SKELETAL MUSCLE: DEVELOPMENTAL HISTORY AND NEURAL ACTIVITY CONTRIBUTE TO THE FIBER-TYPE SPECIFICITY OF TROPONIN I GENES
Identification of TnI Regulatory Elements that Confer Fiber-Type Specificity in Transgenic Mice
Calvo, Buonanno
Multiple DNA motifs in the TnI slow up-stream regulatory enhancer (SURE) are important for the activity of this enhancer. We have generated mutations of the TnI SURE to identify the cis-elements that confer the fiber-type specificity of the TnIs gene. As shown in Figure 17 (left), transgenic mice harboring the 95 bp TnIs basal promoter driven by the 128 bp SURE (extends from –868 to –741 from the transcription start site) express the luciferase reporter gene specifically in the slow-twitch soleus muscle. Transcription in the fast-twitch extensor digitorium longus (EDL), gastrocnemius (Gas), and tibialis anterior (Tib) muscles is extremely low (approximately 10- to 50-fold lower than in soleus). The systematic addition of sequences up to position –827, which contain the E-box, MEF-2, and CACC motifs, confers general muscle-specific transcription but fails to restore fiber-type specificity (Figure 17, middle). Specificity is regained only after a 15-bp region, residing between –842 and –827, is added to the pan muscle enhancer (Figure 17, right). Moreover, SURE is converted into a fast-specific enhancer if the 5'-half of SURE is replaced by the corresponding region of fast intronic regulatory element (FIRE). The results indicate that the TnISURE and FIRE have a modular organization: DNA elements directing fiber-type specificity reside in the 5'-halves and those conferring general muscle specificity are in the 3'-halves. The 5'-half of SURE harbors the CAGG and bicoid-like motifs (BLM), which we previously demonstrated to be necessary for enhancer function. Thus, constructs containing the 15-bp region between –842 and –827 were used on yeast one-hybrid to screen for trans-acting factors regulating slow muscle transcription.

Figure 17

TnI sequences that confer general skeletal muscle and fiber-type specificity of the SURE enhancer. Examples of transgenic mice harboring wild-type SURE(left), and truncations SURE-827(middle) and SURE-842 (right) of the enhancer.

Nuclear Factor GTF3 Interacts with TnI SURE Regulatory Elements
Venepally, Vullhorst, Karavanova, Buonanno
A yeast one-hybrid screen of 2.7 x 107 transformants yielded 10 partial cDNA sequences encoding GTF3, a transcription factor structurally related to TFII-I. Expression vectors for GTF3 encoding either its N- or C-terminal halves were translated in vitro and used on competitive electrophoretic mobility shift assays (EMSAs) to assess the DNA binding activity of both regions of the protein. We found that the C-terminal half of GTF3 specifically binds the BLM in SURE, which conforms to the consensus site for the initiator element (Inr: YYANTYY) that binds TFII-I. Recent reports indicate that GTF3 and TFII-I function as heterodimers.

Northern blots indicate that GTF3 is encoded by a 3.9 kb transcript expressed in many tissues. In situ hybridization shows prominent GTF3 hybridization in forming muscle masses at E15 as well as in skin and bones, which dramatically decreases with maturation. Regenerating adult muscles reactivate GTF3 expression (arrowheads), whereas nondegenerated areas are essentially devoid of GTF3 hybridization signals (arrows).

GTF3 Represses Transcription of the TnI SURE: Possible Implications for Williams-Beuren Syndrome
Vullhorst, Buonanno
We examined the functional role of GTF3 in TnIs transcription by transfecting expression and reporter vectors in adult muscles using in vivo electroporation. GTF3 significantly represses transcription from the TnI SURE. Based on the importance of sequences containing the BLM for fiber-type specificity of the TnI SURE, the early expression of GTF3 in skeletal muscle, and the inhibition of SURE activity by GTF3, we propose that GTF3 contributes to myofiber diversification during early development. The haploinsufficiency of GTF3 and GTF2i may underlie some of the characteristics of persons with WBS who have distinctive physical, cognitive, and behavior abnormalities that may include impaired spatial cognitive skills and myopathies. Alterations in the expression of proteins that regulate the contractile or metabolic properties of skeletal muscles could account either directly or indirectly for the myopathies associated with WBS. We have found that GTF2-I and GTF3 are expressed at high levels in developing musculature and neurons, raising the possibility that an early reduction of these factors could result in the down-regulation or misexpression of target genes later in development.

NEURONAL STUDIES: REGULATION OF NMDA RECEPTORS AND NEUREGULINS DURING DEVELOPMENT
Neuronal Expression of ErbB Receptors and Interactions with PDZ-Domain Proteins
Garcia, Karavanova, Vasudevan, Buonanno in collaboration with S. Carroll, University of Alabama at Birmingham
We analyzed the expression of ErbB receptors throughout the adult brain, using in situ hybridization and immunohistochemistry. In agreement with our earlier work, we found that ErbB-2 is widely expressed by most cell types; ErbB-3 is highest in the myelinated tracks and thalamic nuclei, and ErbB-4 is mostly confined to neurons in the cortex, hippocampus, thalamus, and basal ganglia. Using biochemical techniques, we studied the subcellular distribution of ErbB receptors. We found that ErbB receptors are present in brain fractions enriched for postsynaptic densities (PSD). We used a yeast two-hybrid system to identify proteins that interact with these receptors and that may regulate their subcellular distribution. The C-terminal region of ErbB-4 interacts with three closely related PDZ-domain proteins: PSD-95, CHAPSYN-110, and SAP 102. Using coimmunoprecipitation assays, we confirmed direct interactions between ErbB-4 and PSD-95 in neurons. Immunocytochemical studies confirmed that ErbB-4 colocalizes with NRs and PSD-95 at synaptic puncta of hippocampal neurons apposed to synaptophysin-positive presynaptic terminals. These observations have important implications for the possibility of cross-talk of ErbB and NMDA receptors at synapses and for the role of Nrgs in regulating activity-dependent plasticity at central synapses.

Characterization of NR2A Transcription Regulatory Sequences
Turetsky, Desai, Buonanno
The levels of NMDA receptors harboring the NR2A subunit increase sharply after two weeks of age, a time that coincides with the functional maturation of synapses. We identified the transcription initiation site of NR2A and cloned gene sequences harboring the basal promoter. A fragment extending approximately 9 kb upstream of the promoter was shown to confer neuronal specificity in transgenic mice and transfected primary cortical neurons; transcription in glia was minimal. Given that expression of the NR2A gene increases with maturation, we also analyzed transcription of the 9kb luciferase reporter construct in developing cultured cortical neurons. We found that the reporter is transcribed at low levels during the first week in culture and increases dramatically during the second week. The results indicate that the NR2A up-stream 9 kb fragment harbors sequences that confer both developmental and tissue-specific expression of the gene; experiments are in progress to delineate the regulatory motifs in the NR2A gene (Figure 18).

Figure 18

The upstream 9kb of NR2A gene sequence confer neuronal- and developmental-specific transcription. A. the NR2A-9kb lucierase construct is more selectively transcribed in transfected cortical neurons than in glia. Background levels of transcription are observed with the pGL3-Basic negative control. B. Transcription from the NR2A reporter construct increases during the maturation of cortical neurons in culture.