Biomedical subjects

T Neuman

Publications and source records attributed to T Neuman.

At least 37 records · Page 2Linked to original sources

PubMed1996

The GAP-43 gene is a direct downstream target of the basic helix-loop-helix transcription factors.

The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis-acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.

Animals↗

PubMed1996

Cell type specific regulation of COUP-TF II promoter activity.

COUP-TF family orphan receptors regulate activity of ligand-activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP-TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5' regulatory region of the mouse COUP-TF II gene and demonstrated that the basal promoter is localized in a -200 bp region 5' from the transcription start site. All-trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP-TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all-trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.

Animals↗

PubMed1996

All-trans retinoic acid affects the expression of orphan receptors COUP-TF I and COUP-TF II in the developing neural tube.

The effect of all-trans retinoic acid (RA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) I and COUP-TF II expression in the developing cervical spinal cord and telencephalon was examined using embryonic day 11 and 13 mice. All-trans RA treatment results in changes in expression of COUP-TF I and COUP-TF II genes in the spinal cord and telencephalon. COUP-TF I mRNA levels were reduced in the spinal cord ventricular zone (VZ) after treatment, whereas in the telencephalon, mRNA levels were unaffected in the VZ and increased in the intermediate zone (IZ). COUP-TF II mRNA levels were increased in the spinal cord VZ after treatment, while in the telencephalon, IZ mRNA levels were decreased in the E11 embryos and increased in E13 embryos.

Animals↗

PubMed1995

Helix-loop-helix transcription factors regulate Id2 gene promoter activity.

Id-like helix-loop-helix (HLH) transcription factors are involved in the regulation of proliferation and differentiation of several cell types. We isolated 5' regulatory region of mouse Id2 gene and demonstrated that it contains several E-box clusters. These E-boxes mediate stimulatory effects of basic-HLH (bHLH) transcription factors ME1, ME2, and NSCL1 on Id2 promoter activity. Co-expression of Id2 blocks the stimulatory effect of bHLH transcription factors which suggests the presence of feedback loops in Id2 transcriptional regulation. Overexpression of NSCL1 in F9 cells blocks the downregulation of Id2 gene expression during retinoic acid induced differentiation. Our data demonstrate that bHLH transcription factors regulate Id2 gene expression.

Animals↗

PubMed1995

Expression screening reveals an orphan receptor chick ovalbumin upstream promoter transcription factor I as a regulator of neurite/substrate-cell contacts and cell aggregation.

A rat homologue of chick ovalbumin upstream promoter transcription factor I (COUP-TF I) was isolated using an expression cloning method developed to isolate neurite outgrowth inhibitors. Overexpression of COUP-TF I in 3T3 fibroblasts resulted in reduction of stable contact formation between neurites and transfected cells. Additionally, COUP-TF I enhanced retinoic acid response element-dependent reporter gene expression in 3T3 fibroblasts, indicating that COUP-TF I can modulate transcriptional activation in these cells. Our data suggest that COUP-TF transcription factors are involved in the regulation of cell surface molecules during neruogenesis.

3T3 Cells↗

PubMed1995

Cyclin E is expressed in neurons and forms complexes with cdk5.

Expression of cyclin dependent kinase cdk5 and its regulator p35 has been shown in the cytoplasm of adult neurons. Here we demonstrate that another potential regulator of cdk5, cyclin E, is expressed in the nervous system and forms complexes with cdk5. Western blot analyses identifies expression of two forms of cyclin E in the mouse nervous system with the 56 kDa form mainly expressed in neurons and 51 kDa form expressed in astrocytes and oligodendrocytes.

Animals↗

PubMed1995

A novel initiator regulates expression of the nontissue-specific helix-loop-helix gene ME1.

The mouse ME1 gene (HEB, REB and GE1, homologues in human, rat and chick, respectively) is a member of the nontissue-specific helix-loop-helix (HLH) gene family that includes E2A, E2-2 and Drosophila daughterless. We have examined the factors that control ME1 gene expression. ME1 is a single copy gene that spans > or = 150 kb of DNA and contains > 10 exons. Transcription was directed by an unusual initiator element that contained a 13 bp poly d(A) tract flanked by palindromic and inverted repeat sequences. Both RNase protection and primer extension analyses mapped the ME1 transcriptional start site to the center of the 13 bp poly d(A) tract. The ME1 initiator and its proximal sequences were required for promoter activity, supported basal levels of transcription, and contributed to cell type-specific gene expression. Other cis-elements utilized by the TATA-less ME1 promoter included a cluster of Sp1 response elements, E-boxes and a strong repressor. Collectively, our results suggest that the ME1 initiator and other cis-elements in the proximal promoter play an important role in regulating ME1 gene expression.

Animals↗

PubMed1995

Orphan receptor COUP-TF I antagonizes retinoic acid-induced neuronal differentiation.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TF) are expressed in the developing nervous system and interact with nuclear hormone receptors to regulate expression of different genes. The role of COUP-TF orphan receptors in neurogenesis is virtually unknown. To study the possible function of COUP-TF I during neuronal differentiation, we generated COUP-TF I overexpressing teratocarcinoma PCC7 cell lines and analyzed retinoic acid (RA)-induced neuronal differentiation of these cells. COUP-TF I overexpression results in the blockade of morphological differentiation after induction to differentiate. COUP-TF I represses expression of microtubule-associated protein 2 (MAP2) gene and delays induction of growth-associated protein 43 (GAP43) gene expression. In contrast, expression of the neurofilament light subunit (NF-L) gene is not affected by COUP-TF I overexpression during neuronal differentiation. Also, cells overexpressing COUP-TF I do not stop proliferating after RA and dBcAMP treatment and possess suppressed transcriptional activation from different RA response elements. These results suggest that COUP-TF I plays an important role in regulating RA-induced neuronal differentiation.

Animals↗

PubMed1995

Expression of Rb, E2F1, cdc2, and D, and B cyclins in developing spinal cord.

The spatial and temporal distribution of transcripts for the tumor suppressor gene Rb, transcription factor E2F1, cdc2 kinase, cyclins D1, D2, B1 and B2 during neurogenesis of the spinal cord was determined by in situ hybridization. The Rb and E2F1 transcripts were detectable in proliferating and differentiating cells. By contrast, cdc2, cyclins D1, B1 and B2 are expressed in the ventricular zone where proliferating cells are localized. Cyclin D2 mRNA was detectable only in the marginal zone of the developing neural tube. Electrophoretic mobility shift analyses demonstrated a changing pattern of DNA/protein complexes that bind to E2F binding site. These observations suggest that Rb and E2F1 may be involved in the early stages of neuronal differentiation in addition to the cell cycle regulation.

Animals↗

PubMed1995

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

Animals↗

PubMed1995

Isolation of genes which block neuronal differentiation of teratocarcinoma PCC7 cells.

Since several viral oncoproteins block differentiation and induce proliferation of differentiated cells, we developed an expression screening method to isolate cDNAs which block neuronal differentiation and induce proliferation of teratocarcinoma cells. Mouse E2F1, RNP-1, and RNP-2 (Regulator of Neuronal Proliferation) were isolated using the developed screening method. Overexpression of E2F1, RNP-1, and RNP-2 cDNAs in neuronally differentiated teratocarcinoma PCC7 cells results in blocking differentiation and initiation of proliferation. Also, expression of RNP-1 and RNP-2 blocks the expression of neurofilament-L and GAP-43 genes in PCC7 cells.

Base Sequence↗

PubMed1995

Differential expression and distinct DNA-binding specificity of ME1a and ME2 suggest a unique role during differentiation and neuronal plasticity.

Class A basic-helix-loop-helix (bHLH) proteins have been referred to as ubiquitous and are believed to have redundant functions. They are involved in the control of several developmental pathways, such as neurogenesis and myogenesis. To rationalize the existence of multiple class A bHLH proteins, we evaluated the differences and similarities between ME1a and ME2, two class A bHLH proteins, highly expressed in differentiating neuronal cells. In situ hybridization analyses reveal that ME1a and ME2 are characterized by distinguishable patterns of expression in areas of the adult mouse brain where neuronal plasticity occurs. Also, DNA-binding assays show that both proteins bind to E-boxes as homodimers and heterodimers, and show differences in their DNA-binding specificities, which suggest selective interactions with different binding sites of target genes. In addition, in vitro DNA-binding assays demonstrate that Id2 forms heterodimers with ME1a and ME2. As a result of these interactions, their DNA-binding activity is abolished. Furthermore, overexpression of Id2 in neuronal cells suppresses ME1a and ME2 transcriptional activity. Based on our data, we hypothesize that ME1a and ME2 may activate gene expression of different target genes and therefore are likely to be differently involved during neurogenesis.

Animals↗

PubMed1995

Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression.

Animals↗

PubMed1994

DNA synthesis is induced in adult neurons after expression of E2F1 and E1A.

Differentiated neurons in several brain regions express the retinoblastoma (Rb) tumor suppressor gene. Changing the tumor suppressor function of Rb by expressing transcription factor E2F1 and viral oncoprotein E1A in cerebellar granular neurons in vitro and in cerebral cortical neurons in vivo results in the induction of DNA synthesis in these neurons. Immunoliposome-mediated transfection of E1A and E2F1 cDNAs into the adult cortical neurons of rats in vivo results in initiation of DNA synthesis in 5-15% of the transfected neurons. These results indicate that expression of Rb may be necessary to prevent induction of differentiated neurons to proliferate since many mitogenic growth factors are expressed in the brain.

Adenovirus E1A Proteins↗

PubMed1994

Class A basic helix-loop-helix transcription factors in early stages of chick neural tube development: evidence for functional redundancy.

Basic Helix-Loop-Helix (bHLH) transcription factors play essential role in differentiation of several cell types including neurons. We isolated chick bHLH transcription factor E12 cDNA and demonstrated its expression in developing neural tube interneurons. To examine the function of class A bHLH transcription factors we blocked their expression using antisense oligonucleotides. Simultaneous blocking of class A bHLH transcription factors in cultured neural tube cells results in the reduction of differentiating neurons. Blocking the expression of individual class A bHLH transcription factors has no detectable effect. These results demonstrate that class A bHLH transcription factors have a functional redundancy during neuronal development.

Animals↗

PubMed1994

Expression of basic-helix-loop-helix transcription factor ME2 during brain development and in the regions of neuronal plasticity in the adult brain.

We report the isolation of a cDNA encoding the mouse class A bHLH transcription factor ME2 and the analysis of its expression. ME2 is expressed in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, olfactory neuroepithelium, pyramidal cells of hippocampal layers CA1-CA4, and in the granular cells of the dentate gyrus. The specific expression of ME2 during development and in the regions of neuronal plasticity in the adult brain suggest that ME2 may have a regulatory function in developmental processes as well as during neuronal plasticity.

Amino Acid Sequence↗

PubMed1993

ME1 and GE1: basic helix-loop-helix transcription factors expressed at high levels in the developing nervous system and in morphogenetically active regions.

Several class A basic helix-loop-helix (bHLH) transcription factors have been cloned from the developing mouse and chick nervous system. The cloned cDNAs (ME1, ME2, ME3, ME4, in the mouse and GE1, GE2 in the chick) have HLH coding regions highly homologous to other known class A bHLH genes. The genes corresponding to ME1 and GE1 are abundantly expressed during development of the central nervous system. ME1 and GE1 are expressed in proliferating neuroblasts and in cells at the initial stages of differentiation (for example in the external granule cell layer of the cerebellum and in the lateral region of the ventricular zone in the developing neural tube and cortex). They are also expressed at high levels in morphogenetically active regions such as limb buds, somites and mesonephric tubules. The expression of ME1 and GE1 decreases once cellular differentiation is over. Based on the expression of ME1 and GE1 in regions of active cellular proliferation and differentiation and on the known role of other bHLH factors in development, we suggest that ME1 and GE1 play important roles during development of the nervous system as well as in other organ systems.

Amino Acid Sequence↗

PubMed1993

Neuronal expression of regulatory helix-loop-helix factor Id2 gene in mouse.

Id-like helix-loop-helix (HLH) proteins, which lack a basic DNA binding domain, have been suggested to serve as general inhibitors of differentiation. We present data that Id2 is expressed in specific cell types during neurogenesis and in the adult. At early stages of neurogenesis, Id2 is expressed in the ventricular zone of neuroepithelium. After the first neuronal populations are born, the expression of Id2 is down regulated in neuroepithelial cells and continues to be high in Purkinje cells of the cerebellum, in mitral cells of the olfactory bulb, and in layers 2, 3, and 5 of the cerebral cortex. In neuronally differentiating cell lines, the Id2 expression is up regulated (PCC7), down regulated (NG108), or unchanged (N18) during differentiation. Analyses of promoter sequences of the Id2 gene identify the region which is responsible for the down regulation of transcription during neuronal differentiation. Our data indicate that Id2 has different functions in different cell types during neurogenesis.

Animals↗