Alteration of CaM I mRNA expression in the developing rat superior colliculus following chronic treatment with an NMDA receptor antagonist.

The spatial distribution of CaM I mRNA was investigated in the developing superior colliculus of rats that were chronically treated with the NMDA receptor antagonist AP-5. In control animals, CaM I message was localized in a continuous band of cells that extended across the entire rostro caudal axis of the superficial superior colliculus. Chronic AP-5 treatment resulted in a specific reduction of CaM I message in the caudal colliculus at postnatal day 10. Since normal NMDA receptor function has been implicated in activity dependent synaptic plasticity in the superior colliculus, these results suggest that the regulation of calmodulin may be part of a cascade of events that mediate this plasticity following NMDA receptor activation.

BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers.

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs.

CaM I mRNA is localized to apical dendrites during postnatal development of neurons in the rat brain.

In the rat, a single calmodulin (CaM) protein is encoded by three separate genes which produce five different transcripts. The significance of the multiple CaM genes is not known; however, individual CaM transcripts could be targeted to specific intracellular sites. In this report, the cellular distribution of CaM I mRNAs was analyzed in the postnatal rat brain. The 4.0-kb CaM I transcript was present in neuronal cell bodies and also localized to apical dendritic processes. In cerebral cortical neurons, the 4.0-kb CaM I mRNA was detected in apical dendrites at postnatal day (PD) 5 to 15. In hippocampal neurons, this CaM message was present in dendritic processes from PD S to 20, whereas in Purkinje neurons it was detected in dendrites at PD 15 and 20. The presence of the 4.0-kb CaM I mRNA in dendrites of the rat brain supports the notion of targeting transcripts derived from the CaM multigene family to discrete intracellular destinations.

Analyses of the effects that disease-causing missense mutations have on the structure and function of the winged-helix protein FOXC1.

Five missense mutations of the winged-helix FOXC1 transcription factor, found in patients with Axenfeld-Rieger (AR) malformations, were investigated for their effects on FOXC1 structure and function. Molecular modeling of the FOXC1 forkhead domain predicted that the missense mutations did not alter FOXC1 structure. Biochemical analyses indicated that, whereas all mutant proteins correctly localize to the cell nucleus, the I87M mutation reduced FOXC1-protein levels. DNA-binding experiments revealed that, although the S82T and S131L mutations decreased DNA binding, the F112S and I126M mutations did not. However, the F112S and I126M mutations decrease the transactivation ability of FOXC1. All the FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. These results indicate that the FOXC1 forkhead domain contains separable DNA-binding and transactivation functions. In addition, these findings demonstrate that reduced stability, DNA binding, or transactivation, all causing a decrease in the ability of FOXC1 to transactivate genes, can underlie AR malformations.

Positive and negative regulation of myogenic differentiation of C2C12 cells by isoforms of the multiple homeodomain zinc finger transcription factor ATBF1.

The ATBF1 gene encodes two protein isoforms, the 404-kDa ATBF1-A, possessing four homeodomains and 23 zinc fingers, and the 306-kDa ATBF1-B, lacking a 920-amino acid N-terminal region of ATBF1-A which contains 5 zinc fingers. In vitro, ATBF1-A was expressed in proliferating C2C12 myoblasts, but its expression levels decreased upon induction of myogenic differentiation in low serum medium. Forced expression of ATBF1-A in C2C12 cells resulted in repression of MyoD and myogenin expression and elevation of Id3 and cyclin D1 expression, leading to inhibition of myogenic differentiation in low serum. In contrast, transfection of C2C12 cells with the ATBF1-B isoform led to an acceleration of myogenic differentiation, as indicated by an earlier onset of myosin heavy chain expression and formation of a higher percentage of multinucleated myotubes. The fourth homeodomain of ATBF1-A bound to an AT-rich element adjacent to the E1 E-box of the muscle regulatory factor 4 promoter mediating transcriptional repression. The ATBF1-A-specific N-terminal region possesses general transcription repressor activity. These results suggest that ATBF1-A plays a role in the maintenance of the undifferentiated myoblast state, and its down-regulation is a prerequisite to initiate terminal differentiation of C2C12 cells.