Interactions between hairy/enhancer of split-related proteins and the pancreatic transcription factor Ptf1-p48 modulate function of the PTF1 transcriptional complex - PubMed (original) (raw)

Bidyut Ghosh et al. Biochem J. 2006.

Abstract

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transcription factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix-loop-helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.

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Figures

Figure 1

Figure 1. HES-related proteins and Ptf1-p48 co-immunoprecipitate in COS7 cell lysates

(A) Association between Ptf1-p48 and Hes1. COS7 cells were transfected with expression vectors encoding V5 epitope-tagged Ptf1-p48 and FLAG-tagged Hes1, either alone or in combination. Immunoblot analysis of cell lysates prior to IP (immunoprecipitation) confirms appropriate expression following transient transfection. Following IP with the anti-V5 antibody, FLAG–Hes1 was detectable in the immunoprecipitate only in the presence of V5–Ptf1-p48. (B) Association of Ptf1-p48 with Hey1 and Hey2. COS7 cells were transfected with expression vectors encoding Ptf1-p48, FLAG–Hey1 and FLAG–Hey2, either alone or in combination. Pre-IP samples confirmed appropriate expression. Following IP with the anti-FLAG antibody, immunoreactive Ptf1 was present in the immunoprecipitate only in the presence of either Hey1 or Hey2. Pre-IP samples represented one-tenth of the total cell lysates used in the IP reaction.

Figure 2

Figure 2. Hes1 and the C-terminal domain of Ptf1-p48 interact in a yeast two-hybrid assay

Colonies from SD (synthetic drop-out) media lacking leucine and tryptophan were streaked on X-Gal-containing SD plates lacking leucine, tryptophan and histidine. Vectors containing the Gal4 DNA-binding domain in-frame with either the C-terminal domain of Ptf1-p48 (pGBK-Ptf1 C-terminal) or with a combined bHLH/C-terminal domain (pGBK-Ptf1 bHLH+C-terminal) did not produce viable colonies when combined with an empty Gal4 activation vector (pGAD), but did produce blue colonies when combined with the Gal4 activation domain in-frame with Hes1. No viable colonies were observed when pGAD-Hes1 was transformed with an empty pGBK vector.

Figure 3

Figure 3. Hes1 and Ptf1-p48 interact directly in GST pull-down assays

(A) Schematic representation of GST–Ptf1-p48 fusion proteins utilized in the GST pull-down assays. (B) Coomassie-Blue-stained SDS/15%PAGE gel showing purified GST–Ptf1-p48 fusion proteins (arrowheads), confirming equal loading of proteins on glutathione beads. (C) Retention of 35S-labelled in vitro transcribed and translated Hes1 on glutathione beads pre-loaded with full-length GST–Ptf1-p48, as detected by PhosphorImager detection following resolution on SDS/10% PAGE gel. Truncated versions of Ptf1-p48 involving GST fused to either N-terminal alone [GST–Ptf1(N)], N-terminal + bHLH [GST–Ptf1(N+bHLH)], C-terminal alone [GST–Pft1(C)], or C-terminal + bHLH [GST–Pft1(C+bHLH)] do not retain 35S-labelled Hes1 at a level exceeding that by GST alone. The input lane was loaded with one tenth of the total 35S-labelled Hes1 used in each pull-down assay.

Figure 4

Figure 4. Hes1 binding to Ptf1-p48 correlates with functional inhibition of the PTF1 transcriptional complex

(A) Schematic representation of in vitro transcribed and translated 35S-labelled Hes1 proteins used in the GST pull-down assays with full-length GST–Ptf1-p48. (B) Retention of full-length and truncated versions of 35S-labelled Hes1 by GST–Ptf1-p48 and GST alone, as determined by Phosphorimager analysis following resolution on SDS/15% PAGE gel. The input lanes were loaded with one tenth of the total 35S-labelled Hes1 used in each pull-down assay. Full-length Hes1, Hes1 (bHLH+O+C) and Hes1 (bHLH+O+C+W) were retained by GST–Ptf1-p48, whereas versions of Hes1 lacking either the C-terminal [Hes1 (N+bHLH)] or bHLH [Hes1 (O+C+W)] domains were not retained. (C) Corresponding effects of full-length and truncated versions of Hes1 on the activity of the PTF1 transcriptional complex were assessed by activation of PTF-responsive luciferase reporter (PTF1-luc). Maximal inhibition of PTF1 activity was achieved by full-length Hes1, Hes1 (bHLH+O+C), and Hes1 (bHLH+O+C+W), whereas versions of Hes1 unable to bind Ptf1-p48 had a lesser effect.

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