks distal enhancers in an orientation-independent fashion, and can function when located far from either the distal enhancer or target promoter. Fab-7 may be related to insulator DNAs, which flank genetic loci and functionally isolate neighboring genes. We propose that specialized DNA elements, such as the Fab-7 attenuator, might play a general role in controlling the levels of gene expression by modulating enhancer-promoter interactions.
Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex.
Authors: Hagstrom K , Muller M , Schedl P
Published: June 2007

Fab-7 deletions in the bithorax complex have a novel gain-of-function phenotype, typically transforming parasegment 11 (PS11) into PS12 identity. Genetic analysis indicates that removal of the Fab-7 element results in the fusion of the iab-6 (PS11) and iab-7 (PS12) cis-regulatory domains into a single regulatory domain that inappropriately regulates Abdominal-B in PS11. This has led to the hypothesis that Fab-7 is a chromatin domain boundary that normally functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains. We use several different enhancer blocking assays to demonstrate that Fab-7 has the insulating properties expected of a domain boundary. We define a minimal fragment of Fab-7 sufficient for enhancer blocking, and demonstrate that it is completely distinct from an adjacent Polycomb-dependent silencer. We compare Fab-7 to the su(Hw) insulator element, and show that Fab-7 enhancer blocking activity is intermediate between that of five and twelve reiterated binding sites for the Su(Hw) protein. These results support the model that Fab-7 functions as a domain boundary within the context of the bithorax complex, making Fab-7 one of the first boundary elements that is known to have an essential function in vivo.
A novel, mitogen-activated nuclear kinase is related to a Drosophila developmental regulator.
Authors: Denis G V , Green M R
Published: June 2007

Although the ultimate targets of many signal transduction pathways are nuclear transcription factors, the vast majority of known protein kinases are cytosolic. Here, we report on a novel human kinase that is present exclusively in the nucleus. Kinase activity is increased upon cellular proliferation and is markedly elevated in patients with acute and chronic lymphocytic leukemias. We have identified a human gene that encodes this nuclear kinase and find that it is closely related to Drosophila female sterile homeotic (fsh), a developmental regulator with no known biochemical activity. Collectively, these results suggest that this nuclear kinase is a component of a signal transduction pathway that plays a role in Drosophila development and human growth control.
Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ.
Authors: Arber S , Caroni P
Published: June 2007

The LIM motif defines a double zinc finger structure found in proteins involved in cell fate determination and growth control. LIM proteins, which include LIM homeo domain, LIM kinase, focal adhesion, and LIM-only proteins, usually contain two or more LIM motifs clustered at their amino- or carboxy-terminal end. At present, the mode of action of the LIM domain is not clear. In this study we have analyzed the binding properties of LIM motifs in the cellular environment. We show that MLP, CRP, and betaCRP define a subclass of LIM-only proteins with unique dual subcellular localization in the nucleus and along actin-based filaments in the cytosol. A double MLP construct that accumulated nearly exclusively along actin filaments promoted myogenic differentiation efficiently, arguing for a functional role of cytoskeleton-associated MLP. Binding of MLP to the actin cytoskeleton is specifically attributable to its second LIM motif. An additional LIM motif potentiates binding. Potentiating LIM motifs can be interchanged, resulting in differential targeting of interacting proteins. To analyze LIM-LIM interactions in situ, this property was exploited to develop a hybrid interaction approach based on the relocalization of LIM-containing constructs to the actin cytoskeleton. These experiments revealed the existence of marked selectivity in the interactions of single LIM motifs, and among LIM domains from different LIM-homeo domain and LIM-only proteins. Furthermore, the analysis suggested that the LIM motif has two interacting interfaces. On the basis of these findings, we propose that LIM motifs function as specific adapter elements to promote the assembly and targeting of multiprotein complexes.
Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog.
Authors: Goodrich L V , Johnson R L , Milenkovic L , McMahon J A , Scott M P
Published: June 2007

The signaling protein Hedgehog (Hh) controls cell fate and polarizes tissues in both flies and vertebrates. In flies, Hh exerts its effects by opposing the function of a novel transmembrane protein, Patched, while also locally inducing patched (ptc) transcription. We have identified a mouse homolog of ptc which in many tissues is transcribed near cells making either Sonic or Indian hedgehog. In addition, ectopic Sonic hedgehog expression in the mouse central nervous system induces ptc transcription. As in flies, mouse ptc transcription appears to be indicative of hedgehog signal reception. The results support the existence of a conserved signaling pathway used for pattern formation in insects and mammals.
Analysis of the vestigial tail mutation demonstrates that Wnt-3a gene dosage regulates mouse axial development.
Authors: Greco T L , Takada S , Newhouse M M , McMahon J A , McMahon A P , Camper S A
Published: June 2007

Mice homozygous for the recessive mutation vestigial tail (vt), which arose spontaneously on Chromosome 11, exhibit vertebral abnormalities, including loss of caudal vertebrae leading to shortening of the tail. Wnt-3a, a member of the wingless family of secreted glycoproteins, maps to the same chromosome. Embryos homozygous for a null mutation in Wnt-3a (Wnt-3a(neo)) have a complete absence of tail bud development and are truncated rostral to the hindlimbs. Several lines of evidence reveal that vt is a hypomorphic allele of Wnt-3a. We show that Wnt-3a and vt cosegregate in a high-resolution backcross and fail to complement, suggesting that Wnt-3a(neo) and vt are allelic. Embryos heterozygous for both alleles have a phenotype intermediate between that of Wnt-3a(neo) and vt homozygotes, lacking a tail, but developing thoracic and a variable number of lumbar vertebrae. Although no gross alteration in the Wnt-3a gene was detected in vt mice and the Wnt-3a coding region was normal, Wnt-3a expression was markedly reduced in vt/vt embryos consistent with a regulatory mutation in Wnt-3a. Furthermore, the analysis of allelic combinations indicates that Wnt-3a is required throughout the period of tail bud development for caudal somitogenesis. Interestingly, increasing levels of Wnt-3a activity appear to be necessary for the formation of more posterior derivatives of the paraxial mesoderm.
Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation.
Authors: Doherty D , Feger G , Younger-Shepherd S , Jan L Y , Jan Y N
Published: June 2007

Wing margin formation in Drosophila requires the Notch receptor and, in the dorsal compartment, one of its ligands, Serrate. We provide evidence that Delta, the other known ligand for Notch, is also essential for this process. Delta is required in ventral cells at the dorsal/ventral compartment boundary, where its expression is specifically elevated in second-instar wing discs during wing margin formation. Moreover, ectopic Delta expression induces wingless, vestigial, and cut and causes adult wing tissue outgrowth in the dorsal compartment. The effect is mediated by Notch, because loss of Notch activity suppresses Delta-induced ectopic wing outgrowth. Whereas ectopic expression of Notch or the truncated activated Notch induces cut in both dorsal and ventral compartments, ectopic Delta expression induces cut only in the dorsal compartment and ectopic Serrate induces cut only in the ventral compartment. These observations indicate that Notch-expressing cells in a given compartment have different responses to Delta and Serrate. We propose that Delta and Serrate function as compartment-specific signals in the wing disc, to activate Notch and induce downstream genes required for wing formation.
Multiple functions for Pax6 in mouse eye and nasal development.
Authors: Quinn J C , West J D , Hill R E
Published: June 2007

Mouse embryos, homozygous for the small eye (Sey) mutation die soon after birth with severe facial abnormalities that result from the failure of the eyes and nasal cavities to develop. Mutations in the Pax6 gene are responsible for the Sey phenotype. As a general disruption of eye and nasal development occurs in the homozygous Sey embryos, it is unclear, from the mutant phenotype alone, which tissues require functional Psx6. To examine the roles for Pax6 in eye and nasal development we produced chimeric mouse embryos composed of wild-type and Sey mutant cells. In these embryos we found that mutant cells were excluded from both the lens and nasal epithelium. Both of these tissues were smaller, and in some cases absent, in chimeras with high proportions of mutant cells. The morphology of the optic cup was also severely affected in these chimeras; mutant cells were excluded from the retinal pigmented epithelium and did not intermix with wild-type cells in other regions. The evidence shows that Pax6 has distinct roles in the nasal epithelium and the principal tissue components of the embryonic eye, acting directly and cell autonomously in the optic cup and lens. We suggest that Pax6 may promote cell surface changes in the optic cup and control the fate of the ectoderm from which the lens and nasal epithelia are derived.
CBP as a transcriptional coactivator of c-Myb.
Authors: Dai P , Akimaru H , Tanaka Y , Hou D X , Yasukawa T , Kanei-Ishii C , Takahashi T , Ishii S
Published: June 2007

CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A). CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form. We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator. CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro. The domain of CBP that touches c-Myb is also required for binding to CREB. A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay. CBP stimulates the c-Myb-dependent transcriptional activation. Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation. In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation. Our data thus identify CBP as a coactivator of c-Myb. These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously.
Oxidative stress induced heat shock factor phosphorylation and HSF-dependent activation of yeast metallothionein gene transcription.
Authors: Liu X D , Thiele D J
Published: June 2007

Metallothioneins (MTs) are a class of low-molecular-weight, cysteine- rich metal-binding proteins that function in metal detoxification and oxidative stress protection. We demonstrate that transcription of the Saccharomyces cerevisiae MT gene CUP1 is strongly activated by the superoxide anion generator menadione. This activation is exacerbated in a strain lacking the gene encoding Co, Zn superoxide dismutase (SOD1). CUP1 transcriptional activation by oxidative stress is dependent on a functional CUP1 promoter heat shock element (HSE) and the carboxy-terminal trans-activation domain of heat shock transcription factor (HSF). Furthermore, protection against oxidative stress conferred by CUP1 in a (sod1)delta strain requires HSF-mediated CUP1 transcription. Although in response to heat, HSF-mediated CUP1 transcription and HSF phosphorylation are transient, both CUP1 gene expression and HSF phosphorylation are sustained in response to oxidative stress. Moreover, the patterns of tryptic phosphopeptides resolved from HSF derived from cells subjected to heat shock or oxidative stress are distinct. These results demonstrate that transcription of the S. cerevisiae metallothionein gene under conditions of oxidative stress is mediated by HSF and that in response to distinct activation stimuli, HSF is differentially phosphorylated in a manner that parallels metallothionein gene transcription.
Integration of the yeast retrotransposon Ty1 is targeted to regions upstream of genes transcribed by RNA polymerase III.
Authors: Devine S E , Boeke J D
Published: June 2007

Retroviruses and their relatives, the LTR-containing retrotransposons, integrate newly replicated cDNA copies of their genomes into the genomes of their hosts using element-encoded integrases. Although target site selection is not well understood for this general class of elements, it is becoming clear that some elements target their integration events to very specific regions of their host genomes. Evidence is accumulating that the yeast retrotransposon Ty1 behaves in this manner. Ty1 is found frequently adjacent to tRNA genes in the yeast genome and experimental evidence implicates these regions as preferred integration sites. To determine the basis for Ty1 targeting, we developed an in vivo integration assay using a Ty1 donor plasmid and a second target plasmid that could be used to measure the relative frequency of Ty1 integration into sequences cloned from various regions of the yeast genome. Targets containing genes transcribed by RNA polymerase III (Pol III) were up to several hundredfold more active as integration targets than "cold" sequences lacking such genes. High-frequency targeting was dependent on Pol III transcription, and integration was "region specific," occurring exclusively upstream of the transcription start sites of these genes. Thus, Ty1 has evolved a powerful targeting mechanism, requiring Pol III transcription to integrate its DNA at very specific locations within the yeast genome.
Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo.
Authors: Uemura T , Oda H , Kraut R , Hayashi S , Kotaoka Y , Takeichi M
Published: June 2007

Dynamic epithelial reorganization is essential for morphogenesis of various organs. In Drosophila embryos, for example the Malpighian tubule is generated by cellular rearrangement of a preexisting epithelium and the tracheal network is formed by outgrowth, branching, and fusion of epithelial vesicles. Here we report that the previously identified locus shotgun (shg) encodes DE-cadherin, an epithelial cell-cell adhesion molecule of the classic cadherin type and that zygotic shg mutations rather specifically impair processes of the dynamic epithelial morphogenesis. In the mutants, the Malpighian tubule disintegrated into small spherical structures, and the tracheal network formation was blocked in selected steps. The malformation of these organs could be rescued by overexpression of DE-cadherin cDNA under a heat shock promoter. Unexpectedly, the zygotic null condition did not severely affect general epithelial organization; most epithelial tissues maintained not only their cell-cell associations but also their apicobasal polarity in the mutants. The zygotic null mutant retained a certain level of maternally derived DE-cadherin molecules until the end of embryogenesis. These results suggest that zygotic DE-cadherin expression is critical for the rearrangement processes of epithelial cells, whereas the maternally derived DE-cadherin may serve only for the maintenance of the static architecture of the epithelia.
Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position-independent functions in assembly but not in gene regulation.
Authors: Ling X , Harkness T A , Schultz M C , Fisher-Adams G , Grunstein M
Published: June 2007

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.
Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila.
Authors: Gray S , Levine M
Published: June 2007

The early Drosophila embryo provides a unique system for the analysis of transcriptional repression since a broad spectrum of repressors are distributed in spatially distinct patterns. Krüppel (Kr) and snail (sna), two zinc finger repressors, are essential for segmentation and for the establishment of the mesoderm/neuroectoderm boundary, respectively. Both repressors were examined in the context of synthetic gene complexes containing modular promoters and divergently transcribed reporter genes. These studies indicate that Kr and sna function as short-range repressors, which can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators and permits other enhancers to interact with the transcription complex (enhancer autonomy). In contrast, when bound to promoter-proximal regions the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (<100 bp) of the repressor with either upstream activators or the transcription complex. These studies establish short-range repression as a flexible form of gene regulation and suggest that the key distinction among repressors is their range of action.
Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.
Authors: Joazeiro C A , Kassavetis G A , Geiduschek E P
Published: June 2007

Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription. TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters. To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNATyr gene TFIIIB binding site. Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes. We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC. TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site. Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C. However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly. We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity. The relevance of these findings to alternative initiation of Pol II- and other Pol III-transcribed genes is discussed.
Intercellular C-signaling in Myxococcus xanthus involves a branched signal transduction pathway.
Authors: Søgaard-Andersen L , Slack F J , Kimsey H , Kaiser D
Published: June 2007

C-factor, the product of the csgA gene, is a cell-surface associated short-range intercellular signaling protein in Myxococcus xanthus. C-factor is required for at least four responses during starvation-induced fruiting body morphogenesis: rippling, aggregation, sporulation, and full expression of the csgA gene, all of which fail in a csgA mutant. To analyze the C-factor signaling pathway, eight Tn5 lac insertion mutants that began but failed to complete fruiting body aggregation were characterized. Seven of the insertions identified genes whose products function in the csgA signaling pathway. The seven mutants were differentially deficient in the C-factor responses, and could be divided into two classes on the basis of those differences. On one hand, the four mutants in class I were deficient in rippling and aggregation, but sporulated and produced C-factor at wild-type levels. The Tn5 lac insertions in the class I mutants mapped to the frz locus, which encodes a signal transduction system that controls the frequency of single cell reversals. On the other hand, mutants carrying any of the three closely linked class II Tn5 lac insertions had deficiencies in all four C-factor responses. Because the sporulation defect in the class 11 mutants is cell autonomous, the data suggest that the primary defect in these mutants is an inability to respond to the C-factor signal. All the data can be explained by a model in which the first part of the C-factor signaling pathway is common to all four C-factor-dependent responses. The genes identified by the class 11 insertions would function in the common part. Downstream of class II, the pathway branches. One branch includes the frz genes and leads to aggregation and rippling; the second branch leads to sporulation and controls the level of csgA gene expression. This model was confirmed in epistasis tests with characterized frz mutations, a csgA null mutation, and a class II mutation.
Bifunctional protein required for asymmetric cell division and cell-specific transcription in Bacillus subtilis.
Authors: Feucht A , Magnin T , Yudkin M D , Errington J
Published: June 2007

During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor sigma(F). Three proteins participate in the regulation of sigma(F) activity. The first, SpoIIAB, is an inhibitor of sigma(F), that is, an anti-sigma factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-sigma factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of sigma(F) activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.
Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud and possess small kidneys.
Authors: Godley L A , Kopp J B , Eckhaus M , Paglino J J , Owens J , Varmus H E
Published: June 2007

Transgenic mice expressing wild-type murine p53 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) undergo progressive renal failure due to abnormal kidney development. Similar phenotypes are observed in two transgenic lines that express wild-type p53 within the ureteric bud but not in transgenic animals expressing a dominant-negative p53 mutant allele. Defective differentiation of the ureteric bud, as evidenced by altered marker expression during development, accompanies expression of the p53 transgene. At E17.5-18.5, metanephric mesenchymal cells undergo high rates of apoptosis, and fewer cells than normal are converted to tubular epithelium. As a result, p53 transgenic kidneys grow to only half of their expected size and contain about half of the normal number of nephrons, with compensatory hypertrophy of the glomeruli. In this setting, rather than arrest the cell cycle or induce apoptosis directly, abnormally high levels of wild-type p53 appear to alter cellular differentiation in embryonic ureteric buds and cause secondary effects (apoptosis and inefficient conversion to epithelium) in the adjacent undifferentiated mesenchyme.
Functional interactions between the pelle kinase, Toll receptor, and tube suggest a mechanism for activation of dorsal.
Authors: Norris J L , Manley J L
Published: June 2007

A complex signal transduction pathway functions in the early Drosophila embryo to establish dorsal-ventral polarity. Activation of this pathway results in the nuclear transport of the protein dorsal (dl), a member of the rel/NF-kappaB family of transcription factors. Genetic studies have identified three intracellular components whose activity is required for activation of dl: Toll, a transmembrane receptor; pelle (pll), a serine/threonine protein kinase; and tube, a protein of unknown function. Here we examine the activities of these proteins when coexpressed in Drosophila Schneider cells. Coexpression of pll with dl enhanced dl nuclear localization and resulted in a modest increase in transcriptional activity. However, when pll was coexpressed with a specific mutant derivative of Toll (TlNaeI), although not with wild-type Toll, a striking synergistic activation of dl was detected. Unexpectedly, coexpression of pll plus TlNaeI, in the absence of dl, resulted in a similar synergistic activation of a GAL4-tube fusion protein. Based on these and other results, we propose a model in which pll receives a signal from activated Toll and phosphorylates tube, which then participates directly in dl activation.
Requirement of a corepressor for Dr1-mediated repression of transcription.
Authors: Mermelstein F , Yeung K , Cao J , Inostroza J A , Erdjument-Bromage H , Eagelson K , Landsman D , Levitt P , Tempst P , Reinberg D
Published: June 2007

A Dr1-associated polypeptide (DRAP1) was isolated from HeLa cells and found to function as a corepressor of transcription. Corepressor function requires an interaction between DRAP1 and Dr1. Heterodimer formation was dependent on a histone fold motif present at the amino terminus of both polypeptides. Association of DRAP1 with Dr1 results in higher stability of the Dr1-TBP-TATA motif complex and precluded the entry of TFIIA and/or TFIIB to preinitiation complexes. DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index. Analysis of DRAP1 in the developing brain of rat demonstrated undetectable levels of DRAP1 in actively dividing cells but high levels of DRAP1 expression in differentiated non dividing cells. Dr1 was immunodetected in all cells analyzed. A model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed.
Differential activation of adipogenesis by multiple PPAR isoforms.
Authors: Brun R P , Tontonoz P , Forman B M , Ellis R , Chen J , Evans R M , Spiegelman B M
Published: June 2007

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor expressed predominantly in adipose tissue, where it plays a central role in the control of adipocyte gene expression and differentiation. Because there are two additional PPAR isoforms, PPARalpha and PPARdelta, and these are also expressed at some level in certain adipose depots, we have compared directly the adipogenic potential of all three receptors. Ectopically expressed PPARgamma powerfully induces adipogenesis at a morphological and molecular level in response to a number of PPARgamma activators. PPARalpha is less adipogenic but is able to induce significant differentiation in response to strong PPARalpha activators. Expression and activation of PPARdelta did not stimulate adipogenesis. Of the three PPARs, only PPARgamma can cooperate with C/EBPalpha in the promotion of adipogenesis. To begin to investigate the functional basis for the differential adipogenic activity of the PPAR isoforms, we have examined their ability to bind to several PPAR DNA response sequences. Compared with PPARalpha and PPARdelta, PPARgamma shows preferential binding to two well-characterized regulatory sequences derived from a fat-specific gene, ARE6 and ARE7. These data strongly suggest that PPARgamma is the predominant receptor regulating adipogenesis; however, they also suggest that PPARalpha may play a role in differentiation of certain adipose depots in response to a different set of physiologic activators or in certain disease states.
Suprabasal expression of a dominant-negative RXR alpha mutant in transgenic mouse epidermis impairs regulation of gene transcription and basal keratinocyte proliferation by RAR-selective retinoids.
Authors: Feng X , Peng Z H , Di W , Li X Y , Rochette-Egly C , Chambon P , Voorhees J J , Xiao J H
Published: June 2007

To determine whether 9-cis retinoic acid receptors (RXRs) regulate the biological activity of all-trans retinoic acid (tRA) and its receptors (RARs) in skin, we have targeted a dominant-negative RXR alpha (dnRXR alpha) lacking transactivation function AF-2 to differentiated suprabasal keratinocytes in the epidermis of transgenic mice. Driven by the suprabasal-specific keratin-10 gene promoter, expression of dnRXR alpha severely reduced the ability of RAR-selective ligands tRA and CD367 to induce epidermal mRNA levels of the CRABPII, CRBPI, and CRBPII genes, which contain RA-responsive elements (RAREs) DR1 and/or DR2. It also reduced gene-specific, synergistic induction of CRBPI mRNA by a combination of CD367 and RXR-selective SR11237. Like endogenous RXR alpha, dnRXR alpha in epidermal nuclear extracts from the transgenic mice competitively formed heterodimers with endogenous RAR gamma on RAREs, suggesting that dnRXR alpha impairs retinoid signaling by competing with endogenous RAR gamma-RXR alpha heterodimers. Histologically, the epidermis of dnRXR alpha mice showed no detectable developmental abnormalities. Surprisingly, in adult animals, the suprabasal expression of dnRXR alpha significantly reduced the ability of topically applied tRA to stimulate proliferation of undifferentiated keratinocytes in the basal layer of epidermis. RXR-selective ligands alone had no detectable effects on both normal and transgenic mouse epidermis. Accordingly, we suggest that in vivo: (1) in suprabasal keratinocytes, retinoids regulate gene transcription via RAR-RXR heterodimers in which RAR confers a predominant ligand response, whereas RXR AF-2 is required for liganded RAR AF-2 to efficiently trans-activate target genes, and (2) this suprabasal RXR-assisted mechanism indirectly regulates proliferation of basal keratinocytes likely via intercellular signaling.
Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A.
Authors: Dong X , Zavitz K H , Thomas B J , Lin M , Campbell S , Zipursky S L
Published: June 2007

In the developing eye of Drosophila melanogaster, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, regulator of cyclin A (rca1), encodes a novel protein required for both mitotic and meiotic cell cycle progression. rca1 mutants arrest in G2 of embryonic cell cycle 16 with a phenotype very similar to cyclin A loss of function mutants. Expression of rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion.
Brca2 is required for embryonic cellular proliferation in the mouse.
Authors: Suzuki A , de la Pompa J L , Hakem R , Elia A , Yoshida R , Mo R , Nishina H , Chuang T , Wakeham A , Itie A , Koo W , Billia P , Ho A , Fukumoto M , Hui C C , Mak T W
Published: June 2007

Mutations of the tumor suppressor gene BRCA2 are associated with predisposition to breast and other cancers. Homozygous mutant mice in which exons 10 and 11 of the Brca2 gene were deleted by gene targeting (Brca2(10-11)) die before day 9.5 of embryogenesis. Mutant phenotypes range from severely developmentally retarded embryos that do not gastrulate to embryos with reduced size that make mesoderm and survive until 8.5 days of development. Although apoptosis is normal, cellular proliferation is impaired in Brca2(10-11) mutants, both in vivo and in vitro. In addition, the expression of the cyclin-dependent kinase inhibitor p21 is increased. Thus, Brca2(10-11) mutants are similar in phenotype to Brca1(5-6) mutants but less severely affected. Expression of either of these two genes was unaffected in mutant embryos of the other. This study shows that Brca2, like Brca1, is required for cellular proliferation during embryogenesis. The similarity in phenotype between Brca1 and Brca2 mutants suggests that these genes may have cooperative roles or convergent functions during embryogenesis.
A role for phosphorylation by casein kinase II in modulating Antennapedia activity in Drosophila.
Authors: Jaffe L , Ryoo H D , Mann R S
Published: June 2007

We present evidence that the in vivo activity of the HOX protein Antennapedia (ANTP) is modified because of phosphorylation by the serine/threonine kinase casein kinase II (CKII). Using an in vivo assay a form of ANTP that has alanine substitutions at its CKII target sites has, in addition to wild-type ANTP functions, the ability to alter severely thoracic and abdominal development. The novel functions of this protein suggest that this form of ANTP is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of ANTP that contains acidic amino acid substitutions at its CKII target sites, thereby mimicking a constitutively phosphorylated ANTP protein, is greatly reduced. This hypoactive form of ANTP, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. Our results suggest that phosphorylation of ANTP by CKII is important for preventing inappropriate activities of this homeotic protein during embryogenesis.
Klumpfuss, a putative Drosophila zinc finger transcription factor, acts to differentiate between the identities of two secondary precursor cells within one neuroblast lineage.
Authors: Yang X , Bahri S , Klein T , Chia W
Published: June 2007

The approximately 300 distinct neurons comprising each hemineuromere of the Drosophila embryonic central nervous system are derived from a segmentally reiterated array of approximately 30 progenitor cells, neuroblasts (NBs). Each NB has a unique identity and undergoes repeated cell divisions to produce several smaller secondary precursor cells, ganglion mother cells (GMCs); each GMC divides once to produce two neurons and/or glia, thereby generating a specific lineage of neurons/glia. Understanding the generation of neuronal diversity requires not only elucidation of the molecules and mechanisms that specify NB identity but also those that act to differentiate between the cell types produced within one NB lineage. Here we show that the Drosophila Zn finger protein Klumpfuss (Klu), which shows sequence similarities to the mammalian Wilm's tumor suppressor (WT-1), acts to differentiate between the identities of the first two secondary precursor cells produced from one NB lineage. Klu is expressed in the NB4-2 lineage only after two rounds of NB cell division, in the second born GMC (GMC4-2b). In loss-of-function mutant embryos, the first born GMC (GMC4-2a) as well as its progeny neurons are duplicated; we show that this duplication of the GMC4-2a sublineage arises because GMC4-2b adopts the identity of GMC4-2a and divides to produce the GMC4-2a progeny. Moreover, when Klu is ectopically expressed in GMC4-2a, it fails to acquire its normal identity and fails to produce correctly specified progeny. klu therefore acts to specify the identity of GMC4-2b and to make it distinct from GMC4-2a. Our findings further suggest that the determination of GMC cell fate occurs in two steps; the initial GMC identity is the consequence of inheritance from the maternal NB, however, the subsequent stabilization of this identity requires functions like klu in the GMC.
The role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning.
Authors: Philpott A , Porro E B , Kirschner M W , Tsai L H
Published: June 2007

Cyclin-dependent kinase 5, coupled with its activator p35, is required for normal neuronal differentiation and patterning. We have isolated a novel member of the p35 family, Xp35.1, from Xenopus embryos which can activate cdk5. Xp35.1 is expressed in both proliferating and differentiated neural and mesodermal cells and is particularly high in developing somites where cdk5 is also expressed. Using dominant-negative cdk5 (cdk5 DN), we show that cdk5 kinase activity is required for normal somitic muscle development; expression of cdk5 DN results in disruption of somitic muscle patterning, accompanied by stunting of the embryos. Using explants of animal pole tissue from blastula embryos, which will differentiate into mesoderm in response to activin, we show that blocking cdk5 kinase activity down-regulates the expression of the muscle marker muscle actin in response to activin, whereas the pan-mesodermal marker Xbra is unaffected. Expression of MyoD and MRF4 (master regulators of myogenesis) is suppressed in the presence of cdk5 DN, indicating that these myogenic genes may be a target for cdk5 regulation, whereas the related factor Myf5 is largely unaffected. In addition, overexpression of Xp35.1 disrupts muscle organization. Thus, we have demonstrated a novel role for cdk5 in regulating myogenesis in the early embryo.
The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals.
Authors: de Vetten N , Quattrocchio F , Mol J , Koes R
Published: June 2007

In petunia flowers, the loci an1, an2, and an11 control the pigmentation of the flower by stimulating the transcription of anthocyanin biosynthetic genes. The an1 and an2 locus were recently cloned and encode a basic helix-loop-helix (bHLH) and MYB-domain transcriptional activator, respectively. Here, we report the isolation of the an11 locus by transposon tagging. RNA gel blot experiments show that an11 is expressed independently from an1 and an2 throughout plant development, as well as in tissues that do not express the anthocyanin pathway. It encodes a novel WD-repeat protein that is highly conserved even in species that do not produce anthocyanins such as yeast, nematodes, and mammals. The observation that the human an11 homolog partially complements the an11 petunia mutant in transient assays shows that sequence similarity reflects functional conservation. Overexpression of an2 in an11- petals restored the activity of a structural anthocyanin gene in transient assays, indicating that AN11 acts upstream of AN2. Cell fractionation experiments show that the bulk of the AN11 protein is localized in the cytoplasm. Taken together, this indicates that AN11 is a cytoplasmic component of a conserved signal transduction cascade that modulates AN2 function in petunia, thereby linking cellular signals with transcriptional activation.
Mitogenic stimulation of resting T cells causes rapid phosphorylation of the transcription factor LSF and increased DNA-binding activity.
Authors: Volker J L , Rameh L E , Zhu Q , DeCaprio J , Hansen U
Published: June 2007

The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.
pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes.
Authors: Hurford R K , Cobrinik D , Lee M H , Dyson N
Published: June 2007

The activity of the E2F transcription factor is controlled by physical association with the retinoblastoma protein (pRB) and two related proteins, p107 and p130. The pRB family members are thought to control different aspects of E2F activity, but it has been unclear what the respective functions of these proteins might be. To dissect the specific functions of pRB, p107, and p130 we have investigated how the expression of E2F-regulated genes is changed in cultures of primary cells lacking each of these family members. Whereas no changes were found in the expression of E2F-target genes in cells lacking either p107 or p130, deregulated expression of E2F targets was seen in cells lacking pRB and in cells lacking both p107 and p130. Surprisingly, the genes that were disregulated in these two settings were completely different. These findings show that pRB and p107/p130 indeed provide different functions in E2F regulation and identify target genes that are dependent on pRB family proteins for their normal expression.
Cyclin E-induced S phase without activation of the pRb/E2F pathway.
Authors: Lukas J , Herzinger T , Hansen K , Moroni M C , Resnitzky D , Helin K , Reed S I , Bartek J
Published: June 2007

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G1 arrest imposed by either the p16INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.
ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.
Authors: Pasero P , Braguglia D , Gasser S M
Published: June 2007

We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.