Inhibition of cortical astrocyte differentiation by Hes6 requires amino‐ and carboxy‐terminal motifs important for dimerization and phosphorylation

Abstract

Hairy/Enhancer of split (Hes) 6 is a basic helix-loop-helix protein that interacts with the transcriptional co-repressor, Groucho, and antagonizes the neural functions of the Notch pathway. More specifically, mouse Hes6 regulates cerebral corticogenesis by promoting neurogenesis and suppressing astrocyte differentiation. The molecular mechanisms underlying the anti-astrogenic function of Hes6 are poorly defined. Here we describe studies aimed at testing whether Hes6 inhibits astrocyte differentiation by antagonizing the transcription repression activity of Notch-activated Hes family members like Hes1. It is reported that Hes6 preferentially forms homodimers. Heterodimerization with Hes1 is antagonized in part by a conserved N-terminal patch of negatively charged residues. Mutation of this motif enhances heterodimerization with Hes1 and increases Hes6 ability to antagonize Hes1-mediated transcriptional repression. However, this mutation does not increase, but instead decreases, the antiastrogenic activity of Hes6. It is shown further that Hes6 harbors a second conserved sequence, a C-terminal SPXXSP motif. This sequence is phosphorylated by the mitogen activated protein kinase pathway and its mutation disrupts the anti-astrogenic activity of Hes6 without affecting its ability to suppress Hes1. Together, these observations suggest that Hes6 homodimers regulate astrocyte differentiation through mechanisms that depend on the phosphorylation of Hes6 C-terminal domain but are independent of its ability to suppress Hes1-mediated transcriptional repression. Keywords: astrocyte differentiation, cerebral cortex, Groucho, Hes, neurogenesis, Notch. In contrast to Hes1/Hes5, a related family member, Hes6, is not activated in response to Notch signaling and promotes, rather than inhibiting, neuronal differentiation in murine retinal explants (Bae et al. 2000), Xenopus neural plate (Koyano-Nakagawa et al. 2000), and cortical neural progenitor cells (Gratton et al. 2003;Jhas et al. 2006 These authors contributed equally to this study.Abbreviations used: bHLH, basic helix-loop-helix; CK2, protein kinase CK2; EMSA, electrophoretic mobility shift assay; ESE, Enhancer of split E; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; Gro, Groucho; HA, hemagglutinin; HEK, human embryonic kidney; Hes, Hairy/Enhancer of split; MAPK, mitogen activated protein kinase; Ngn, neurogenin; TLE, transducin-like Enhancer of split.

Inhibition of Cortical Neuron Differentiation by Groucho/TLE1 Requires Interaction with WRPW, but Not Eh1, Repressor Peptides

“…In each experiment, cells were co-transfected with 1.0 g of pCMV2-FLAG-Gro/TLE1 (WT, V486S, C488R, R534A, E550K, or L743F) and 1.0 g of either pEBG-HES1 (or pEBG-HES1(⌬WRPW) as control), pCMV2-HA-FoxG1, pMycTcf3, or pCMV2-HA-En1. Cell lysates were prepared and GST co-precipitations or co-immunoprecipitations using either anti-HA (Covance, Berkeley, CA) or anti-Gro/TLE1 (14) antibodies were performed as described (14,20,27). This was followed by Western blotting analysis using anti-FLAG (1:10,000; Sigma), anti-GST (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (1:5,000), or anti-Myc (1:200; BD Pharmingen, San Diego, CA) antibodies.…”

Interaction and Antagonistic Roles of NF-κB and Hes6 in the Regulation of Cortical Neurogenesis

“…The following expression and reporter plasmids have been described previously: pCAGGS-GFP (where GFP is green fluorescent protein) (19); pcDNA3-IB␣M (20); pAd-Track-IKK␤-DN (where IKK␤-DN is dominant negative form of IB kinase subunit beta) (21,22); pCMV2-FLAG-Hes6WT, pCMV2-FLAG-Hes6⌬55-95, pCMV2-FLAG-Hes6⌬WRPW, pCMV2-FLAG-Hes1WT, and pEGFP (23,24); pCMV-p65/RelA (pCMV-RelA) and pNF-B-luciferase (25); and pRSV-␤-gal (7,24). The IB␣M coding sequence was subcloned as an EcoRI fragment into the EcoRI site of the pCIG2 vector, which contains a cDNAinternal ribosome entry site (IRES)-enhanced GFP (EGFP) expression cassette under the control of a cytomegalovirus (CMV) enhancer and chicken ␤-actin promoter (26).…”

“…Human embryonic kidney 293 (HEK293) cells were transfected with plasmids encoding FLAG epitope-tagged Hes1 or Hes6 proteins (200 ng/transfection) using the SuperFect reagent (Qiagen). Lysates from transfected cells were subjected to immunoprecipitation with either anti-FLAG (Sigma) or anti-RelA antibody as described previously (23,31). This step was followed by Western blotting of immunoprecipitates, together with 1/10 of each input lysate, with anti-FLAG (1/5,000) or anti-RelA antibody.…”

“…In each case, a pRSV-␤-gal plasmid (250 ng/transfection) was used to normalize for transfection efficiency. Twenty-four hours after transfection, cell lysates were subjected to determination of luciferase and ␤-Gal activities as described previously (7,23,24).…”

The novel BTB/POZ and zinc finger factor Zbtb45 is essential for proper glial differentiation of neural and oligodendrocyte progenitor cells

“…Double mutant mice harboring deletions in Olig1/Olig2 or Mash1/ Ngn2 show increased astrocytic differentiation in the spinal cord and forebrain, respectively, 42,43 and the bHLH factor Hes6 has been shown to repress astrocytic differentiation and promote neuronal differentiation. 44 In addition, NSCs isolated from mice harboring gene deletion for the BTB/POZ domaininteracting co-repressors NCoR or SMRT show spontaneous astrocytic differentiation even in the presence of FGF2. 17,19 The results from the present study suggest that Zbtb45 is a novel factor essential for proper glial differentiation as Zbtb45 mRNA knockdown results in increased astrocytic gene expression and Zbtb45 siRNA and mitogen withdrawal (Fig.…”