Sakano D et al. (2010), BCL6 canalizes Notch-dependent transcription, e...

XB-ART-41299

Dev Cell 2010 Mar 16;183:450-62. doi: 10.1016/j.devcel.2009.12.023.

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BCL6 canalizes Notch-dependent transcription, excluding Mastermind-like1 from selected target genes during left-right patterning.

Sakano D , Kato A , Parikh N , McKnight K , Terry D , Stefanovic B , Kato Y .

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Although the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6 forms a complex with BCL6 corepressor (BCoR) on the promoters of selected Notch target genes such as enhancer of split related 1. BCL6 also inhibits the transcription of these genes by competing for the Notch1 intracellular domain, preventing the coactivator Mastermind-like1 (MAM1) from binding. These results define a mechanism restricting Notch-activated transcription to cell-type-appropriate subsets of target genes, and elucidate its relevance in vivo during left-right asymmetric development.

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Species referenced: Xenopus
Genes referenced: bcl6 bcor esr1 gal.2 hes4 hes5 maml1 nodal1 notch1 pitx2
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	Figure 2. Related Functions of BCL6 and Notch Signaling during LR Patterning(A) 40 ng BCL6 MO, 40 ng Control MO, or/and 2 ng mBCL6-GR was injected for each experiment. An arrow or a spiral indicates the orientation of the heart or the gut coiling, respectively. Ventral views are shown.(B) The normal left-specific expression of Xnr1 or Pitx2 is indicated by an arrow in the nucÎ²-gal-injected embryo.(C) 80 ng Notch1 MO or 80 ng Control MO was injected for each experiment. An arrow or a spiral indicates the orientation of the heart or the gut coiling, respectively. Ventral views are shown.(D and E) 150 ng Notch1 MO, 150 ng Control MO, or/and 1 ng GR-NICD RNA was injected for each experiment. The dotted line indicates the embryonic midline. The injected side is indicated by â€œLâ€ (left) or â€œRâ€ (right) beside the names of the injected samples. L, left; R, right; a, anterior; p, posterior.
	Figure 3. BCL6 Maintains Pitx2 Expression by Inhibiting Notch Signaling(Aâ€“D) 50 pg GR-NICD, 100 pg GR-at-Su(H), 100 pg MAM1-GR, 2 ng GR-dn-Su(H), 2 ng dn-MAM1-GR, or/and 2 ng mBCL6-GR was injected for each experiment.(E and F) The expression of Pitx2 at stage 25 was tested by whole-mount in situ hybridization, and the ratios of the Pitx2-expressing embryo number versus the total tested embryo number are shown. The total numbers of each injection are shown as â€œnâ€ on the top of each bar.(G) 2 ng NBD-S-GR or/and mBCL6-GR was injected for each experiment. The injected side is indicated by â€œLâ€ (left) beside the names of the injected samples. L, left; R, right; a, anterior; p, posterior.
	Figure 5. ESR1 Is a Target of BCL6 during LR Patterning and Neural Development(A) BCL6 MO was injected into the left side of embryos, and left LPM tissues were dissected from 20 embryos at stage 25 for quantitative RT-PCR. The injection side was traced by the coinjection of GFP (data not shown). p < 0.05, n = 3.(B) BCL6 MO was injected into a dorsal blastomere of four-cell-stage embryos, and embryos were fixed at stage 14. Increased ESR1 expression and decreased N-tubulin expressions are indicated by white and black arrows, respectively.(C) BCL6 MO or/and Notch1 MO was injected into the left side of embryos, and left LPM tissues were dissected from 20 embryos at stage 25 for quantitative RT-PCR. p < 0.01, n = 3; p < 0.01, n = 3; *p < 0.01, n = 3.(D) 1 ng ESR1-GR or Hairy2-GR was injected for each experiment.(E) 40 ng ESR1 MO or/and BCL6 MO was injected for each experiment. The injected side is indicated by â€œLâ€ (left) beside the names of the injected samples. L, left; R, right; a, anterior; p, posterior.All error bars shown are standard deviation (SD) from the mean of triplicates.
	Figure 6. The Mechanisms by Which BCL6 Shuts Down the Expression of Selected Notch Target Genes(A) ChIP assays were performed with nuclear extracts from stage-25 embryos by using Î±-BCL6 antibody, Î±-Notch1 antibody, or mouse IgG. Mouse IgG was used for a mock ChIP assay.(B) 2 ng dn-Su(H) was injected into two-cell-stage embryos, and nuclear extracts were isolated at stage 10 for ChIP assays.(C) BCL6 MO or/and 1 ng Flag-MAM1 was injected into two-cell-stage embryos, and nuclear extracts were isolated at stage 10 for ChIP assays. The levels of input proteins were confirmed by immunoblotting.(D) Deleted fragments of the X. tropicalis ESR1 gene were linked to the luciferase reporter. Numbers indicate the position of nucleotides from the initiation site.(E) Incubation of GST-BCL6 or GST-ZF with a probe corresponding to a 134 bp (âˆ’1030/âˆ’897) element yielded one distinct retarded band indicated by an arrow.(F) 2 ng BCL6 ZF-GR or/and mBCL6-GR was injected for each experiment. The injected side is indicated by â€œLâ€ (left) beside the names of the injected samples. L, left; R, right; a, anterior; p, posterior.

References [+] :

Adachi, Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer. 1999, Pubmed