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Cited by 38Genomics and Chromatin DynamicsEpigenetics and DNA MethylationHistone Deacetylase Inhibitors Research

Protein Acetylation Regulates Both PU.1 Transactivation and Igκ 3′ Enhancer Activity

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Bai, Yuchen, Srinivasan, Lakshmi, Perkins, Leslie, Atchison, Michael L·2005·The Journal of Immunology
DOI 10.4049/jimmunol.175.8.5160
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Abstract

Igκ gene expression and chromatin structure change during B cell development. At the pre-B cell stage, the locus is relatively hypoacetylated on histone H3, whereas it is hyperacetylated at the plasma cell stage. We find in this study that the histone deacetylase inhibitor, trichostatin A (TSA) stimulated 3′ enhancer activity through the PU.1 binding site. TSA also stimulated PU.1 transactivation potential. PU.1 activity was increased by the coactivator acetyltransferase protein, p300, and p300 physically interacted with PU.1 residues 7–30. PU.1 served as a substrate for p300 and was acetylated on lysine residues 170, 171, 206, and 208. Mutation of PU.1 lysines 170 and 171 did not affect PU.1 DNA binding, but did lower the ability of PU.1 to activate transcription in association with p300. Lysine 170 was acetylated in pre-B cells and plasmacytoma cells, but TSA treatment did not stimulate PU.1 acetylation at this residue arguing that a second mechanism can stimulate 3′ enhancer activity. Using chromatin immunoprecipitation assays we found that TSA caused preferential acetylation of histone H3 at the 3′ enhancer. The relevance of these studies for PU.1 function in transcription and hemopoietic development is discussed.

Integrity signals

Registry

Crossref · 10.4049/jimmunol.175.8.5160

Cites

59 of 59 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 16 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § TSA induces 3Ј enhancer activity and PU.1 transactivation

    TSA treatment raises PU.1 transcriptional activity in NIH-3T3 cells expressing PU.1.

    “TSA treatment resulted in a very dramatic increase in PU.1 transcriptional activity (10-to 20-fold; Fig. 1B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: TSA specifically inhibits histone deacetylases, and its effects on enhancer activity and PU.1 transactivation are due to this inhibition.
  • Findingown data
    § PU.1 is acetylated in vivo

    PU.1 is acetylated on lysine 170 at both pre-B and plasma cell stages.

    “PU.1 was acetylated on lysine 170 at both developmental stages (Fig. 5B).”
    Quote in the paperSubstantial claim
  • Findingown data
    § PU.1 transactivation domain can physically interact with p300

    PU.1 and p300 interact in vivo.

    “Therefore, PU.1 and p300 can interact in vivo.”
    Quote in the paperSubstantial claim
  • Findingown data
    § PU.1 transactivation domain can physically interact with p300

    PU.1 residues 7-30 are required for physical interaction with p300.

    “Deletion of PU.1 residues 7-30 greatly reduced PU.1-p300 interaction, whereas deletion of PU.1 residues 33-100 had no effect”
    Quote in the paperSubstantial claim
  • Findingown data
    § PU.1 is a substrate for p300 acetylation

    p300 acetylates PU.1 in vitro.

    “p300 also acetylated GST-PU.1 nearly as efficiently as p53”
    Quote in the paperSubstantial claim
  • May combine two claims. 13
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 12

  • May combine two claims.

    Findingown data
    § Functional consequences of PU.1 acetylation

    PU.1 mutant K206,208R binds DNA poorly and is inefficient at recruiting Pip.

    “In contrast, mutant K206,208R bound to DNA poorly and was inefficient at recruiting Pip to DNA (lanes 5 and 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Introduction

    PU.1 is a substrate for p300 acetylation with acetylation sites at lysines 170, 171, 206, and 208.

    “We find that PU.1 physically interacts with p300 and is a substrate for p300 acetylation with acetylation sites at lysines 170, 171, 206, and 208.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: p300 is the acetyltransferase responsible for PU.1 acetylation in vivo.
    • Rests on an assumption: The acetylation of PU.1 at lysines 170, 171, 206, and 208 in vitro reflects the functionally relevant acetylation in vivo.

  • May combine two claims.

    Findingown data
    § Functional consequences of PU.1 acetylation

    PU.1 mutant K170,171R binds DNA and recruits Pip as efficiently as wild-type PU.1.

    “Mutant K170,171R bound to DNA and recruited Pip as efficiently as wildtype PU.1 (Fig. 6A, lanes 1-4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    The transactivation function of PU.1 is increased by either TSA treatment or cotransfection with p300.

    “First, the PU.1 transactivation function was increased by either inhibition of histone deacetylases via TSA treatment or cotransfection with acetyltransferase protein p300.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § TSA induces H3 acetylation preferentially at the 3Ј enhancer

    TSA increases H3 acetylation by about 6-fold at the intron enhancer and by about 3-fold at the constant region.

    “H3 acetylation increased ϳ6-fold at the intron enhancer and ϳ3-fold at the constant region (lanes 1, 2, 5, and 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Introduction

    PU.1 transactivation potential is increased in the presence of TSA or when cotransfected with p300.

    “The PU.1 transactivation potential was also increased in the presence of TSA or when cotransfected with the histone acetyltransferase (HAT) protein, p300.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: TSA specifically inhibits histone deacetylases, and its effects on enhancer activity and PU.1 transactivation are due to this inhibition.

  • May combine two claims.

    Interpretationown data
    § PU.1 is a substrate for p300 acetylation

    PU.1 lysines 170, 206, and 208 are major p300 acetylation target sites, and lysine 171 is a minor site.

    “The above results indicate that PU.1 lysine residues 170, 206, and 208 are major p300 acetylation target sites, and lysine 171 is a minor site.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: p300 is the acetyltransferase responsible for PU.1 acetylation in vivo.
    • Rests on an assumption: The acetylation of PU.1 at lysines 170, 171, 206, and 208 in vitro reflects the functionally relevant acetylation in vivo.

  • The quote only partly states this claim.

    Interpretationown data
    § TSA induces 3Ј enhancer activity and PU.1 transactivation

    Most of the TSA-driven rise in 3' enhancer activity depends on the PU.1/Pip composite element.

    “However, the activity of the PU.1/Pip reporter was very dramatically increased by TSA treatment (10-fold; Fig. 1, lanes 5 and 6).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: TSA specifically inhibits histone deacetylases, and its effects on enhancer activity and PU.1 transactivation are due to this inhibition.

  • May combine two claims.

    Interpretationown data
    § Introduction

    Either other PU.1 lysines are targeted or a second mechanism is needed for TSA induction of enhancer activity.

    “arguing that either other PU.1 lysines are targeted, or a second mechanism is needed for TSA induction of enhancer activity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: TSA specifically inhibits histone deacetylases, and its effects on enhancer activity and PU.1 transactivation are due to this inhibition.

  • Classified as probably not a substantial claim.

    Interpretationcitation
    § Discussion

    PU.1 may respond to TSA treatment by differentially recruiting coactivators or corepressors to the enhancer.

    “Because PU.1 DNA binding in vivo is not appreciably changed by TSA treatment (36), PU.1 may respond to TSA treatment by differentially recruiting coactivators or corepressors to the enhancer.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § FIGURE 7.

    Low PU.1 expression levels in progenitor cells generally give rise to B cell development, whereas higher levels yield macrophage development.

    “Low PU.1 expression levels in progenitor cells generally give rise to B cell development, whereas higher levels yield macrophage development (23).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] 2000

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Pax5 can repress Ig 3' enhancer function in cooperation with PU.1.

    “We similarly previously showed that Pax5 can repress Ig 3Ј enhancer function in cooperation with PU.1 (53).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] 2000

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Interpretationreasoning
    § Discussion

    Competition between Grg4 and p300 for interaction with PU.1 could constitute a molecular switch between repression and activation.

    “Because Pax5 and PU.1 can recruit the corepressor Grg4 to DNA (52), competition between Grg4 and p300 for interaction with PU.1 could constitute a molecular switch between repression and activation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Discussion

    PU.1 can inhibit CBP HAT activity in vivo, leading to changes in global acetylation patterns.

    “Interestingly, PU.1 can inhibit CBP HAT activity in vivo, leading to changes in global acetylation patterns (33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] 2002

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § FIGURE 7.

    Changing PU.1 activity by expression level or by function via protein interactions or post-translational modifications could have a dramatic impact on biological processes.

    “Thus, changing PU.1 activity by either expression level or, perhaps, its function via protein interactions or post-translational modifications could have a very dramatic impact on biological processes.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationown data
    § Functional consequences of PU.1 acetylation

    TSA induction of 3' enhancer activity involves a second acetylation mechanism.

    “However, our results are also con-sistent with the possibility that TSA induction of 3Ј enhancer activity involves a second acetylation mechanism.”
    • Also stated in: § FIGURE 7. · plain
      “Lysine 170 was acetylated in pre-B cells and plasmacytoma cells, but TSA treatment did not stimulate PU.1 acetylation at this residue arguing that a second mechanism can stimulate 3 enhancer activity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    CBP requires PU.1 sequences 74-122 for physical interaction.

    “Interaction with PU.1 residues 7-30 was somewhat surprising because the p300-related protein CBP requires PU.1 sequences 74 -122 for physical interaction (32).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] 1999

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Discussion

    Pax5 and PU.1 can recruit the corepressor Grg4 to DNA.

    “Because Pax5 and PU.1 can recruit the corepressor Grg4 to DNA (52), competition between Grg4 and p300 for interaction with PU.1 could constitute a molecular switch between repression and activation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] 2004

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § In vitro acetylation assays

    Gels were stained with Coomassie Blue to verify protein quantity.

    “Gels were stained with Coomassie Blue to verify protein quantity, then subjected to autoradiography to evaluate acetylation activity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning
    § Discussion

    p300 is likely first recruited via PU.1 residues 7-30 and then acetylates PU.1 on lysine residues 170, 171, 206, and 208.

    “It is likely that p300 is first recruited via PU.1 residues 7-30, and then p300 subsequently acetylates PU.1 on lysine residues 170, 171, 206, and 208.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning
    § Discussion

    Acetylation of PU.1 can increase PU.1 transactivation and consequently enhancer activity.

    “Thus, acetylation of PU.1 can increase PU.1 transactivation and, as a result, enhancer activity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Cell culture and transfections

    Transfections were performed three to five times.

    “In each case, transfections were performed three to five times.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Cell culture and transfections

    Transfection efficiencies were normalized using β-galactosidase activity.

    “Transfection efficiencies were normalized using ␤-galactosidase activity”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § FIGURE 7.

    Reducing PU.1 expression levels in vivo to 20% of wild-type levels leads to a high incidence of acute myeloid leukemia.

    “In addition, reducing PU.1 expression levels in vivo to 20% of wild-type levels leads to a high incidence of acute myeloid leukemia (31).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] 2004

      Not checked in the free run.

24 of 24

  • The paper relies on this without support.

    TSA specifically inhibits histone deacetylases, and its effects on enhancer activity and PU.1 transactivation are due to this inhibition.

    “TSA treatment was performed for 24 h at a concentration of 33 nM.”
    “an agent that inhibits histone deacetylase activity.”

  • The paper relies on this without support.

    p300 is the acetyltransferase responsible for PU.1 acetylation in vivo.

    “PU.1 physically interacts with p300 and is a substrate for p300 acetylation”
    “PU.1 is acetylated in vivo”

  • The paper relies on this without support.

    The acetylation of PU.1 at lysines 170, 171, 206, and 208 in vitro reflects the functionally relevant acetylation in vivo.

    “We find that PU.1 physically interacts with p300 and is a substrate for p300 acetylation with acetylation sites at lysines 170, 171, 206, and 208.”
    “PU.1 was acetylated on lysine 170 at both developmental stages (Fig. 5B).”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • TSA treatment duration differs between methods and Figure 1

    Major

    The methods specify 24 hours of TSA treatment, while the Figure 1 caption says the treated transfections received TSA for 18 hours.

  • Figure 6C sample type differs between results and caption

    Minor

    The results describe Western blots of whole-cell lysates for the Figure 6C experiment, but the caption says the samples were nuclear extracts.

  • Lysines 206 and 208 are classified as both weak and major sites

    Minor

    The same results section calls lysines 206 and 208 weak p300 acetylation sites, then includes them among the major target sites.

3 of 3

Cites

Flagged first, then by citations

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Cited by

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Authors
Bai, Y., Srinivasan, L., Perkins, L., & Atchison, M. L.
Affiliations
University of Pennsylvania (US)
ORCID
Venue
The Journal of Immunology
Publisher
Oxford University Press (OUP)
Published
2005-10
Volume
175
Issue
8
Pages
5160-5169
ISSN
  • 0022-1767 (ISSN-L)
  • 1550-6606
DOI
10.4049/jimmunol.175.8.5160
OpenAlex
W2135558550
Cited by
38
References
59
Citations by year
2026: 2 · 2023: 2 · 2022: 1 · 2021: 2 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 1 · 2016: 2 · 2015: 5 · 2014: 2 · 2013: 3 · 2012: 4
FWCI
0.46
Type
journal article
Language
en
Open access
Licence
academic.oup.com/pages/standard-publication-reuse-rights (vor)
Locations
  • The Journal of Immunology (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Histone Deacetylase Inhibitors Research
Keywords
protein acetylation, PU.1, P300, histone deacetylase inhibition, trichostatin A, histone H3 acetylation, transcriptional activation, pre-B cells
MeSH terms
p300-CBP-Associated Factor, Proto-Oncogene Protein Spi-1, Acetylation, Amino Acid Sequence, Animals, Enhancer Elements, Genetic, Histones, Hydroxamic Acids, Immunoglobulin kappa-Chains

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