Expression of the bHLH transcription factor Tcf12 (ME1) gene is linked to the expansion of precursor cell populations during neurogenesis.

In this study, we focused on the potential function of the murine gene Tcf12 (also known as ME1 or HEB) encoding the bHLH E-protein ME1 during brain development. An exencephaly phenotype of low penetrance has consistently been observed in both Tcf12 null mice and Tcf12(dm) homozygous mice. Thus, to address the possible underlying mechanism of the Tcf12 gene during the early steps of brain development, we performed a detailed analysis of its spatio-temporal expression pattern at distinct steps of gastrulation and neurogenesis. We found that Tcf12 transcripts are detected in the embryonic ectoderm prior to neural induction during gastrulation. During neurulation, Tcf12 transcripts are evident at high levels in the proliferating neuroepithelium of the neural folds and the cephalic mesenchyme. Thus, Tcf12 gene expression coincides with the massive proliferation occurring in the forming neuroepithelium and cephalic mesenchyme during neural tube formation, which is consistent with the exencephaly phenotype of Tcf12 null mice. In the developing cortex and spinal cord, Tcf12 expression is restricted to the proliferative ventricular zones, indicating that Tcf12 expression is down regulated when these neuronal cells undergo their final differentiation. Interestingly, we found that the postnatal Tcf12 expression parallels the ongoing adult neurogenesis in the mitotically active subventricular zone. Thus, the timing and location of Tcf12 expression combined with this severe neurulation defect support our hypothesis that the Tcf12 gene may be involved in the control of proliferating neural stem cells and progenitor cells and that it may be critical to sustain their undifferentiated state during embryonic and adult neurogenesis.

Differential expression patterns of the basic helix-loop-helix transcription factors during aging of the murine brain.

In this study, we investigated the expression pattern of the basic Helix-Loop-Helix transcription factors during brain aging. We provide the first evidence that NeuroD and ME2 are differentially expressed during brain aging. Modulation of their expression is specific to distinct areas of the aging brain. NeuroD expression is sustained at high levels in aging cerebellum, whereas it severely declines in aging hippocampus. In contrast, the bHLH E-protein ME2 remains expressed in both aged cerebellum and hippocampus, although at lower levels. These observations support the idea that a shift in the transcriptional dynamics controlling gene expression is associated with the progressive functional decline observed during brain aging.

Expression of the basic Helix-Loop-Helix ME1 E-protein during development and aging of the murine cerebellum.

Genesis of cerebellar granule cells is controlled by key transcription factors, such as the lineage-specific basic Helix-Loop-Helix (bHLH) transcription factor MATH-1, whose activity is dependent upon dimerization with bHLH E-proteins. In an effort to understand the molecular mechanisms of bHLH proteins orchestrating cerebellar development, we explored the spatio-temporal expression of the ME1 E-protein. Our results reveal that ME1 expression is first detected in the cerebellar primordium and then in the rhombic lip cells at E12.5. Its expression persists in the emerging external germinal layer during embryonic expansion. In adult cerebellum, prominent ME1 expression is detected in mature granule cells located in the internal granular layer. However, ME1 expression is not sustained in aged cerebellum. A similar declined pattern of expression is also observed in the aging hippocampus.

Expression of the bHLH gene NSCL-1 suggests a role in regulating cerebellar granule cell growth and differentiation.

We report that the neuronal-specific basic helix-loop-helix (bHLH) gene NSCL-1 is expressed at multiple and distinct stages of cerebellar granule cell differentiation. During embryonic development, NSCI-1 expression is initially evenly distributed in the cerebellar primordium and then becomes restricted to the ventricular zone. At the early steps of granule cell development, NSCL-1 is not expressed in rhombic lip cells, but instead in migrating granule cell precursors. Its expression culminates during postnatal proliferation of the external germinal layer, and remains only transiently in the newly formed internal granular layer, and at a much lower level. Thus, NSCL-1 expression is linked to the onset of granule cell differentiation, but is not involved in the maintenance of the differentiated state. These findings suggest that NSCL-1 does not behave as a specification factor, but rather as a factor promoting expansion of progenitor external germinal layer (EGL) cells. Gel mobility shift assays show that NSCL-1 only binds DNA as a heterodimeric complex with the ME1a E-protein. We also provide the first evidence that NSCL-1 functions as a transcriptional activator when heterodimerized with the ME1a E-protein. Taken together, these results suggest that NSCL-1 participates in the regulatory network controlling gene expression during cerebellar granule cell differentiation.

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.

Transient expression of the basic helix-loop-helix protein NSCL-2 in the mouse cerebellum during postnatal development.

The spatio-temporal expression of the basic helix-loop-helix transcription factor NSCL-2 was analyzed in the postnatal development of the murine brain by in situ hybridization. We found that NSCL-2 is transiently expressed in the cerebellum. NSCL-2 was found exclusively in the premigratory zone of the external granule layer where postmitotic neurons undergo initial stages of neuronal differentiation. NSCL-2 expression was not detected in mature neurons. This pattern of expression suggests that NSCL-2 is critical for the onset of neuronal differentiation, but not for the maintenance of the differentiated state.

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.

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.

Human T-cell leukemia virus type I Tax protein represses gene expression through the basic helix-loop-helix family of transcription factors.

The human T-cell leukemia virus type I (HTLV-I) oncoprotein Tax is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but utilizes cellular transcription factors to mediate activation. In this report, we examine the role of the basic helix-loop-helix (bHLH) proteins in Tax deregulation of gene expression, as these proteins play a critical role in progression through the cell cycle and have been implicated in neoplastic disease. We show that the bHLH proteins do not mediate activation, but instead mediate repression of gene expression in the presence of Tax. We further show that a consensus bHLH binding site in the promoter of the beta-polymerase gene, which encodes an enzyme involved in DNA repair, mediates the previously reported repression of beta-polymerase gene expression by Tax. Together, these results suggest that Tax may induce malignant transformation, at least in part, through bHLH-mediated repression of key cellular regulatory genes.

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.

Coupling of DNA replication to growth rate in Escherichia coli: a possible role for guanosine tetraphosphate.

Two promoters for the Escherichia coli operon that contains the four genes dnaA, dnaN, recF, and gyrB were found to be growth rate regulated and under stringent control. Transcript abundance relative to total RNA increased with the growth rate. Changes in transcription from the dnaAp1 and dnaAp2 promoters that were induced by amino acid starvation and chloramphenicol and were relA dependent were correlated with the stringent response. The abundance of these transcripts per total RNA also decreased in spoT mutants as the severity of the mutation increased (guanosine 5'-diphosphate 3'-diphosphate [ppGpp] basal levels increased). Because expression of these promoters appears to be inhibited by ppGpp, it is proposed that one mechanism for coupling DNA replication to the growth rate of bacteria is through ppGpp synthesis at the ribosome.

Expression of Escherichia coli dnaA and mioC genes as a function of growth rate.

The synthesis of specific cellular components related to the initiation process of DNA replication was correlated with changes in growth rate. The concentrations of DnaA protein and mioC mRNA were determined for cells grown at six different growth rates; both increased relative to either total protein or total RNA, respectively, as the growth rate increased. Expression from the chromosomal mioC promoter, which contains a DnaA protein-binding site, was not repressed when the DnaA protein concentration was increased and was not derepressed in a dnaA46 mutant at 42 degrees C. The mioC transcript had a characteristic mRNA-type half-life of 1.51 min.

Transcription termination within the Escherichia coli origin of DNA replication, oriC.

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.