Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Sep;21(17):6080-9.
doi: 10.1128/MCB.21.17.6080-6089.2001.

HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling

T Iso  1 , V Sartorelli, C Poizat, S Iezzi, H Y Wu, G Chung, L Kedes, Y Hamamori
Affiliations

HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling

T Iso et al. Mol Cell Biol. 2001 Sep.

Abstract

HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding-but modified-tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Alignment of HERP1, HERP2, and HES1 amino acid sequences. (A) Schematic diagram of mouse HES1, HERP1, and HERP2 amino acid sequences. The values are the percentages of protein sequence similarity in the bHLH domain, the Orange domain, and a region between the bHLH and Orange domains. Note that the HERP1 tetrapeptide is YQPW in mice and YRPW in humans. (B and C) Amino acid sequences of the basic domain (B) and the carboxyl terminus including the tetrapeptide motif (C) from mouse HES1, HERP1, and HERP2 and Drosophila Hesr are aligned by using ClustalW. Identical amino acids are in black, and conserved residues are in gray. An arrowhead indicates the invariant amino acid residues in the basic domain of HERP1, HERP2, and Drosophila Hesr (glycine) and HES1 (proline). Asterisks indicate the tetrapeptide motifs.
FIG. 2
FIG. 2
(A and B) Both HERP1 (A) and HERP2 (B) are transcriptional repressors. C3H10T1/2 cells were transfected with UAS-tk-luc reporter constructs (2 μg) and the indicated GAL fusion expression vectors. The means of luciferase activities with standard deviations are shown. (C and D) The bHLH domain mediates the repressor activity of HERP1. C3H10T1/2 cells were transfected with UAS-tk-luc reporter constructs (2 μg) and the indicated GAL fusion expression vectors (1 μg). (C) Fold repression was calculated as a ratio of reduction in luciferase expression mediated by GAL-HERP1 fusion constructs relative to the empty GAL4-DBD. (D) Repressor activity is expressed as a percentage of fold repression with full-length HERP1 or HES1 set at 100% and GAL4-DBD alone set at 0%. Repressor activity is directly compared between HERP1 and HES1. All the GAL fusion constructs showed equal expression levels as judged by Western blot analysis (data not shown).
FIG. 3
FIG. 3
HERP1 forms a complex with mSin3A, N-CoR, and HDAC. (A) HERP1 associates with mSin3A. Cells were transfected with the myc-mSin3A vector as well as with either pcDNA3.1(−)-Flag-HERP1 or pcDNA3.1(−) empty vector. The extracts from the transfected cells were incubated with an equal amount of anti-Flag M2 antibody or control normal mouse IgG. Bound proteins were separated by SDS-PAGE followed by Western blot analysis with anti-myc antibody. (B) HERP1 associates with the corepressor, N-CoR. 293T cells were transfected with pCEP4 Flag-N-CoR or pcDNA3.1(−) empty vector plus pcDNA3.1(−) HA-HERP1. The interaction was studied as described above using anti-HA antibody. (C) HERP1 associates with HDAC1. Cells were transfected with pcDNA3-HDAC1-cFlag or pcDNA3 empty vector plus pcDNA3.1(−) HA-HERP1. The
FIG. 3
FIG. 3
HERP1 forms a complex with mSin3A, N-CoR, and HDAC. (A) HERP1 associates with mSin3A. Cells were transfected with the myc-mSin3A vector as well as with either pcDNA3.1(−)-Flag-HERP1 or pcDNA3.1(−) empty vector. The extracts from the transfected cells were incubated with an equal amount of anti-Flag M2 antibody or control normal mouse IgG. Bound proteins were separated by SDS-PAGE followed by Western blot analysis with anti-myc antibody. (B) HERP1 associates with the corepressor, N-CoR. 293T cells were transfected with pCEP4 Flag-N-CoR or pcDNA3.1(−) empty vector plus pcDNA3.1(−) HA-HERP1. The interaction was studied as described above using anti-HA antibody. (C) HERP1 associates with HDAC1. Cells were transfected with pcDNA3-HDAC1-cFlag or pcDNA3 empty vector plus pcDNA3.1(−) HA-HERP1. The
FIG. 4
FIG. 4
DNA binding properties of HES and HERP. (A) HES1 and HERP1 show distinct DNA binding preferences. Electrophoretic mobility shift assays were performed using various DNA probes. Autoradiographs after short and long exposures are shown. Top-strand oligonucleotide sequences used in this assay are indicated (bottom panel). Consensus binding elements are underlined. Sequences of classes A and B are derived from those described previously (24). The C-1, C-2, C-3, and C-4 oligonucleotide sequences are from the regulatory region of the known HES/E(spl) target genes; achaete (41), E(spl) genes (45), hASH (9), and HES1 itself (46), respectively. Radioactivities of each band were measured by a PhosphorImager with ImageQuant software to assess the relative binding activities shown in the bottom panel. The radiolabeling efficiency of the probes was comparable among the different probes, and excess amounts of the free probes were confirmed in all lanes in a parallel experiment with the same results (data not shown). (B) HES1 and HERP1 form a heterodimer. HES1 and HERP1 proteins were either individually translated (lanes 2 to 7, 12 to 17, 22 to 27, 32 to 37, and 42 to 47) or cotranslated (lanes 8 to 10, 18 to 20, 28 to 30, 38 to 40, and 48 to 50). In vitro-translated proteins prepared in parallel using [35S]methionine are shown to verify equal protein product (lanes 42 to 50). (C) A gel shift assay was performed using either in vitro-translated proteins (lanes 1 to 5) or nuclear extract from the cells transfected with the expression vectors for HERP1 and HES1 (lanes 6 to 17). These proteins were incubated with the DNA probe C-1 at room temperature for 20 min, and the indicated antibodies (lanes 4, 5, 7, 9, 11 to 13, and 15 to 17) were added to the mixture, followed by additional incubation for 30 min on ice. DNA-protein complexes were resolved by electrophoresis on a 5% acrylamide gel. Note that nuclear extracts from the cells expressing both HES1 and HERP1 showed a dramatic increase of DNA binding activity. Symbols: thin arrow, nonspecific band; thin open arrow, supershifted homodimer band; thick open arrow, heterodimer band; thick solid arrow, supershifted heterodimer band. The amount of HES1 protein in the nuclear extract from the cotransfected cells (lanes 10 and 14) was half of that from singly transfected cells (lane 6), as confirmed by Western blot analysis, and the same was true for HERP1 (data not shown).
FIG. 4
FIG. 4
DNA binding properties of HES and HERP. (A) HES1 and HERP1 show distinct DNA binding preferences. Electrophoretic mobility shift assays were performed using various DNA probes. Autoradiographs after short and long exposures are shown. Top-strand oligonucleotide sequences used in this assay are indicated (bottom panel). Consensus binding elements are underlined. Sequences of classes A and B are derived from those described previously (24). The C-1, C-2, C-3, and C-4 oligonucleotide sequences are from the regulatory region of the known HES/E(spl) target genes; achaete (41), E(spl) genes (45), hASH (9), and HES1 itself (46), respectively. Radioactivities of each band were measured by a PhosphorImager with ImageQuant software to assess the relative binding activities shown in the bottom panel. The radiolabeling efficiency of the probes was comparable among the different probes, and excess amounts of the free probes were confirmed in all lanes in a parallel experiment with the same results (data not shown). (B) HES1 and HERP1 form a heterodimer. HES1 and HERP1 proteins were either individually translated (lanes 2 to 7, 12 to 17, 22 to 27, 32 to 37, and 42 to 47) or cotranslated (lanes 8 to 10, 18 to 20, 28 to 30, 38 to 40, and 48 to 50). In vitro-translated proteins prepared in parallel using [35S]methionine are shown to verify equal protein product (lanes 42 to 50). (C) A gel shift assay was performed using either in vitro-translated proteins (lanes 1 to 5) or nuclear extract from the cells transfected with the expression vectors for HERP1 and HES1 (lanes 6 to 17). These proteins were incubated with the DNA probe C-1 at room temperature for 20 min, and the indicated antibodies (lanes 4, 5, 7, 9, 11 to 13, and 15 to 17) were added to the mixture, followed by additional incubation for 30 min on ice. DNA-protein complexes were resolved by electrophoresis on a 5% acrylamide gel. Note that nuclear extracts from the cells expressing both HES1 and HERP1 showed a dramatic increase of DNA binding activity. Symbols: thin arrow, nonspecific band; thin open arrow, supershifted homodimer band; thick open arrow, heterodimer band; thick solid arrow, supershifted heterodimer band. The amount of HES1 protein in the nuclear extract from the cotransfected cells (lanes 10 and 14) was half of that from singly transfected cells (lane 6), as confirmed by Western blot analysis, and the same was true for HERP1 (data not shown).
FIG. 4
FIG. 4
DNA binding properties of HES and HERP. (A) HES1 and HERP1 show distinct DNA binding preferences. Electrophoretic mobility shift assays were performed using various DNA probes. Autoradiographs after short and long exposures are shown. Top-strand oligonucleotide sequences used in this assay are indicated (bottom panel). Consensus binding elements are underlined. Sequences of classes A and B are derived from those described previously (24). The C-1, C-2, C-3, and C-4 oligonucleotide sequences are from the regulatory region of the known HES/E(spl) target genes; achaete (41), E(spl) genes (45), hASH (9), and HES1 itself (46), respectively. Radioactivities of each band were measured by a PhosphorImager with ImageQuant software to assess the relative binding activities shown in the bottom panel. The radiolabeling efficiency of the probes was comparable among the different probes, and excess amounts of the free probes were confirmed in all lanes in a parallel experiment with the same results (data not shown). (B) HES1 and HERP1 form a heterodimer. HES1 and HERP1 proteins were either individually translated (lanes 2 to 7, 12 to 17, 22 to 27, 32 to 37, and 42 to 47) or cotranslated (lanes 8 to 10, 18 to 20, 28 to 30, 38 to 40, and 48 to 50). In vitro-translated proteins prepared in parallel using [35S]methionine are shown to verify equal protein product (lanes 42 to 50). (C) A gel shift assay was performed using either in vitro-translated proteins (lanes 1 to 5) or nuclear extract from the cells transfected with the expression vectors for HERP1 and HES1 (lanes 6 to 17). These proteins were incubated with the DNA probe C-1 at room temperature for 20 min, and the indicated antibodies (lanes 4, 5, 7, 9, 11 to 13, and 15 to 17) were added to the mixture, followed by additional incubation for 30 min on ice. DNA-protein complexes were resolved by electrophoresis on a 5% acrylamide gel. Note that nuclear extracts from the cells expressing both HES1 and HERP1 showed a dramatic increase of DNA binding activity. Symbols: thin arrow, nonspecific band; thin open arrow, supershifted homodimer band; thick open arrow, heterodimer band; thick solid arrow, supershifted heterodimer band. The amount of HES1 protein in the nuclear extract from the cotransfected cells (lanes 10 and 14) was half of that from singly transfected cells (lane 6), as confirmed by Western blot analysis, and the same was true for HERP1 (data not shown).
FIG. 5
FIG. 5
HERP1 and HES1 associate in solution. (A) HERP1 directly associates with HES1 in the absence of DNA in vitro. In vitro protein-protein interaction assays were carried out as described for Fig. 3E. (B) HERP1 associates with HES1 in cells. 293T cells were transfected with pcDNA3.1(−)-Flag-HERP1 or pcDNA3.1(−) empty vector plus pc3Gal-HES1. The extracts from the transfected cells were incubated with an equal amount of anti-Flag M2 antibody or normal mouse IgG. Bound proteins were separated by SDS-PAGE followed by Western blot analysis with anti-GAL4-DBD antibody. Ip, immunoprecipitation; Ab, antibody. Numbers to the right of the gels are molecular masses in kilodaltons. interaction was studied as described above using anti-HA antibody. (D) Both Flag-HERP1 and -HERP2 associate with endogenous HDAC1. Cells were transfected with either pcDNA3.1(−)-Flag empty, pcDNA3.1(−)-Flag-HERP1, or pcDNA3.1(−)-Flag-HERP2. The interaction was studied as described above using anti-HDAC1 antibody. (E) HERP1 interacts with mSin3A via its bHLH domain. In vitro-translated 35S-myc-mSin3A was incubated with equal amounts of the indicated GST-HERP1 fusion proteins for 1 h at 4°C. Bound proteins were analyzed by autoradiography after SDS-PAGE. (F) HERP1 interacts with N-CoR via its bHLH domain. Assays were performed as described above using 35S-N-CoR. (G) Summary of the domain mapping studies. Ip, immunoprecipitation; Ab, antibody. Numbers to the right of the gels are molecular masses in kilodaltons.
FIG. 6
FIG. 6
HERP1 and HES1 synergistically repress transcription. C3H10T1/2 cells were transfected with a reporter plasmid containing multimerized C-1 sites (C1×4-βactin-luc), together with expression vectors for HES1 and HERP1. The data are means of duplicate data from three independent experiments with similar results.
FIG. 7
FIG. 7
Model for HES and HERP cooperation in Notch signaling. Upon Notch stimulation, HES and HERP expression might both be induced. In tissues where only HES or HERP is expressed, the respective homodimer binds promoters of target genes. The HES homodimers recruit TLE via their WRPW motif, whereas the HERP homodimers recruit the mSin3A–HDAC–N-CoR complex via their bHLH domain. In tissues where both HES and HERP are coexpressed, the HES-HERP heterodimers become the predominant complex that binds a specific DNA site newly defined by the basic domains of HES and HERP. Because of the higher DNA binding affinity of the heterodimers, a lower concentration of them may be sufficient to achieve repression. Repression by HES-HERP heterodimers may be reinforced by their ability to recruit a more diverse set of repressors. The model is based on the experimental data largely from HES1 and HERP1. Because of the significant similarity among members of each family, however, such a model may be apt for other members including those in humans, mouse, and Drosophila.
See this image and copyright information in PMC

References

    1. Alland L, Muhle R, Hou H, Jr, Potes J, Chin L, Schreiber-Agus N, DePinho R A. Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature. 1997;387:49–55. - PubMed
    1. Andrews N C, Faller D V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. - PMC - PubMed
    1. Apelqvist A, Li H, Sommer L, Beatus P, Anderson D J, Honjo T, Hrabe de Angelis M, Lendahl U, Edlund H. Notch signalling controls pancreatic cell differentiation. Nature. 1999;400:877–881. - PubMed
    1. Artavanis-Tsakonas S, Rand M D, Lake R J. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–776. - PubMed
    1. Ayer D E. Histone deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol. 1999;9:193–198. - PubMed

Publication types

MeSH terms

Substances