An in vivo selection system for isolating targets of DNA binding proteins in yeast was developed and used to identify the DNA binding site for the NGFI-B protein, a member of the steroid-thyroid hormone receptor superfamily. The feasibility of the technique was verified by selecting DNA fragments that contained binding sites for GCN4, a well-characterized yeast transcriptional activator. The DNA binding domain of NGFI-B, expressed as part of a LexA-NGFI-B-GAL4 chimeric activator, was then used to isolate a rat genomic DNA fragment that contained an NGFI-B binding site. The NGFI-B response element (NBRE) is similar to but functionally distinct from elements recognized by the estrogen and thyroid hormone receptors and the hormone receptor-like proteins COUP-TF, CF1, and H-2RIIBP. Cotransfection experiments in mammalian cells demonstrated that NGFI-B can activate transcription from the NBRE with or without its putative ligand binding domain.
The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRalpha1-GFRalpha4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRalpha3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.
We examined in detail the DNA interaction of the nuclear receptors NGFI-B and steroidogenic factor 1 (SF-1) by using a series of gain-of-function domain swaps. NGFI-B bound with high affinity as a monomer to a nearly linear DNA molecule. The prototypic zinc modules interacted with a half-site of the estrogen receptor class, and a distinct protein motif carboxy terminal to the zinc modules (the A box) interacted with two AIT base pairs 5' to the half-site. SF-1 bound in the same manner as NGFI-B, with an overlapping but distinct sequence requirement 5' to the half-site. The key features that distinguished the NGFI-B and SF-1 interactions were an amino group in the minor groove of the SF-1 binding sequence and an asparagine in the SF-1 A box. These results define a common mechanism of NGFI-B and SF-1 DNA binding, which may underlie a competitive mechanism of gene regulation in steroidogenic tissues that express these proteins. This monomer-DNA interaction represents a third paradigm of DNA binding by nuclear receptors in addition to direct and inverted dimerization.It is well established that the receptors for steroid hormones are intracellular proteins that are themselves transcription factors. Binding of steroid to the conserved carboxy-terminal domains results in a conformational change that allows shedding of heat shock proteins, phosphorylation, translocation of the protein to the nucleus, and binding to specific DNA sites via the conserved central domain of the proteins (1,13,18,24,31,42,45). Proteins with these characteristic, conserved functional domains also mediate the effects of vitamin D, all-trans-and 9-cis-retinoic acid, thyroid hormone, the insect molting hormone ecdysone, and probably various fatty acid compounds (5,9,12,17,22,33,37,63). The term nuclear receptor is now generally applied to this superfamily of proteins to broaden the consideration of their functions while recognizing their general receptorlike characteristics. This term may still be inadequate, since molecular cloning techniques have revealed many proteins that share these structural features but whose function is unknown (the orphan receptors), which may serve as transcription factors regulated by other means (27,41). At least one protein of this class, NGFI-B (also called nur77), is closely regulated at the transcriptional level by the actions of growth factors and membrane depolarization, but once expressed NGFI-B can activate transcription without exogenously added ligand (6,16,38,43,61,64,65).The first demonstrations of DNA binding by nuclear receptors were made by examining the promoters of known glucocorticoid-and estrogen-regulated genes (19,20,44). The identified response elements function as inverted repeats of protein-specific 6-bp elements (termed half-sites) separated by three nonconserved base pairs, each half-site interacting with one molecule of a steroid receptor dimer (23, 55). Many nonsteroid receptors have since been shown to bind inverted half-site repeats with different spacings (11) or direct rather than inve...
A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.
In the peripheral nervous system, Schwann cells (SCs) surrounding damaged axons undergo an injury response that is driven by an intricate transcriptional program and is critical for nerve regeneration. To examine whether these injury-induced changes in SCs are also regulated posttranscriptionally by miRNAs, we performed miRNA expression profiling of mouse sciatic nerve distal segment after crush injury. We also characterized the SC injury response in mice containing SCs with disrupted miRNA processing due to loss of Dicer. We identified 87 miRNAs that were expressed in mouse adult peripheral nerve, 48 of which were dynamically regulated after nerve injury. Most of these injury-regulated SC miRNAs were computationally predicted to inhibit drivers of SC dedifferentiation/proliferation and thereby re-enforce the transcriptional program driving SC remyelination. SCs deficient in miRNAs manifested a delay in the transition between the distinct differentiation states required to support peripheral nerve regeneration. Among the miRNAs expressed in adult mouse SCs, miR-34a and miR-140 were identified as functional regulators of SC dedifferentiation/proliferation and remyelination, respectively. We found that miR-34a interacted with positive regulators of dedifferentiation and proliferation such as Notch1 and Ccnd1 to control cell cycle dynamics in SCs. miR-140 targeted the transcription factor Egr2, a master regulator of myelination, and modulated myelination in DRG/SC cocultures. Together, these results demonstrate that SC miRNAs are important modulators of the SC regenerative response after nerve damage.
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