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Results for “ectopic gene conversion”

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PubMed1999

Role of PPAR gamma in regulating adipocyte differentiation and insulin-responsive glucose uptake.

Adipocyte differentiation is regulated by at least two families of transcription factors, CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs). Induction of PPAR gamma gene transcription during the differentiation of preadipocytes into adipocytes in vitro occurs following an initial phase of cell proliferation and requires a direct involvement of C/EBP beta, C/EBP delta, and glucocorticoids. Ectopic expression of PPAR gamma in non-adipogenic, Swiss 3T3 fibroblasts promotes their conversion into adipocytes as indicated by the accumulation of lipid droplets and the induction of C/EBP alpha, aP2, insulin-responsive aminopeptidase (IRAP), and glucose transporter 4 (GLUT4) expression. These PPAR gamma-expressing Swiss cells also exhibit a high level of insulin-responsive glucose uptake that is comparable to that expressed in 3T3-L1 adipocytes. In contrast, PPAR gamma-expressing NIH-3T3 fibroblasts, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not synthesize C/EBP alpha and fail to acquire insulin sensitivity. In Swiss 3T3 cells ectopically expressing PPAR gamma, the development of insulin-responsive glucose uptake correlates with C/EBP alpha expression. Furthermore, ectopic expression of C/EBP alpha in NIH-3T3 cells induces PPAR gamma expression and adipogenesis, but also restores insulin-sensitive glucose transport. These results suggest that although PPAR gamma is sufficient to trigger the adipogenic program, C/EBP alpha is required for establishment of insulin-sensitive glucose transport in adipocytes.

3T3 Cells↗

PubMed2025

Heterochromatin-based silencing of a foreign tandem repeat in Drosophila melanogaster shows unusual biochemistry and temperature sensitivity.

Eukaryotic genomes are packaged into chromatin, a regulatory nucleoprotein assembly. Establishment, maintenance, and interconversion of chromatin states is required for correct patterns of gene expression, genome integrity, and survival. Transcriptionally repressive heterochromatin minimizes mobilization of transposable elements and limits expansion of other repetitive DNA, but mechanisms for recognition of the latter sequences are not well established. We previously demonstrated in Drosophila melanogaster that transcripts derived from 1360 and Invader4 transposon insertions can trigger local conversion of transcriptionally permissive euchromatin to heterochromatin through the piRNA system, but only in a subset of genomic locations near existing blocks of heterochromatin. Here we show that a ~9 kb tandem array of the 36-nucleotide lac operator (lacO) sequence of Escherichia coli can form ectopic heterochromatin at a similar subset of sites, resulting in variegating expression of an adjacent reporter gene. Heterochromatin Protein 1a (HP1a) and histone deacetylation are required for lacO repeat-induced silencing, but, contrasting with previously described Position Effect Variegation (PEV), we do not observe increased histone H3 lysine 9 methylation. Silencing is effective at 25°C and suppressed at 18°C (in contrast to canonical PEV, which is enhanced at 18°C), indicating involvement of a temperature-sensitive component. Temperature switching experiments show that lacO repeat-induced heterochromatin formation is reversible throughout larval development following an HP1a-dependent initiation step in the early embryo. We conclude that the Drosophila nucleus can recognize a completely foreign tandem repeat as a target for heterochromatin formation, and that the heterochromatin structure established is distinct from that of endogenous tandem arrays.

HP1a↗

PubMed2005

CDX2-regulated expression of iron transport protein hephaestin in intestinal and colonic epithelium.

BACKGROUND & AIMS: The homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. Mice heterozygous for Cdx2 inactivation develop colonic polyps with epithelial cells showing gastric or squamous differentiation. Loss of CDX2 expression is seen in some poorly differentiated colon carcinomas in humans. Conversely, ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression often is seen in intestinal metaplasia in stomach and esophagus. To enhance knowledge of CDX2 function, we sought to define CDX2-regulated genes. METHODS: HT-29 and WiDr colorectal cancer (CRC) cells with low endogenous CDX2 expression were transduced with a CDX2 expression vector, and gene expression changes were assessed by microarrays. RESULTS: The gene for ceruloplasmin-related iron transport protein hephaestin (HEPH) was induced by CDX2 in HT-29 and WiDr. In other CRC lines and human and mice tissues, endogenous HEPH expression was linked to CDX2 expression. Activation of CDX2 rapidly induced HEPH expression, and RNA interference-mediated inhibition of CDX2 led to lower HEPH expression. Studies with HEPH reporter gene constructs and chromatin-immunoprecipitation approaches suggested that CDX2 directly regulates HEPH transcription. In CRC cells, CDX2 induction suppressed intracellular iron levels, consistent with the view that HEPH regulates iron export. CDX2 expression was modulated in response to changes in intracellular iron levels, implying a regulatory pathway in which increased iron levels lead to increased expression of CDX2 and HEPH and enhanced iron export. CONCLUSIONS: CDX2 has a key role in regulating HEPH expression and iron levels in intestinal cells.

Animals↗

PubMed1996

The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function.

The class B organ identity genes, APETALA3 and PISTILLATA, are required to specify petal and stamen identity in the Arabidopsis flower. We show here that the activities of these two genes are sufficient to specify petals and stamens in flowers, in combination with the class A and C genes, respectively. Flowers of plants constitutively expressing both PISTILLATA and APETALA3 under the control of the 35S promoter from cauliflower mosaic virus consist of two outer whorls of petals and inner whorls of stamens. These plants also exhibit vegetative phenotypes that are not present in either of the singly (APETALA3 or PISTILLATA) overexpressing lines. These phenotypes include leaf curling and the partial conversion of later-arising cauline leaves to petals. The presence of additional floral whorls in flowers ectopically expressing APETALA3 and PISTILLATA and the rescue of missing organs in class A mutants by ectopic B function suggest that APETALA3 and PISTILLATA play an additional role in proliferation of the floral meristem.

Arabidopsis↗

PubMed1997

Efficient gap repair in Drosophila melanogaster requires a maximum of 31 nucleotides of homologous sequence at the searching ends.

Double-strand breaks (DSB) were generated in the Drosophila melanogaster white gene by excision of the P-w(hd) element. An ectopic P-element vector carrying a modified white gene was used as a template for DSB repair. All template-dependent repair events were examined, and four different classes of events were recovered. The two most common products observed were gene conversions external to the P-w(hd) element and gene conversions (targeted transpositions) internal to the P-w(hd) element. These two events were equally frequent. Similar numbers for both orientations of internal conversion events were recovered. The results suggest that P-element excision occurs by a staggered cut that leaves behind at least 33 nucleotides of single-stranded sequence. Our results further demonstrate that an efficient homology search is conducted by the broken end with less than 31 nucleotides.

Animals↗

PubMed2006

Drosophila CRYPTOCHROME is a circadian transcriptional repressor.

BACKGROUND: Although most circadian clock components are conserved between Drosophila and mammals, the roles assigned to the CRYPTOCHROME (CRY) proteins are very different: Drosophila CRY functions as a circadian photoreceptor, whereas mammalian CRY proteins (mCRY1 and 2) are transcriptional repressors essential for molecular clock oscillations. RESULTS: Here we demonstrate that Drosophila CRY also functions as a transcriptional repressor. We found that RNA levels of genes directly activated by the transcription factors CLOCK (CLK) and CYCLE (CYC) are derepressed in cry(b) mutant eyes. Conversely, while overexpression of CRY and PERIOD (PER) in the eye repressed CLK/CYC activity, neither PER nor CRY repressed individually. Drosophila CRY also repressed CLK/CYC activity in cell culture. Repression by CRY appears confined to peripheral clocks, since neither cry(b) mutants nor overexpression of PER and CRY together in pacemaker neurons significantly affected molecular or behavioral rhythms. Increasing CLK/CYC activity by removing two repressors, PER and CRY, led to ectopic expression of the timeless clock gene, similar to overexpression of Clk itself. CONCLUSIONS: Drosophila CRY functions as a transcriptional repressor required for the oscillation of peripheral circadian clocks and for the correct specification of clock cells.

ARNTL Transcription Factors↗

PubMed2003

Selective reactions of cutaneous and muscle afferent neurons to peripheral nerve transection in rats.

To determine whether peripheral nerve injury has similar effects on all functional types of afferent neuron, we retrogradely labeled populations of neurons projecting to skin and to muscle with FluoroGold and lesioned various peripheral nerves in the rat. Labeled neurons were counted after different periods and related to immunohistochemically identified ectopic terminals and satellite cells in lumbar dorsal root ganglia. After 10 weeks, 30% of cutaneous afferent somata labeled from transected sural nerves had disappeared but, if all other branches of the sciatic nerve had also been cut, 60% of cutaneous neurons were lost. Small-diameter sural neurons preferentially disappeared. In contrast, the number of muscle afferent somata was not affected by transection of various nerves. p75 was downregulated in axotomized cutaneous neurons but in not axotomized muscle neurons. Conversely, p75 was upregulated in satellite cells around cutaneous but not muscle neurons. Consistent with this, perineuronal rings containing tyrosine hydroxylase, calcitonin gene-related peptide, galanin, or synaptophysin were formed preferentially around cutaneous neurons. Selective lesions of predominantly cutaneous nerves triggered the formation of rings, but none were detected after selective lesions of muscle nerves. We conclude that cutaneous neurons are both more vulnerable and more associated with ectopic nerve terminals than muscle neurons in dorsal root ganglia after transection and ligation of peripheral nerves.

Animals↗

PubMed2006

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development.

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK-dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, we further demonstrated that the RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila.

Active Transport, Cell Nucleus↗

PubMed2000

Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis.

The control of cell proliferation during organogenesis plays an important role in initiation, growth, and acquisition of the intrinsic size of organs in higher plants. To understand the developmental mechanism that controls intrinsic organ size by regulating the number and extent of cell division during organogenesis, we examined the function of the Arabidopsis regulatory gene AINTEGUMENATA (ANT). Previous observations revealed that ANT regulates cell division in integuments during ovule development and is necessary for floral organ growth. Here we show that ANT controls plant organ cell number and organ size throughout shoot development. Loss of ANT function reduces the size of all lateral shoot organs by decreasing cell number. Conversely, gain of ANT function, via ectopic expression of a 35S::ANT transgene, enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number in both Arabidopsis and tobacco plants. This hyperplasia results from an extended period of cell proliferation and organ growth. Furthermore, cells ectopically expressing ANT in fully differentiated organs exhibit neoplastic activity by producing calli and adventitious roots and shoots. Based on these results, we propose that ANT regulates cell proliferation and organ growth by maintaining the meristematic competence of cells during organogenesis.

Arabidopsis↗

PubMed1988

Familial ectopic ossification.

We report a family with dominantly inherited ectopic ossification. It is characterised by childhood onset of multifocal subcutaneous ossifications (primary osteoma cutis), which are of trivial clinical significance. One family member had extensive ectopic ossification involving one limb. We speculate that this may reflect somatic mutation having caused conversion to homozygosity.

Bone and Bones↗

PubMed1998

Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

AGAMOUS Protein, Arabidopsis↗

PubMed1999

Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to notch signalling.

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. We report here the role of the COE transcription factor Collier in the formation of a single muscle, muscle DA3([A])(DA4([T])). Col expression is first observed in two promuscular clusters (in segments A1-A7), the two corresponding progenitors and their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3([A]). This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signalling. In col mutant embryos, the DA3([A]) founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3([A]) phenotype. In wild-type embryos, each of the DA3([A])-recruited myoblasts turns on col transcription, indicating that the conversion, by the DA3([A]) founder cell, of 'naive' myoblasts to express its distinctive pattern of gene expression involves activation of col itself. We find that muscles DA3([A]) and DO5([A]) (DA4([T]) and DO5([T])) derive from a common progenitor cell. Ectopic expression of Col is not sufficient, however, to switch the DO5([A]) to a DA3([A]) fate. Together these results lead us to propose that specification of the DA3([A]) muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles.

Animals↗

PubMed1998

Myc suppresses induction of the growth arrest genes gadd34, gadd45, and gadd153 by DNA-damaging agents.

The growth arrest and DNA damage inducible (gadd) genes are induced by various genotoxic and non-genotoxic stresses such as serum starvation, ultraviolet irradiation and treatment with alkylating agents. Their coordinate induction is a growth arrest signal which may play an important role in the response of cells to DNA damage. Conversely, c-myc is a strong proliferative signal, and overexpression of Myc is frequently observed in cancer cells. We have found that ectopic expression of v-myc in RAT-1 cells results in an attenuated induction of the three major gadd transcripts by methyl methanesulfonate (MMS), and almost completely blocks the response to ultraviolet (UV) radiation. Myc acts in part by reducing the stress-responsiveness of the gadd45 promoter, as a c-myc expression vector strongly suppressed activation of gadd45-reporter constructs. This activity of Myc localizes to a recently described GC-rich binding site within the gadd45 promoter. These results indicate that a coordinate down-regulation of the gadd gene response is one mechanism by which Myc can circumvent growth arrest and contribute to the neoplastic phenotype.

Animals↗

PubMed1995

Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis.

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Notch signaling is required for arterial-venous differentiation during embryonic vascular development.

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line

Animals↗

PubMed2001

A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny.

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from a proneural cluster, and undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. We show that phyllopod (phyl), previously identified to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters, leading to the fate transformation of neurons and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. We further show that seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. It has been shown previously that Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. We further demonstrate that phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development.

Animals↗

PubMed2002

Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns.

Body coloration affects how animals interact with the environment. In insects, the rapid evolution of black and brown melanin patterns suggests that these are adaptive traits. The developmental and molecular mechanisms that generate these pigment patterns are largely unknown. We demonstrate that the regulation and function of the yellow and ebony genes in Drosophila melanogaster play crucial roles in this process. The Yellow protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. Conversely, Ebony is required to suppress some melanin formation, and is expressed in cells that will produce both melanized and non-melanized cuticle. Ectopic expression of Ebony inhibits melanin formation, but increasing Yellow expression can overcome this effect. In addition, ectopic expression of Yellow is sufficient to induce melanin formation, but only in the absence of Ebony. These results suggest that the patterns and levels of Yellow and Ebony expression together determine the pattern and intensity of melanization. Based on their functions in Drosophila melanogaster, we propose that changes in the expression of Yellow and/or Ebony may have evolved with melanin patterns. Consistent with our hypothesis, we find that Yellow and Ebony are expressed in complementary spatial patterns that correlate with the formation of an evolutionary novel, male-specific pigment pattern in Drosophila biarmipes wings. These findings provide a developmental and genetic framework for understanding the evolution of melanin patterns.

Animals↗

PubMed1998

Dorsoventral patterning in the Drosophila central nervous system: the vnd homeobox gene specifies ventral column identity.

The Drosophila CNS develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. In this and the accompanying paper, we investigate the role of two homeobox genes, vnd and ind, in establishing ventral and intermediate cell fates within the Drosophila CNS. During early neurogenesis, Vnd protein is restricted to ventral column neuroectoderm and neuroblasts; later it is detected in a complex pattern of neurons. We use molecular markers that distinguish ventral, intermediate, and dorsal column neuroectoderm and neuroblasts, and a cell lineage marker for selected neuroblasts, to show that loss of vnd transforms ventral into intermediate column identity and that specific ventral neuroblasts fail to form. Conversely, ectopic vnd produces an intermediate to ventral column transformation. Thus, vnd is necessary and sufficient to induce ventral fates and repress intermediate fates within the Drosophila CNS. Vertebrate homologs of vnd (Nkx2.1 and 2.2) are similarly expressed in the ventral CNS, raising the possibility that DV patterning within the CNS is evolutionarily conserved.

Animals↗