Interaction of MTG family proteins with NEUROG2 and ASCL1 in the developing nervous system

Abstract

During neural development, members of MTG family of transcriptional repressors are induced by proneural basic helix-loop-helix (bHLH) transcription factors and in turn inhibit the activity of the bHLH proteins, forming a negative feedback loop that regulates the normal progression of neurogenesis. Three MTG genes, MTG8, MTG16 and MTGR1, are expressed in distinct patterns in the developing nervous system. Various bHLH proteins are also expressed in distinct patterns. We asked whether there is a functional relationship between specific MTG and bHLH proteins in developing chick spinal cord. First, we examined if each MTG gene is induced by specific bHLH proteins. Although expression of NEUROG2, ASCL1 and MTG genes overlapped, the boundaries of gene expression did not match. Ectopic expression analysis showed that MTGR1 and NEUROD4, which show similar expression patterns, are regulated differently by NEUROG2 and ASCL1. Thus, our results show that expression of MTG genes is not regulated by a single upstream bHLH protein, but represents an integration of the activity of multiple regulators. Next, we asked if each MTG protein inhibits specific bHLH proteins. Transcription assay showed that NEUROG2 and ASCL1 are inhibited by MTGR1 and MTG16, and less efficiently by MTG8. Deletion mapping of MTGR1 showed that MTGR1 binds NEUROG2 and ASCL1 using multiple interaction surfaces, and all conserved domains are required for its repressor activity. These results support the model that MTG proteins form a higher-order repressor complex and modulate transcriptional activity of bHLH proteins during neurogenesis.

Figure 1. Expression pattern of chicken MTGR1, MTG8 and MTG16 relative to NEUROG2 and ASCL1

Chromogenic (A, B) and fluorescent double (C–E) in situ hybridization. Cross sections of the spinal cord at embryonic (E) days 3–7 at the brachial level are shown. The probes used are indicated according to color in the figures. Insets are enlargements of the boxed areas in the same figure. Bar: 100μm.

Figure 2. Induction of MTG genes by NEUROG2 and ASCL1

E2 chick spinal cords were electroporated with expression vectors of NERUOG2 (A–E) or ASCL1 (F–J) along with the GFP expression vector and analyzed 24h later. Adjacent sections were stained by in situ hybridization using MTGR1 (A, F), MTG8 (B, G), MTG16 (C, H) or NEUROD4 (D, I) probes. Electorporated areas were visualized by staining the sections with anti GFP immunohistochemistry. D, E and G, J are from the same sections. Large arrowheads indicate ectopic expression of the markers and small arrowheads point to areas where markers are down-regulated. Unelectroporated sides (left side) serve as controls. Bar: 100μm.

Figure 3. Relative inhibitory activity of MTG proteins

P19 cells were transfected with the indicated vectors and analyzed 24 h later. Numbers indicate the μg amount of MTG expression vectors transfected. Data represent average of triplicate transfections and bars show standard deviation.

Figure 4. Deletion assay of MTGR1

A: Truncation mutants used in this experiment. B, C: Reporter gene assay. Del1 showed inhibitory activity comparable to the wild-type molecule, but any other deletion constructs had weaker inhibitory activity to NEUROG2+E47 (B) or ASCL1+E47 (C). D: All constructs were tagged with the myc epitope and expression levels in P19 cells were examined by Western blotting against the myc epitope tag. Western blot using anti α-tubulin antibody is shown as a loading control. E: Q-PCR was carried out to examine the endogenous expression level of MTG genes in P19 cells. F–H: GST pull-down assay. Deletion mutants were radiolabeled by in vitro translation and tested for binding to GST fusion molecules of NEUROG2 (F), ASCL1 (G) or E47 (H). Recovered radioactivity was quantified and shown as percent recovery of the input in bar graphs. Numbers in F–H correspond to the numbers of deletion constructs and asterisks indicate the positions of the bands. TAF: TATA box binding protein associated factor, DD: dimerization domain, NHR3: nervy homology region 3, Znf: zinc finger-like motif.