XB-IMG-76512
Xenbase Image ID: 76512
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Fig. 4. Meis3 and Tsh1 act cooperatively on the Meis3 promoter; recruited Tsh1 represses transcription. (A)Meis3-5'UTR expression
analysis in mid-neurula embryos injected at the one-cell stage with Meis3 mRNA (1 ng; b) or Tsh1-MO (5 pmol; a), or both (c). (B)Meis3 expression in mid-neurula embryos injected at the one-cell stage with Tsh1 mRNA (0.4 ng; b) or Meis3-MO (28 ng; d), or both (c). (C)A schematic representation of the Meis3 proximal promoter region. Arrow indicates start of transcription; green ovals indicate two Meis consensus sites (A and B), indicated in light-blue text; white oval indicates ChIP-negative control site (C); numbers indicate positions relative to the transcription start site (+1); ATG (+585) is the translation initiation site. (D)ChIP-QPCR analysis on the Meis3 promoter in Meis3-Myc injected embryos (0.8 ng) at mid-neurula stages. MeisA and MeisB are Meis consensus site amplicons, MeisC and Mlc2 are negative control sites. IgG ChIP is a negative IP control. Pooled data from three independent experiments are represented as the percentage of input chromatin. Error bars are s.e.m. from three independent experiments. For amplicon primers, see supplementary material Table S2. (E)ChIP-QPCR analysis on the Meis3 promoter in Myc-Tsh1- injected embryos (0.4 ng), either wild type or Meis3-MO (28 ng) co-injected, at mid-neurula stages. IgG-IP, MeisC and Mlc2 are negative controls as in D. Pooled data from three independent experiments are represented as the percentage of input chromatin. Error bars are s.e.m. from three independent experiments. Tsh1-ChIP on Meis3-MO embryos (yellow) completely abolished the Tsh1-ChIP signal seen in wild-type embryos (dark blue). Tsh1 ChIP to both wild-type and Meis3-MO embryos were performed together in the same experiment and ChIP run; thus, Tsh1 ChIP on wild-type embryos is a positive control for Tsh1 ChIP on Meis3-MO embryos. (F)In vitro co-IP of Tsh1 and Meis3 proteins. In vitro synthesized HA- Meis3, S35-Met-Tsh1 and S35-Met-Cyp26 were mixed. HA-Meis3 IP was performed. IgG-IP and Cyp26 are a negative controls. (G)Kinetics of Tsh1 and Meis3 transcription in response to increasing levels of a forced Meis3 transcriptional activator. RT-PCR was performed on pools of 18 mid- neurula AC explants from VP16-Meis3 mRNA-injected embryos (0.25-1 ng). Results were quantitated, normalized to the Ef1-alpha control and plotted as fold change. Bars are s.e.m. from three independent experiments. T samples showed no DNA contamination. A x-value is the non-injected controls, with a y-value as a basal expression level. (H)RT-PCR to Meis3-5'UTR and Krox20 in mid-neurula embryos injected with VP16-Meis3 mRNA (250 pg). Robust Krox20 expression controls for a strong Meis3 protein activity. Image published in: Elkouby YM et al. (2012) Copyright © 2012. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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