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PreprintCited by 242Genomics and Chromatin Dynamicsinterferon and immune responsesT-cell and B-cell Immunology

Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1.

Open access
Brass, A L, Kehrli, E, Eisenbeis, C F, Storb, U, Singh, H·1996·Genes & Development
DOI 10.1101/gad.10.18.2335
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Abstract

Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.1. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses alpha/beta-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of alpha/beta-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.1-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.

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  • A C-terminal regulatory domain in Pip is required for ternary complex assembly.

    “A C-terminal regulatory domain within Pip itself is also required for ternary complex assembly (Brass et al., 1996).”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Brass et al., 1996

    The deletion analysis reports that Pip amino acids 410–439 are necessary for high-affinity ternary complex formation with PU.1. However, mutants lacking that segment still formed weak ternary complexes, so the paper does not show that the C-terminal domain is required for assembly altogether. The relevant passages are in the section on Pip repression of IFN-inducible transcription; page numbers are unavailable in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Pip deletion mutants 1-439 and 1-419 retained ternary complex formation with PU.1 and stimulated transcription comparably to full-length Pip.

    “Pip deletion mutants which retained the ability to form a ternary complex with PU.1 (1-439 and 1-419; Brass et al., 1996; data not shown) stimulated transcription comparable with full-length Pip.”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Brass et al., 1996

    In the section on Pip amino acids 410–439 (page not supplied), Brass et al. report that deleting Pip to residue 439 did not affect ternary complex formation with PU.1. Our copy does not report the 1–419 mutant or show that these two mutants stimulated transcription comparably to full-length Pip; the citing sentence labels the transcription result “data not shown.” (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Full-length Pip cannot bind DNA without PU.1, whereas the C-terminal deletion mutant Pip 1-410 can bind DNA independently.

    “As shown previously, full-length Pip cannot bind to DNA in the absence of PU.1; however, a C-terminal deletion mutant, Pip 1-410, can bind DNA independently (Figure 2A, compare lanes 2 and 3; Brass et al.,1996).”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Brass et al., 1996

    The deletion-analysis section reports that full-length Pip did not bind DNA independently in its gel-shift assay, while Pip 1–410 did. But the ‘Pip functions as a dichotomous regulator’ section says full-length Pip could bind DNA without PU.1 in cell extracts under different conditions, so the unqualified ‘cannot’ overstates the result. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • A predicted α-helix in Pip's C-terminus (aa 395-413) is critical for ternary complex formation and autoinhibition of DNA binding.

    “we identified a predicted α-helix within Pip's C-terminus (amino acids 395-413) that is critical for both formation of a ternary complex and autoinhibition of DNA binding”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Brass et al., 1996

    The Structural basis section postulates that a putative α-helical region may play a key role in ternary complex formation and autoinhibition; the Model section identifies that region as amino acids 399–413, not 395–413. The paper’s deletion results establish the importance of a broader segment, amino acids 410–439, but do not establish that the predicted helix itself is critical. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Other uncharacterized helices in Pip's region 150-340 may pack with helix 395-413 to form a fold competent for PU.1 association.

    “it appears that other, as yet uncharacterized helices within this region may pack together with Pip's helix amino acids 395-413 to form a tertiary fold which is competent for PU.1 association.”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Brass et al., 1996

    In the deletion-analysis passage (page not available), removing Pip residues 150–340 weakened ternary complex formation with PU.1; the authors suggest that region may position the carboxy-terminal domain or contain another required region. In “Structural basis for PU.1-dependent DNA-binding of Pip” (page not available), they propose a role for a putative helix near residues 399–413. Our copy does not say that uncharacterized helices in residues 150–340 pack with it to form a tertiary fold. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

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Cites

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Research works printed but not linked: 1

  1. McKnight, S. and Y. Yamamoto. 1992. Transcriptional regula- tion. Cold Spring Harbor Laboratory Press, Cold Spring Har- bor, NY.

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Related

Authors
Brass, A. L., Kehrli, E., Eisenbeis, C. F., Storb, U., & Singh, H.
Affiliations
Howard Hughes Medical Institute (US)
ORCID
Singh, H
Venue
Genes & Development
Publisher
Cold Spring Harbor Laboratory
Published
1996-09-15
Volume
10
Issue
18
Pages
2335-2347
ISSN
  • 0890-9369 (ISSN-L)
  • 1549-5477
DOI
10.1101/gad.10.18.2335
OpenAlex
W2005606581
Cited by
242
References
49
Citations by year
2026: 4 · 2025: 8 · 2024: 4 · 2023: 3 · 2022: 5 · 2021: 5 · 2020: 6 · 2019: 7 · 2018: 4 · 2017: 6 · 2016: 4 · 2015: 6 · 2014: 7 · 2013: 15 · 2012: 11
Type
posted content
Language
en
Open access
Locations
  • Genes & Development (publishedVersion)
  • PubMed (publishedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • York University
  • National Institutes of Health
  • U.S. Public Health Service (FM07183)
  • National Institute of General Medical Sciences (T32GM007281, GM-07281)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD-07009)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
Pip, interferon regulatory factors, PU.1, ETS transcription factors, ternary complex formation, autoinhibition, B cell activation, transcriptional repression, p48, DNA-binding domain
MeSH terms
Interferon Regulatory Factor-8, Interferon Regulatory Factor-4, Proto-Oncogene Protein Spi-1, Animals, B-Lymphocytes, Binding Sites, Carrier Proteins, DNA-Binding Proteins, Gene Expression Regulation, Interferon-gamma, Models, Molecular, Nucleic Acid Conformation, Protein Conformation, Proto-Oncogene Proteins, Regulatory Sequences, Nucleic Acid, Repressor Proteins, Transcription Factors, Transcription, Genetic, Trans-Activators, Interferon-alpha, Interferon-Stimulated Gene Factor 3, Interferon-Stimulated Gene Factor 3, gamma Subunit, Interferon Regulatory Factors, Mice

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