Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers

Ectopic co-expression of PU.1 and Pip synergistically activates a reporter containing multiple copies of the IgL lambda composite element E lambda 2-4.

“When co-expressed ectopically, PU.1 and Pip synergistically activate transcription of a reporter gene containing multiple copies of the composite element from the IgL λ enhancer, E λ2-4 (Eisenbeis et al., 1995).”

• cites Eisenbeis et al., 1995 sourceSupported

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).”

• cites Brass et al., 1996 sourcePartly supported

  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's regulatory domain autoinhibits Pip DNA binding in the absence of PU.1.

“In addition, this regulatory domain autoinhibits binding of Pip to DNA in the absence of PU.1.”

• cites Brass et al., 1996 sourceSupported

A putative alpha-helical region in Pip's C-terminus (amino acids 399-413) was previously suggested to be important for ternary complex formation with PU.1.

“Previously, using deletion analysis and secondary structure predictions, we suggested that a putative α-helical region (amino acids 399-413) in Pip's C-terminus is important for ternary complex formation with PU.1 (Figure 1A and B; Brass et al., 1996).”

• cites Brass et al., 1996 sourceSupported

PU.1 and Pip are major components of the ternary complex formed with nuclear extract from J558L cells.

“PU.1 and Pip represent the major components of the ternary complex formed with nuclear extract from these cells”

• cites Brass et al., 1996 sourceSupported

On lower-affinity sites, the linker's chelating effect may lower entropic cost, allowing PipPU to bind as efficiently as wild-type proteins.

“On lower affinity sites, however, the chelating effect provided by the linker may lower the overall entropic cost associated with binding, allowing PipPU to bind as efficiently as the wildtype proteins”

• cites Klemm and Pabo, 1996 sourcePartly supported

  The “Contribution of the linker to cooperative binding” section (page unavailable) says the linker produces a chelate effect and reduces the entropic cost of complex formation. The paper studies Oct-1 POU-domain binding; it does not establish that PipPU binds lower-affinity sites as efficiently as wild-type proteins. (LLM, conf=0.87)

  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.”

• cites Brass et al., 1996 sourcePartly supported

  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

J chain inhibition is presumably due to displacement of endogenous PU.1 from an isolated Ets site in the J chain promoter.

“This is presumably due to displacement of endogenous PU.1 from an isolated Ets site in the J chain promoter”

• cites Shin and Koshland, 1993 sourceSupported

This is consistent with increased Pip expression in plasma cell lines such as J558L.

“This is consistent with increased expression of Pip in plasma cell lines such as J558L”

• cites Brass et al., 1996 sourceSupported

Each enhancer contains an essential composite site (λB) bound by PU.1 and Pip.

“Each enhancer contains an essential composite site (λB) bound by PU.1 and Pip”

• cites Hagman et al., 1990 sourceCould not verify

  The Abstract identifies two lambda enhancers, and “Cloning of a second enhancer and comparison with” discusses conserved sequence motifs. Neither passage identifies an essential λB composite site or reports binding by PU.1 and Pip; searches for those terms found no supporting passage in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites Rudin and Storb 1992Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Eisenbeis et al., 1993 sourceNot checked — no full text of the source

Full-length Pip's affinity for the PU.1-DNA complex is more than 800-fold greater than that of the Pip DBD (2.6 nM versus 2.1 micromolar).

“However, the affinity of full-length Pip for the PU.1-DNA complex is Ͼ800-fold greater than that observed for the Pip DBD (2.6 nM versus 2.1 µM).”

The isolated Pip DBD has intrinsic weak DNA affinity with Kd about 96 micromolar.

“Unlike full-length Pip, the isolated Pip DBD possessed intrinsic, albeit weak, DNA affinity (K d ~96 µM, compare Figures 3B and 4A), consistent with removal of the autoinhibitory domain.”

PU.1-Pip interaction involves the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip.

“PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative α-helix in Pip.”

PU.1-Pip interaction involves the ets and IRF DNA-binding domains.

“PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative α-helix in Pip.”

Glutaraldehyde cross-linking detected a Pip-containing adduct of about 80-85 kDa, consistent with a Pip-PU.1 heterodimer.

“A glutaraldehyde-dependent adduct containing Pip was detected, with a molecular mass of ~80-85 kDa, in excellent agreement with the predicted size of a crosslinked species comprising a heterodimer of Pip and PU.1 983 (Figure 5B, compare lanes 1 and 2).”

Chimeric proteins containing two or three copies of the linker bound to the lambdaB site with highest affinity.

“Chimeric proteins containing either two or three copies of the linker bound to the λB site with highest affinity.”

Alanine substitution of Pip amino acids 397-400 (PipAS397) impaired ternary complex formation to about 1% of wild-type binding.

“The alanine substitution of amino acids 397-400 (PipAS397) and 401-404 (PipAS401) within the predicted helix impaired ternary complex formation, resulting in ~1 or 26% binding, respectively, as compared with wild-type Pip (Figure 1D, compare lane 3 with lanes 6 and 7).”

Substitution of arginine for lysine at position 399 retained 40% of wild-type binding.

“A substantial retention of wild-type binding (40%) was seen after substitution of an arginine for lysine at that position (Figure 1E, compare lanes 9 and 3).”

Pip's DNA-binding activity is completely dependent on PU.1 at all protein concentrations tested.

“We observed that the DNA-binding activity of Pip was completely dependent on the presence of PU.1 at all protein concentrations tested (Figure 3B and C).”

Formation of the PU.1-Pip protein adduct depends on lambdaB DNA containing intact PU.1- and Pip-binding sites.

“Formation of the PU.1-Pip protein adduct was dependent on: (i) phosphorylation of Ser148 in PU.1; (ii) Lys399 in Pip's interaction helix; and (iii) λB DNA containing intact PU.1-and Pip-binding sites (Figure 5B, lanes 3-8, probes designated B1 and B2 contain severe mutations in either the PU.1-or Pip-binding sites; detailed in Figure 6B).”

Alanine substitution of Pip amino acids 389-392 (PipAS389) impaired ternary complex formation to 17% of wild-type binding.

“Furthermore, the region immediately preceding the helix, amino acids 389-392 (PipAS389), was also important for complex formation (17% of wild-type Pip binding; Figure 1D, lanes 3 and 4).”

IRF-1 DBD bound the lambdaB site as both a monomer and a dimer.

“(ii) the IRF-1 DBD bound the λB site as both a monomer and a dimer;”

The increasing ability of PipAS401, PipAS405, and PipAS409 to stimulate transcription correlated with increasing ternary complex formation.

“The increasing ability of the PipAS401, 405 and 409 proteins to stimulate transcription correlated with increasing propensity for ternary complex formation (Figure 1D; Table I).”

PipAS397 was least effective among the alanine substitution mutants in stimulating transcription.

“Consistent with the biochemical analysis, PipAS397 was least effective in stimulating transcription (Table I).”

Alanine substitution of Pip amino acids 401-404 (PipAS401) impaired ternary complex formation to 26% of wild-type binding.

“The alanine substitution of amino acids 397-400 (PipAS397) and 401-404 (PipAS401) within the predicted helix impaired ternary complex formation, resulting in ~1 or 26% binding, respectively, as compared with wild-type Pip (Figure 1D, compare lane 3 with lanes 6 and 7).”

The isolated ets domain of PU.1 (amino acids 160-272) recruited Pip DBD to lambdaB comparably to PU.1 containing the PEST region (amino acids 119-272).

“Furthermore, similar experiments revealed that the isolated ets domain of PU.1 (amino acids 160-272) recruited the Pip DBD to the λB site comparably with the PU.1 protein containing the PEST region (amino acids 119-272, Figure 4C).”

The cross-linked protein adduct also contained PU.1.

“Western blotting using anti-PU.1 antisera showed that this protein adduct also contained PU.1 (data not shown).”

Formation of the PU.1-Pip protein adduct depends on Lys399 in Pip's interaction helix.

“Formation of the PU.1-Pip protein adduct was dependent on: (i) phosphorylation of Ser148 in PU.1; (ii) Lys399 in Pip's interaction helix; and (iii) λB DNA containing intact PU.1-and Pip-binding sites (Figure 5B, lanes 3-8, probes designated B1 and B2 contain severe mutations in either the PU.1-or Pip-binding sites; detailed in Figure 6B).”

PU.1 and full-length Pip synergistically activated transcription 17-fold in NIH 3T3 cells with the B4-TKCAT reporter.

“Consistent with previous data, PU.1 and Pip (FLPip) synergistically activated transcription (17-fold).”

No changes in the rate or pattern of proteolysis were detected when Pip was recruited by PU.1 on lambdaB DNA.

“No changes in the rate or pattern of proteolysis were detected when Pip was recruited by PU.1 on λB DNA (Figure 3A, compare lanes 1-6 with lanes 7-12).”

Formation of the PU.1-Pip protein adduct depends on phosphorylation of Ser148 in PU.1.

“Formation of the PU.1-Pip protein adduct was dependent on: (i) phosphorylation of Ser148 in PU.1; (ii) Lys399 in Pip's interaction helix; and (iii) λB DNA containing intact PU.1-and Pip-binding sites (Figure 5B, lanes 3-8, probes designated B1 and B2 contain severe mutations in either the PU.1-or Pip-binding sites; detailed in Figure 6B).”

IRF-1 DBD has higher DNA affinity than the Pip DBD.

“These data demonstrated: (i) the high DNA affinity of IRF-1's DBD compared with that of the Pip DBD (compare Figure 4D with A, taking note of protein concentrations);”

PU.1-Pip interaction is template directed.

“PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative α-helix in Pip.”

PipE406P severely reduced interaction with PU.1 to 2% of wild-type levels.

“PipE406P showed severely reduced interaction with PU.1 (2% of wild-type levels; Figure 1E, compare lane 10 with lane 3).”

Alanine substitution mutagenesis of residues 389-420 showed that amino acids 401-408 are specifically required for autoinhibition.

“Alanine substitution mutagenesis of residues 389-420 showed that amino acids 401-408 were specifically required for autoinhibition (Figure 2A, compare lane 2 with lanes 7 and 8).”

Increasing concentrations of IRF-1 DBD displaced either PU.1 or PU.1 DBD from the lambdaB site.

“(iii) increasing concentrations of the IRF-1 DBD displaced either PU.1 or PU.1 DBD from the λB site.”

Mutation of E406 to proline generated a protein that bound DNA independently 32-fold above background.

“Mutation of E406 to proline also generated a protein which bound DNA independently (32-fold above background), suggesting that overall helix integrity is necessary for both autoinhibition and ternary complex formation (Figure 2A, compare lanes 2 and 12).”

Pip binding depends on pSer148, as shown by the phosphorylation-defective PU.1S148A mutant.

“In addition, gel shift assays using the phosphorylation-defective PU.1 mutant, PU.1S148A, showed that the binding of Pip was dependent on the presence of pSer148 (data not shown).”

The interaction between isolated Pip DBD and PU.1 does not depend on Ser148 phosphorylation.

“We note that the interaction between the isolated DBD of Pip and PU.1 is not dependent on phosphorylation of Ser148 since the phosphorylation-deficient PU.1S148A interacted with the Pip DBD equivalently to PU.1 (data not shown).”

Substitution of Lys399 by alanine reduced ternary complex assembly to 8% of wild-type.

“This experiment showed that Lys399 was important for ternary complex formation, as its substitution by alanine resulted in 8% of wild-type complex assembly (Figure 1E, lane 7).”

The equilibrium dissociation constant of Pip in the presence of PU.1 is about 2.6 nM.

“The data from gel shift assays allowed us to calculate the equilibrium dissociation constant (K d ) of Pip in the presence of PU.1 to be ~2.6 nM (Figure 3D).”

Pip DBD bound the lambdaB template with higher affinity in the presence of PU.1, Kd about 2.1 micromolar.

“Pip DBD bound the λB template with higher affinity in the presence of PU.1, K d ~2.1 µM (Figure 4A and B).”

Mutation of Glu406 to alanine had no effect on ternary complex formation.

“Importantly, mutation of this Glu406 to an alanine had no effect on ternary complex formation, indicating that the nature of the side chain was not critical (data not shown).”

Alanine substitutions of Arg398 and Leu400 diminished complexation to 45% or 31% of wild-type binding.

“Alanine subsitutions of Arg398 and Leu400 also diminished complexation (45 or 31% of wild-type binding levels; Figure 1E, compare lane 3 with lanes 6 and 8).”

Comparative analysis of the wild-type dimer with mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone.

“Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone.”

When stably expressed, the wild-type fused dimer strongly repressed expression of a rearranged immunoglobulin lambda gene.

“When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin λ gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression.”

On the CD20 element, PipPU bound with affinity comparable to PU.1 and Pip.

“On the CD20 element, PipPU bound with comparable affinity to PU.1 and Pip”

Substitution of Lys399 by arginine partially restored complex formation, indicating a positively charged residue at this site is important.

“Substitution of Lys399 by arginine partially restored complex formation, showing that a positively charged residue at this site is an important structural component of the complex.”

The double mutant fused dimer did not detectably interact with composite sites.

“the double mutant (DM) fused dimer did not interact detectably with the composite sites”

PU.1 bound the CD20 composite site with lower affinity than the λB site.

“PU.1 bound with lower affinity to the CD20 site as compared with λB”

• cites Himmelman et al., 1997Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

On the λB site, PU.1 and Pip bound about fourfold more strongly than the PipPU chimera.

“the binding of PU.1 and Pip to λB was greater than that of the chimera (4-fold)”

The wild-type chimera PipPU repressed endogenous IgL λ gene expression 17-fold.

“the wild-type chimera (PipPU) potently repressed endogenous IgL λ gene expression (17-fold).”

Pipmut and PUmut inhibited IgL λ expression less effectively, with 5.5-fold and 1.8-fold repression, respectively.

“the Pipmut or PUmut mutant chimeras inhibited less effectively (5.5-and 1.8-fold repression, respectively).”

The J chain gene was repressed equivalently by wild-type and Pipmut repressors.

“the J chain gene was repressed equivalently by the wild-type and Pipmut repressors.”

Binding of PUmut to the composite element depended strictly on the Pip binding site.

“the binding of PUmut to the composite element was strictly dependent on the Pip binding site”

The Eλ2-4 element stimulated transcription 12-fold.

“the presence of E λ2-4 stimulated transcription 12-fold.”

Alanine substitution showed Arg398, Lys399 and Leu400 in the predicted helix are critical for PU.1-Pip interaction in vitro and in vivo.

“The alanine substitution study demonstrated that Arg398, Lys399 and Leu400, located within this predicted helix and conserved between Pip and ICSBP, are critical for PU.1-Pip interaction in vitro and in vivo”

The PipPU fused dimer bound the wild-type CD20 site with eightfold higher affinity than the C2 mutant site, which has a mutation in the Pip-binding site.

“the PipPU fused dimer bound the wild-type CD20 site with an 8-fold higher affinity than the C2 mutant site”

• AssumptionRests on an assumption: The CD20 composite element contains a lower-affinity PU.1-binding site (AAAGAA).

PipPU was somewhat more effective at blocking reporter gene activity than PUmut or Pipmut.

“The PipPU protein was somewhat more effective at blocking reporter gene activity as compared with the PUmut or Pipmut proteins.”

The PU.1 mutant bound λB and CD20 sites with affinity similar to the wild-type fused dimer.

“the PU.1 mutant (PUmut) bound the λB and CD20 sites with affinity similar to the wild-type fused dimer”

In gel shift assays with the λB site, PipPU preferred the composite element over a probe containing only a PU.1 site by about threefold.

“PipPU preferred the composite element to one containing only a PU.1 site by a factor of 3”

The half-life of the PU.1-Pip ternary complex was nearly identical to that of PU.1 alone.

“the half-life of the PU.1-Pip ternary complex was nearly identical to that of PU.1 alone”

PUmut had DNA specificity different from PipPU.

“Although the DNA affinity of PUmut was comparable with that of PipPU, its DNA specificity was quite different.”

The wild-type PipPU chimera inhibited J chain gene transcription 3.5-fold.

“Transcription of the J chain gene was inhibited 3.5-fold by the wild-type PipPU chimera.”

Interdependent protein-protein and protein-DNA contacts regulate PU.1-Pip ternary complex assembly.

“Here we undertake a series of biochemical studies to elucidate this mechanism, and find that interdependent protein-protein and protein-DNA contacts regulate PU.1-Pip ternary complex assembly.”

The PUmut chimera did not inhibit J chain gene transcription.

“J chain gene transcription was not inhibited by the PUmut chimera.”

We detected an additional, much weaker cooperative interaction between Pip's DBD and PU.1's DBD.

“We also detected an additional, albeit much weaker, cooperative interaction between Pip's DBD and PU.1's DBD.”

Glutaraldehyde cross-linking showed PU.1-Pip adducts are detectable only in the presence of cognate DNA, indicating template-directed interaction.

“glutaraldehyde cross-linking demonstrated that the protein-protein interaction is template directed, because PU.1-Pip cross-linked adducts are detectable only in the presence of cognate DNA.”

The DBD-DBD interaction was specific because IRF-1 bound competitively with PU.1 on the λB site.

“Subsequent assays suggested that this interaction was specific, because a Pip homolog, IRF-1, bound competitively with PU.1 on the λB site.”

Elimination of Pip's regulatory domain results in an 800-fold drop in affinity of Pip for the PU.1-DNA complex.

“elimination of the regulatory domain results in an 800-fold drop in the affinity of Pip for the PU.1-DNA complex.”

Mutations in a hydrophobic patch at the C-terminus of the helix (aa 401-408) alleviated autoinhibition.

“Mutations in a predominantly hydrophobic patch at the C-terminus of the helix (amino acids 401-408) resulted in alleviation of autoinhibition.”

Proteins containing the POU domain linker were stable in transient and stable expression systems.

“proteins containing this linker were found to be stable in both transient and stable expression systems”

Fusion of the two DBDs greatly enhanced DNA-binding stability compared with wild-type PU.1 or PU.1-Pip.

“kinetic analysis demonstrated that fusion of the two DBDs greatly enhanced DNA-binding stability as compared with wildtype PU.1 or PU.1-Pip”

Prototypical IRF family members IRF-1 and IRF-2 bind DNA with high affinity.

“the prototypical members of the IRF family, IRF-1 and IRF-2, bind to DNA with high affinity.”

Two chymotrypsin cleavage sites in Pip were mapped to Tyr169 and Trp178 in the authors' assays.

“The asterisk denotes two chymotrypsin cleavage sites mapped in our assays (Tyr169 and Trp178).”

When stably expressed, PipPU repressed IgL λ more efficiently than mutant chimeras.

“When stably expressed, PipPU repressed an EICE-containing gene, IgL λ, more efficiently than the mutant chimeras”

Cross-linking between PU.1 and Pip depends on the DNA template.

“PU.1 and Pip cross-linking is DNA template dependent.”

The PUmut dimer formed a less stable protein-DNA complex (half-life 6 min) than complexes with PipPU or Pipmut (half-life 60 min).

“The PUmut dimer formed a protein-DNA complex that was considerably less stable (half-life ϭ 6 min) as compared with those complexes containing PipPU or Pipmut (half-life ϭ 60 min).”

When stably expressed, the wild-type PipPU chimera strongly inhibits endogenous immunoglobulin λ gene expression.

“The wild-type PipPU chimera potently inhibits endogenous immunoglobulin λ gene expression when stably expressed.”

The structural determinants that mediate autoinhibition of Pip DNA binding overlap with those needed for ternary complex formation.

“Structural determinants required for autoinhibition of Pip DNA binding overlap with, but can be uncoupled from, those required for ternary complex formation.”

In transient assays, Pipmut and PUmut repressed transcription similarly to wild-type chimera.

“in transient assays, the Pipmut and PUmut chimeras repressed transcription similarly to the wild-type chimera.”

J chain gene transcription was inhibited by wild-type PipPU chimera.

“Transcription of the J chain gene was also inhibited by the wild-type PipPU chimera.”

PU.1 binds cooperatively with the DNA-binding domain of Pip.

“PU.1 binds cooperatively with the DBD of Pip but competitively with the related DBD of IRF-1.”

Both wild-type and mutant PipPU chimeras repress λ enhancer activity in transient transfection assays.

“Both wild-type and mutant PipPU chimeras repress λ enhancer function in transient transfection assays.”

Several residues within Pip's interaction helix are required for the formation of a ternary complex with PU.1 in vitro.

“Multiple residues in Pip's interaction helix are important for ternary complex formation with PU.1 in vitro and transcriptional synergy in vivo.”

Both DBDs contribute to the higher combined affinity and selectivity of PipPU compared with either mutant.

“Our gel shift studies revealed that both DBDs contribute to the higher combined affinity and selectivity of the PipPU chimera as compared with either of the mutants.”

The structural determinants that mediate autoinhibition of Pip DNA binding can be separated from those needed for ternary complex formation.

“Structural determinants required for autoinhibition of Pip DNA binding overlap with, but can be uncoupled from, those required for ternary complex formation.”

J chain gene was repressed equivalently by wild-type and Pipmut repressors.

“the J chain gene was repressed equivalently by the wild-type and Pipmut repressors.”

PU.1 binds competitively with the related DNA-binding domain of IRF-1.

“PU.1 binds cooperatively with the DBD of Pip but competitively with the related DBD of IRF-1.”

Several residues within Pip's interaction helix are required for transcriptional synergy in vivo.

“Multiple residues in Pip's interaction helix are important for ternary complex formation with PU.1 in vitro and transcriptional synergy in vivo.”

In stably expressing cells, maximal repression of endogenous IgL λ gene depended on both wild-type Pip and PU.1 DBDs.

“upon testing the effects of these proteins on endogenous IgL λ gene transcription in stably expressing cells, significant differences in their function were found, with maximal repression depending on the presence of both wild-type Pip and PU.1 DBDs.”

PU.1 and Pip form complexes in B cells on composite elements in immunoglobulin light chain gene enhancers.

“The lymphoid-restricted interferon regulatory factor, Pip (IRF-4), and the hematopoietic-specific Ets protein, PU.1, form complexes in B cells on composite elements present in immunoglobulin light chain (IgL) gene enhancers (Pongubala et al., 1992;Eisenbeis et al., 1993).”

• cites Pongubala et al., 1992Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Eisenbeis et al., 1993 sourceNot checked — no full text of the source

Pip binding to DNA requires DNA-bound PU.1 phosphorylated at Ser148.

“In each instance, the binding of Pip to DNA is dependent on DNA-bound PU.1, which must be phosphorylated on Ser148 (pSer148; Pongubala et al., 1993).”

• cites Pongubala et al., 1993 sourceNot checked — no full text of the source

Multiple side chains within Pip amino acids 397-400 may contact PU.1.

“Together, these data suggest that multiple side chains within this region may contact PU.1.”

The crystal structure of PU.1 DBD bound to DNA shows that the N-terminus of PU.1 faces the binding site for Pip.

“The crystal structure of the PU.1 DBD bound to DNA shows that the N-terminus of PU.1 faces the binding site for Pip (Kodandapani et al., 1996).”

• cites Kodandapani et al., 1996 sourceNot checked — no full text of the source

Protein-protein and protein-DNA interactions function coordinately to establish a specific and stable ternary complex.

“These results suggest that protein-protein and protein-DNA interactions function coordinately in establishing a specific and stable ternary complex.”

Pip structural determinants important for ternary complex assembly are also required for transcriptional activation in vivo.

“Therefore, the structural determinants of Pip that are important for ternary complex assembly are also required for transcriptional activation in vivo.”

The predicted alpha-helix is essential for PU.1-Pip interaction.

“Taken together, these observations suggest that the predicted α-helix is essential for PU.1-Pip interaction.”

A functional Pip regulatory domain containing ternary complex and autoinhibition regions resides within an independently folded module (amino acids 170-450).

“Therefore, these experiments establish that a functional regulatory domain, containing the regions necessary for ternary complex formation and autoinhibition, resides within an independently folded protein module (amino acids 170-450).”

The Pip DBD's low intrinsic affinity for lambdaB is increased 20- to 40-fold by PU.1.

“Thus, the Pip DBD has a low intrinsic affinity for the λB sequence which is increased 20-to 40-fold by PU.1.”

The structural determinants in Pip required for autoinhibition overlap with, but can be uncoupled from, those required for ternary complex formation.

“Interestingly, these experiments showed that the structural determinants in Pip required for autoinhibition of DNA binding overlap with, but can be uncoupled from, those required for ternary complex formation.”

This regulatory domain is tethered to the DBD by a protease-sensitive linker.

“This domain is tethered to the DBD by a proteasesensitive linker (Figure 3A, bottom).”

Partial transcriptional activation by PipAS397 may result from weak association with PU.1 and DNA through an IRF-ets DBD interaction.

“Partial activation of transcription by this mutant protein may be due to its ability to associate weakly with PU.1 and DNA through an interaction between the IRF and ets DBDs (see below).”

PU.1 and Pip additionally interact through their DBDs.

“These data suggest that PU.1 and Pip additionally interact through their DBDs.”

Any PU.1-induced conformational change within Pip occurs by pivoting of Pip's regulatory domain about the flexible linker.

“These data suggest that any PU.1-induced conformational change within Pip occurs by pivoting of Pip's regulatory domain about the flexible linker.”

The PU.1-Pip interaction detected by cross-linking has the same requirements as ternary complex assembly by gel shift.

“Therefore, the PU.1-Pip interaction detected by glutaraldehyde cross-linking displayed the same requirements as those determined for ternary complex assembly using the gel shift assay (Figure 5A and B).”

The Kd value is likely an underestimate because at saturation about 75% of recombinant PU.1 interacts with Pip and is phosphorylated appropriately.

“This K d value is likely to be an underestimate, because the binding curve shows that at saturation, ~75% of the recombinant PU.1 interacts with Pip and is therefore phosphorylated appropriately.”

A coordinated set of protein-protein and protein-DNA contacts is essential for PU.1-Pip ternary complex assembly.

“Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly.”

Interaction of Pip's regulatory domain with the PEST region of PU.1 greatly enhances DNA-binding cooperativity.

“Thus, the interaction of Pip's regulatory domain with the PEST region of PU.1 greatly enhances DNAbinding cooperativity.”

Nuclear extracts from stably transduced lines formed complexes with λB DNA to similar levels for PipPU and mutant chimeras.

“the PipPU and mutant chimeras formed complexes with λB DNA to similar levels”

J558L cells express a functionally rearranged Vλ1-Jλ1 IgL allele.

“it expresses a functionally rearranged Vλ1-Jλ1 IgL allele”

• cites Oi et al., 1983Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

This helix is also required for transcriptional co-activation.

“This helix is also required for transcriptional co-activation.”

On some composite elements, fusing the two DBDs can reproduce the affinity of the PU.1-Pip complex.

“thus demonstrating that on certain composite elements, 984 fusion of the two DBDs can recapitulate the affinity of the PU.1-Pip complex.”

Individual J558L clones expressed similar levels of the chimeric proteins, as shown by Western blotting.

“Individual J558L clones that expressed similar levels of the chimeric proteins were identified by Western blotting”

PU.1-Pip complexes are functionally important in B cell gene expression.

“When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin λ gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression.”

PipPU's binding stability depended on Arg232 and Arg235 of the PU.1 DBD.

“suggests that PipPU's binding stability depended on Arg232 and Arg235 of the PU.1 DBD.”

The PipPU fused dimer and its variants can distinguish genes regulated by PU.1 and Pip from those regulated by PU.1 alone.

“the overall analysis establishes the utility of using the PipPU fused dimer and its variants to distinguish genes regulated by PU.1 and Pip from those regulated by PU.1 alone.”

PU.1 alone is required for J chain gene activity.

“these data demonstrate that PU.1 alone is required for J chain gene activity.”

Similar residues in ICSBP and p48 may play a general role in IRF-containing complexes, including IRF-3.

“Therefore, such residues may play a more general role in IRF-containing complexes, including those involving IRF-3”

• cites Au et al., 1995Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Wathelet et al., 1998 sourceNot checked — no full text of the source

Comparable levels of all four chimeric proteins were expressed in transfected cells, as determined by Western blotting.

“Comparable levels of all four chimeric proteins were expressed in transfected cells as determined by Western blotting”

The higher DNA-binding affinity of the ternary complex compared with PU.1 is due to a faster association rate.

“suggesting that the elevated DNA-binding affinity of the ternary complex, as compared with PU.1, is due to an increased rate of association.”

The ternary complex may also be stabilized by hydrophobic and/or polar contacts between Pip's Leu400, Ile401, Thr402 and His403 and PU.1.

“The ternary complex may also be stabilized by hydrophobic and/or polar contacts between Pip's Leu400, Ile401, Thr402 and His403, and PU.1.”

Fusing the Pip and PU.1 DBDs produced a protein that bound DNA more stably than wild-type PU.1 or its DBD.

“These results demonstrate that fusion of the Pip and PU.1 DBDs created a protein which bound DNA with much greater stability than wildtype PU.1 or its DBD.”

The chimeric proteins were expressed at considerably higher levels than endogenous PU.1.

“the chimeric proteins were expressed at considerably higher levels than endogenous PU.1”

PU.1-Pip association may involve an interaction between PU.1's pSer148 and Pip's Arg398 and Lys399.

“our data suggest a possible mechanism for PU.1-Pip association involving an interaction between PU.1's pSer148 and Pip's Arg398 and Lys399.”

Repression of the endogenous IgL λ gene depended on both the PU.1 and Pip DBDs.

“This repression was dependent on both the PU.1 and Pip DBDs”

PU.1-Pip complexes are essential for expression of a productively rearranged immunoglobulin λ gene in a plasma cell.

“These results establish that PU.1-Pip complexes are essential for the expression of a productively rearranged immunoglobulin λ gene in a plasma cell.”

On the λB site, covalent fusion did not fully reproduce wild-type binding affinity.

“illustrating that on this site the covalent linkage did not fully recapitulate wild-type affinity”

Expression of the fusion proteins did not alter endogenous PU.1 levels.

“determine that expression of the fusion proteins had not altered endogenous PU.1 levels”

Cooperative binding of Pip and PU.1 largely depends on interaction between the phosphorylated PEST region of PU.1 and Pip's regulatory domain.

“The cooperative binding of Pip and PU.1 is largely dependent on the interaction between the phosphorylated PEST region of PU.1 and the regulatory domain of Pip”

A mutation in Pip's regulatory domain should not perturb the structure of its DBD.

“Thus, a mutation in Pip's regulatory domain should not perturb the structure of its DBD.”

The enhanced stability is most likely due to increased protein-DNA contacts elevating the free energy of interaction.

“most likely due to the increased protein-DNA contacts elevating the free energy of interaction.”

Upon interaction with PU.1 and DNA, Pip undergoes a conformational change that moves its regulatory domain away from its DBD and into contact with PU.1's PEST region.

“Upon interaction of Pip with PU.1 and DNA, Pip undergoes a conformational change involving the swiveling of its regulatory domain (amino acids 170-450) away from its own DBD, and into direct contact with PU.1's PEST region.”

Pip's regulatory domain autoinhibits DNA binding by masking the DBD via direct hydrophobic contacts.

“(i) Pip's regulatory domain autoinhibits DNA binding by masking the DBD via direct hydrophobic contacts.”

Dependence of this IRF subgroup on a DNA-binding partner creates additional specificity in gene regulation.

“The dependence of this subgroup on a DNAbinding partner creates additional specificity in gene regulation, thereby expanding the utility of the IRF DBD.”

PipAS397's binding properties are unlikely to result from misfolding because Pip consists of two independent structural domains separated by a flexible linker.

“It is unlikely that the binding properties of PipAS397 are the result of misfolding of this polypeptide, because our proteolytic analysis shows that Pip is composed of two independent structural domains, a DBD domain and a regulatory domain, separated by a flexible linker.”

In both instances, transcription-activating complexes form only upon interaction of appropriate partners and cognate DNA, ensuring specific gene regulation.

“In both of these instances, transcription-activating complexes form only upon interaction of the appropriate partners and cognate DNA, thus ensuring the specific regulation of a distinct set of genes.”

Independent binding of PUmut may arise from phosphate backbone contacts from PU.1's DBD stabilizing the weak DNA-binding activity of the Pip DBD.

“The independent binding of PUmut may arise from phosphate backbone contacts provided by PU.1's DBD, which stabilize the weak DNAbinding activity of the Pip DBD.”

The biological function of autoinhibition is to prevent Pip and ICSBP from binding inappropriately to IFN-regulated promoters and enhancers.

“we propose that the biological function of autoinhibition is to prevent Pip and ICSBP from binding inappropriately to IFN-regulated promoters and enhancers.”

The C-terminal region inhibits binding by masking the DBD.

“We propose that this region inhibits binding by masking the DBD”

Binding interference between PU.1 and IRF-1 results from competition for overlapping sites.

“suggesting that the binding interference between PU.1 and IRF-1 results from the two proteins competing for binding to overlapping sites.”

In the wild-type ternary complex, both PU.1's DBD N-terminus and PEST region would be positioned to contact Pip.

“It follows then that in the wild-type ternary complex, both the N-terminus of PU.1's DBD (amino acids 160-272) and its PEST region (amino acids 119-159) would be in position to contact Pip.”

The model provides general insight into concerted action of Ets-IRF complexes in lymphoid and myeloid lineages.

“we believe this model provides general insight into the concerted action of these transcription factors in both lymphoid and myeloid lineages.”

The inability of our cross-linking assays to detect interaction is consistent with template DNA stabilizing weak PU.1-Pip protein-protein contacts.

“The inability of our cross-linking assays to detect such an interaction is consistent with template DNA stabilizing weak protein-protein contacts between PU.1 and Pip.”

Cross-linking experiments showed NFAT-AP-1 adducts detectable only with specific DNA, arguing protein-protein interactions alone are insufficient.

“cross-linking experiments have shown that NFAT-AP-1 adducts are detectable only in the presence of specific DNA, arguing that protein-protein interactions alone are of insufficient strength for productive complexation”

• cites Chen et al., 1995 sourceNot checked — no full text of the source

Autoinhibition is alleviated upon interaction with a specific partner, PU.1 or Spi-B, in the context of a composite regulatory element.

“This autoinhibition is alleviated upon interaction with a specific partner, i.e.PU.1 or Spi-B, in the context of a composite regulatory element.”

The DBD-DBD interaction is likely an important component of ternary complex formation.

“it is likely that the interaction between the DBDs will be found to be an important component of ternary complex formation.”

In the PU.1 DBD-DNA co-crystal structure, the N-terminus of PU.1 is adjacent to the predicted Pip binding site.

“in the PU.1 DBD-DNA co-crystal structure, the N-terminus of PU.1 is adjacent to the predicted binding site of Pip.”

• cites Kodandapani et al., 1996 sourceNot checked — no full text of the source

The IRF-1-DNA co-crystal structure shows IRF-1 makes extensive contacts with DNA backbone phosphates up to six nucleotides upstream of the GAAA core.

“The recent IRF-1-DNA co-crystal structure (Escalante et al., 1998) reveals that in addition to the specific base contacts made by the recognition helix, IRF-1 makes extensive contacts with DNA backbone phosphates, even as far as six nucleotides upstream of the GAAA recognition core.”

• cites Escalante et al., 1998 sourceNot checked — no full text of the source

Additional conformational changes needed for cooperativity may occur in the native complex but not in PipPU.

“These results suggest that additional conformational changes necessary for cooperativity may occur in the context of the native complex that are not recapitulated within PipPU.”

Partial loss of IRF-1-DNA contacts may account for attenuated binding of Pip, ICSBP and p48.

“then partial loss of these interactions may account for the attenuated binding of Pip, ICSBP and p48.”

The POU domain linker may be generally resistant to proteolysis in vivo.

“suggesting that this linker may be generally resistant to proteolysis in vivo.”

The PU.1-DNA co-crystal structure shows numerous protein-DNA backbone contacts.

“This last suggestion is supported by the numerous protein-DNA backbone contacts observed in the PU.1-DNA co-crystal structure”

• cites Kodandapani et al., 1996 sourceNot checked — no full text of the source

The selectivity of a chimeric transcription factor depends on intermediate affinity and high specificity of isolated subdomains.

“This is consistent with the overall selectivity of a chimeric transcription factor depending on the intermediate affinity and high specificity of the isolated sub-domains”

• cites Pomerantz et al., 1995 sourceNot checked — no full text of the source

PU.1-Pip complexes are essential for IgL λ expression in vivo.

“demonstrating that PU.1-Pip complexes are essential for IgL λ expression in vivo.”

Additional contacts between the DNA-binding domains of Pip and PU.1 stabilize the ternary complex.

“Additional contacts between Pip's DBD and PU.1's DBD stabilize the ternary complex.”

Differential DNA-binding properties of the chimeric proteins likely account for their varying abilities to repress endogenous IgL λ transcription.

“Thus, it is likely that the differential DNA-binding properties of the chimeric proteins account for their varying abilities to repress endogenous IgL λ gene transcription.”

Transcription of rearranged IgL λ gene appears regulated by two enhancers Eλ2-4 and Eλ3-1, which share high sequence identity.

“Transcription of a rearranged IgL λ gene appears to be regulated by two enhancers, E λ2-4 and E λ3-1 , which share high sequence identity.”

Unlike IgL λ, PU.1 alone is required for J chain gene activity.

“unlike the case with the IgL λ gene, PU.1 alone is required for J chain gene activity.”

The study establishes a functional requirement for PU.1-Pip complexes for expression of the IgL λ locus in a plasma cell.

“Our study establishes the functional requirement for PU.1-Pip complexes for expression of the IgL λ locus in a plasma cell.”

Pip's regulatory domain, spanning amino acids 170-450, autoinhibits DNA binding by masking the Pip DNA-binding domain.

“Pip's regulatory domain (RD, amino acids 170-450) autoinhibits DNA binding by masking the Pip DBD.”

When Pip interacts with a PU.1-DNA template complex, its regulatory domain swivels around the flexible linker and contacts the PEST region of PU.1.

“Interaction of Pip with a PU.1-DNA template complex causes the regulatory domain to swivel about the flexible linker region (L) and contact PU.1's PEST region.”

Combined analysis of wild-type dimer with mutants allowed distinguishing genes regulated by PU.1 and Pip from those regulated by PU.1 alone.

“the combined analysis of the wildtype dimer with a series of mutants allowed us to distinguish genes regulated by PU.1 and Pip from those regulated by PU.1 alone.”

Eλ2-4 and Eλ3-1 contain juxtaposed binding sites occupied by multiple factors.

“E λ2-4 and E λ3-1 also contain juxtaposed binding sites occupied by multiple factors”

• cites Rudin and Storb, 1992Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Stable binding of both DBDs may be necessary for PipPU to compete with endogenous Ets and IRF proteins for site occupancy.

“the stable binding of both its DBDs may be necessary to compete with endogenous Ets and IRF proteins for site occupancy.”

Gel shift analyses confirmed purified proteins had similar specific activities to their mammalian counterparts.

“Gel shift analyses of these purified proteins confirmed that they possesed similar specific activities to their mammalian counterparts.”

In stably transduced cells, PipPU's enhanced DNA-binding properties may allow it to locate endogenous EICEs more effectively.

“PipPU's enhanced DNA-binding properties may allow it to locate more effectively a small number of endogenous EICEs”

The kinetic stability of fused dimer-DNA complexes correlates with their ability to repress transcription in vivo.

“Thus the kinetic stability of the fused dimer-DNA complexes correlates with their ability to repress transcription in vivo”

PipPU probably represses IgL λ transcription by disrupting both λ enhancers via displacement of PU.1-Pip complexes.

“The PipPU fused dimer probably represses IgL λ gene transcription by disrupting the function of both λ enhancers via the displacement of PU.1-Pip complexes.”