Input:
The core promoter of A3G is located within the region -114/+66 relative to the TSS
For characterization of the A3G promoter, we cloned the 1025 bp located at position -959/+66 relative to the identified transcription start into the promoterless pGL3-Basic luciferase reporter plasmid and designated the plasmid pGL3-Basic-APOprom1025. Similarly, pGL3-APOprom502 (containing sequence -436/+66), pGL3-APOprom225 (containing sequence -159/+66) and further 5' deletion reporter constructs containing 180, 150, 120 or 60 bp upstream of position +65 were generated (Figure 3A). In order to analyze transcriptional activity, the luciferase reporter plasmids were transiently transfected into A3.01 T cells. Luciferase assays revealed a approximately20-fold increased transcriptional activity of the 1025 bp sequence as compared to the empty vector, indicating that we had identified an active A3G promoter sequence (Figure 3B). This transcription rate was not significantly altered by the 5' deletions leading to the 502, 225 and 180 bp fragments (Figure 3B). In contrast, a drop in luciferase activity was observed in the case of the 150 bp fragment. This construct only retained 28% of the transcriptional activity of the 180 bp promoter in A3.01 T cells and activity of the 120 bp fragment was further reduced. Comparable reductions relative to the activity of 180 bp fragment were observed in the myeloid cell line U937 and the hepatic cell lines HepG2 and Huh7 (Figure 3C), indicating that the core promoter of A3G is located within the region -114/+66 relative to the TSS.

1025 bp sequence

luciferase

luciferase

Luciferase

luciferase

drop

transcriptional activity

increased

transcriptional activity

1025 bp sequence

altered

increased

transcriptional activity

Luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
The A3G promoter is not inducible in T cells
According to the current knowledge, APOBEC3G plays a role in the innate defence against pathogens like HIV-1. The latter has evolved a mechanism to counteract A3G activity by expressing the regulatory protein Vif (4). Vif induces proteasomal degradation of A3G and additionally inhibits A3G activity by further mechanisms (8, 37-43). To investigate whether the overexpression of HIV-1 proteins also influences A3G promoter activity, we either expressed HIV-1 Vif or the HIV-1 Tat protein, the latter has been shown to activate different viral and cellular promoters (44,45). In addition, increasing amounts of the plasmid pNL4-3, containing the full-length genome of HIV-1, were transfected. These constructs or the empty vector pcDNA3.1 were cotransfected with the 1025 bp construct into A3.01 T cells. Luciferase assays showed that the transcriptional activity of the A3G promoter was not significantly altered in the presence of Vif, Tat or HIV-1NL4-3 (Figure 4A), suggesting that HIV-1 is not modulating A3G expression on the transcriptional level.
It has been shown that the amount of A3G mRNA in T cells is increased in response to mitogenic stimulation with phorbol ester (46-48). In addition, interferons have been described to upregulate A3G expression in hepatocytes and macrophages (46,48). To investigate whether these stimuli interfere with A3G promoter activity in T cells, we transfected A3.01 T cells with the 1025 bp promoter or the empty vector pGL3-Basic (vector) and treated the cells with phorbol ester (TPA). The luciferase assay showed that the approximately15-fold increased transcriptional activity of the 1025 bp promoter relative to the empty vector was not further enhanced by TPA treatment (Figure 4B), although the functional activity of TPA was confirmed by induction of the SV40 promoter-containing reporter plasmid pGL3-Control (data not shown). Similarly, treatment of A3.01 T cells transfected with the A3G promoter deletion constructs with IFN-alpha or IFN-gamma showed no effect (Figure 4C). Interestingly, a control plasmid (pGL2-CVX) containing twoIFN-responsive GAS ( gamma activated sequence) elements upstream of the luciferase reporter gene was only induced by IFN-alpha in these cells (Figure 4C). Since two reports describe A3G upregulation by interferons in hepatocytes (47,48), we additionally performed the experiment in the hepatic cell line HepG2. In line with these publications, we observed an induction of the A3G promoter by approximately 2-fold after IFN-alpha or IFN-gamma stimulation (Figure 4D) with IFN-gamma being slightly more potent. For both interferon types, induction of A3G promoter activity was observed for all deletion constructs except for the 60 bp fragment, indicating that the responsible region is located within the 60 nt present in the 120 bp, but not in the 60 bp fragment.

promoter

promoter

Vif

Vif

Vif

Tat

Luciferase

Vif

Tat

luciferase

IFN-alpha

IFN-gamma

luciferase

IFN-alpha

IFN-alpha

IFN-gamma

IFN-gamma

expressing

Vif

expressed

Vif

expressed

Tat

expression

expression

counteract

expressing

inhibits

Vif

inducible

promoter

induces

Vif

degradation

increased

upregulate

expression

increased

transcriptional

activity

transcriptional

enhanced

increased

upregulation

induction

promoter

degradation

modulating

expression

transcriptional

luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The word or phrase that expresses the event.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
A GC-box located at position -87/-78 of the A3G promoter is important for transcriptional activity
The reporter studies shown in Figure 3 had demonstrated a drop in luciferase activity after deletion of the 30 nt at the 5' end of the 180 bp core promoter. We therefore inspected the 30 bp sequence deleted in the 150 bp fragment and identified a GC-box at position -87/-78 (see Figure 1). The sequence TGGGCGGGAC, which is interrupted in the 150 bp fragment, represents a variant of the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif recognized by Sp1 and Sp3 transcription factors. To analyze whether this putative Sp1/Sp3-binding site mediates transcriptional activity of the 180 bp core promoter, we introduced two point mutations which changed the sequence from TGGGCGGGAC to TGTTCGGGAC (mutations shown in bold). This resulted in a 71% reduction of the transcriptional activity compared to the unmodified 180 bp promoter (Figure 5A) and this value was only marginally higher than the luciferase activity of the 150 bp fragment, indicating that the identified motif is essential for basal activity of the A3G core promoter. To further examine the transcriptional potency of the 30 nt present in the 180 bp promoter, we cloned the region -114/-85 (containing all nucleotides which are deleted in the 150 bp fragment, designated E1) or the region -92/-63 (containing the putative Sp1/Sp3 motif, designated E2) into the vector pGL3-Promoter (see Figure 1). This vector contains a luciferase reporter gene under the control of an SV40 promoter without enhancer sequences, and putative transcriptionally active sequences can be cloned upstream of the SV40 promoter. Luciferase assays showed that the E1 element increased SV40 promoter activity only by approximately2-fold (Figure 5B). In contrast, the 30 nt of E2 enhanced the transcriptional activity of the SV40 promoter by approximately4.3-fold, indicating that the intact GC-box present in the E2 element was responsible for the strongly enhanced transcriptional activity of the SV40 promoter.

the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif

30 nt

luciferase

luciferase

luciferase

Luciferase

recognized

the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif

recognized

the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif

enhanced

30 nt

transcriptional activity

responsible

enhanced

enhanced

transcriptional activity

transcriptional activity

Luciferase

under the control

luciferase

transcriptional activity

Luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Sp1 and Sp3 transcription factors bind to the GC-box at position -87/-78 of the A3G promoter
To investigate whether the GC-box represents a binding site for the transcription factors Sp1 and Sp3, we performed EMSA analyses with nuclear extracts isolated from A3.01 T cells. As probes, we radioactively labeled the unmodified E2 sequence (nucleotides -92/-63 of the A3G promoter, probe designated APO-Sp1/3) or the same region carrying the two point mutations described above (probe designated APO-Sp1/3mut). As control, we used a commercially available Sp1 consensus oligonucleotide (Sp1cons), which is known to be recognized by Sp1 transcription factors. Four DNA-protein complexes were observed in the presence of the APO-Sp1/3 probe (Figure 6A). The upper two complexes were specific since they disappeared in the presence of a 30-fold molar excess of unlabeled APO-Sp1/3 probe (Figure 6A, lane 7). Further confirmation of the specificity of these DNA-protein complexes was demonstrated by the inability of the unlabeled APO-Sp1/3mut probe to abolish binding (Figure 6A, lanes 12 and 13). As expected, the two specific complexes were also present in the case of the Sp1 cons control probe (Figure 6A, lanes 2 and 3). In contrast, none of these specific complexes was observed when the mutated probe was used (Figure 6A, lanes 10 and 11), confirming that protein binding was dependent on the identified GC-box. In order to characterize the complexes, we performed supershift experiments using Sp1- and Sp3-specific antibodies. The upper of the complexes that appeared in combination with the Sp1cons or APO-Sp1/3 probes, shifted in the presence of the Sp1 antibody (Figure 6B, lanes 3 and 7), whereas the lower complex shifted in the presence of the Sp3 antibody (Figure 6B, lanes 4 and 8). Taken together, the EMSA demonstrated that the GC-box located on the A3G core promoter serves as a binding site for Sp1 and Sp3.
To show binding of Sp1 and Sp3 factors also in the context of the endogenous A3G promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Sp1 and Sp3 antibodies, but not an actin antibody, immunoprecipitated the A3G promoter in A3.01 T cells (Figure 6C, upper panel). In contrast, no PCR signal was received with a primer pair recognizing a region in the A3G gene approximately4000 bp downstream of the Sp1/Sp3-binding site (Figure 6C, lower panel). Only the positive controls showed a DNA band, with an A3G expression plasmid or the sheared and cross-linked input DNA used as template. This demonstrates the binding of Sp1 and Sp3 transcription factors to the endogenous A3G promoter.

GC-box

GC-box

GC-box

promoter

promoter

actin

bind

GC-box

bind

GC-box

represents a binding site

GC-box

represents a binding site

GC-box

recognized

serves as a binding site

GC-box

serves as a binding site

GC-box

binding

promoter

binding

promoter

binding

promoter

binding

promoter

expression

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Silencing of Sp1 and Sp3 reduces A3G promoter activity
To confirm the role of Sp1 and Sp3 in regulation of A3G promoter activity, we silenced their translation via RNA interference. Functionality of the siRNAs directed against Sp1 or Sp3 was confirmed by western blot analysis: protein levels of Sp1 as well as the long and short isoforms of Sp3 were strongly reduced in the presence of 150 or 300 ng specific siRNA (Figure 7A). An unspecific control siRNA had no influence (Figure 7A). We then cotransfected the luciferase reporter plasmid containing the 180 bp A3G promoter together with 100 ng siRNA using an optimized protocol for the cotransfection of plasmid plus siRNA. This resulted in a 31-43% reduction of luciferase activity in the presence of Sp1- or Sp3-specific siRNA compared to the control siRNA. In contrast, no influence on transcriptional activity of the 150 bp fragment, which does not contain the Sp1/Sp3-binding motif, was observed. Thus, both Sp1 and Sp3 factors are mediating transcriptional activity of the A3G promoter.

luciferase reporter plasmid containing the 180 bp A3G promoter

protein levels

protein levels

cotransfected

luciferase reporter plasmid containing the 180 bp A3G promoter

Silencing

Silencing

reduced

protein levels

reduction

luciferase reporter plasmid containing the 180 bp A3G promoter

siRNA

siRNA

siRNA

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
DISCUSSION
Shortly after the discovery of APOBEC3G, it became clear that this human gene plays an important role in antiretroviral defense (4). Originally identified as a restriction factor of HIV-1 infection, expression of APOBEC3G was found in human peripheral blood lymphocytes and macrophages, which represent the main target cells for HIV-1 (32). In addition, the protein was detected in lung, liver, spleen, testis and ovary, but little is known about its transcriptional regulation (29,32,47). To address this question, we cloned the human APOBEC3G promoter and analyzed its regulation in T cells. Applying 5'-RACE, we identified multiple start sites for transcription of the A3G gene. This observation is consistent with the fact that the gene appears to lack canonical CCAAT and TATA boxes (32), a condition often associated with multiple transcriptional start sites (49-51). However, one single start site was detected in 6 of 19 clones and was designated as +1. This TSS is located 66 bp upstream of the start of the published mRNA sequence (GenBanktrade NM021822).
We cloned a 1025 bp promoter, which ranges from position -959/+66 relative to the identified TSS. Luciferase reporter assays showed that this promoter was transcriptionally active in A3.01 T cells. Treatment with the phorbol ester TPA did not further enhance transcriptional activity, although upregulation of A3G mRNA levels in T cells by TPA has been described (33). Rose et al. used actinomycin D to block further transcription and did not find evidence for enhanced mRNA stability. Therefore, the authors suggested that an enhanced transcription rate could be responsible for the increased amount of A3G mRNA after TPA treatment. However, the promoter analysis we performed indicates a different mechanism that is independent of transcriptional regulation. In addition, we observed that the A3G promoter was not inducible by IFN-alpha or IFN-gamma in A3.01 T cells, whereas in the hepatic cell line HepG2, a moderate induction was measured. It has been described previously that A3G gene expression is upregulated by interferons in hepatocytes and macrophages (46-48, 52). In this context, two interferon-responsive elements have been identified at the positions -6/+9 and +1/+15 relative to the TSS (48). Consistent with this data, we observed an interferon induction in hepatocytes for the fragments ranging between 1025 and 120 bp length. The activity of the 60 bp fragment that does not contain these motifs was not affected. Thus, the induction we observed was most likely mediated by the described interferon-responsive elements. However, according to our results, these motifs can enhance transcription in hepatic cells, but not in T cells. This is surprising, since IFN-alpha-inducible signaling cascades are present in A3.01 T cells: we showed that the control plasmid harboring interferon-responsive GAS elements was markedly induced by IFN-alpha treatment. In contrast, the GAS element was not responsive to IFN-gamma treatment in these cells, suggesting that our T cell line was not able to mediate IFN-gamma-induced signals. So far, enhanced A3G promoter activity by interferons is clearly described in hepatocytes and macrophages, whereas the situation in T cells is still unclear. A recent study found no influence of IFN-alpha or IFN-gamma on A3G expression in resting primary blood lymphocytes (52). Another publication describes an enhanced expression of A3G after IFN-alpha treatment in resting primary CD4 T cells, but not in activated T cells (53). This observation is not contradictory to our results since T cell lines are mitotically active and therefore rather in an activated than in a resting state. In conclusion, the current data suggests that regulation of the A3G promoter activity by interferons is dependent on the cell type and possibly also from the cellular activation status.
We additionally analyzed the influence of HIV-1 proteins on A3G promoter activity. The HIV-1 Vif protein is neutralizing the antiviral function of A3G by a multitude of ways. Targeting the A3G protein for proteasomal degradation is considered to be the main mode of action (37-41), but it becomes more and more clear that other mechanisms are also involved. There is evidence that Vif also promotes exclusion of A3G from the virus particles and an influence on the A3G translation process is discussed (8,42,43). The question whether Vif alters transcription controlled by the A3G promoter has not been analyzed so far. Our analysis indicates that transcription from the A3G promoter is unaffected by Vif or other HIV-1 proteins. Taken together, in T cell lines, the A3G promoter appears constitutively active.
By generating a series of 5' deletions, we showed that the core promoter is located within the region -114/+66 relative to the TSS. This 180 bp promoter was transcriptionally active in the lymphoid, myeloid and hepatic cell lines we tested, indicating that ubiquitous transcription factors are involved in regulation of A3G gene transcription. A GC-box with the sequence TGGGCGGGAC was identified at position -87/-78 of the A3G promoter. This GC-box is essential for basal promoter activity, since changing the motif to TGTTCGGGAC by introducing two point mutations strongly reduced A3G promoter activity. The transcriptional potency of the GC-box was further demonstrated by cloning it upstream of an SV40 promoter. This resulted in a 4-fold enhanced transcription rate. The hypothesis that a general transcription factor is involved in regulation of A3G transcription was confirmed by EMSA. Supershift analysis showed that the transcription factors Sp1 and Sp3 which are ubiquitously expressed in mammalian cells, bind specifically to the identified motif. In addition, the binding of Sp1 and Sp3 to the endogenous A3G promoter was demonstrated by a chromatin immunoprecipitation assay. This observation is consistent with the results of the 5'-RACE, since Sp1 is known to play an important role for RNA polymerase II to bind to the transcription initiation site in TATA-boxless promoters and is associated with multiple transcription initiation sites (54-56). We also tested whether overexpression of Sp1 or Sp3 proteins had an influence on A3G promoter activity. Luciferase assays revealed that A3G promoter activities remained unchanged (data not shown), most probably due to the high basal expression level of the endogenous Sp1 and/or Sp3 proteins. The Sp family of transcription factors is involved in transcriptional regulation of many housekeeping, tissue-specific, viral and inducible genes (57). Whereas Sp1 typically acts as an activator, Sp3 can serve as a repressor or activator (57). However, in the context of the A3G promoter, both transcription factors serve as activators, as shown by siRNA-mediated silencing of the single factors. Although siRNAs directed against Sp1 or Sp3 significantly reduced transcription controlled by the 180 bp promoter, transcriptional activity was not reduced to the level of the 150 bp fragment. This can be explained by the remaining low amounts of Sp1/Sp3 proteins which can be seen in the western blot analysis. Alternatively, other transcription factors could be involved in A3G regulation.
Our results also provide information about the regulation of APOBEC3F (A3F), another member of the human APOBEC3 family. A3F is expressed in many human tissues that also express A3G and both proteins have been shown to form heteromultimers, which are most likely generated through binding to an RNA intermediate (26,29,30). A3F only differs in 16 nt from the 560 nt upstream of the TSS of A3G (GenBanktrade DQ146365 and DQ147772). The 180-bp core promoter region, which we identified for A3G has 100% identity with the A3F sequence. Therefore, Sp1/Sp3 transcription factors are most likely also mediating basal transcription of the A3F gene.
Our study revealed that the human A3G gene is controlled by a promoter with multiple transcriptional start sites. In A3.01 T cells, the A3G promoter appears constitutively active and is not inducible by TPA, type I or II interferons or by HIV-1 proteins. The core promoter is located within a region of 180 bp at position -114/+66 relative to the TSS. A GC-box is crucial to the function of the core promoter and represents a binding site for Sp1 and Sp3 transcription factors. Our results can serve as a basis for future studies aimed at understanding how A3G and A3F expression is controlled in different tissues and how these restriction factors can be used to develop novel therapeutic strategies against HIV-1 infection.

promoter

promoter

60 bp fragment

the described interferon-responsive elements

promoter

promoter

promoter

promoter

promoter

180 bp promoter

promoter with multiple transcriptional start sites

promoter

GC-box

core promoter

Luciferase

IFN-alpha

IFN-gamma

IFN-alpha

IFN-alpha

IFN-gamma

IFN-gamma

IFN-alpha

IFN-gamma

IFN-alpha

Vif

Vif

Vif

Vif

TGTTCGGGAC

RNA polymerase II

Luciferase

bind

bind

binding

promoter

binding

promoter

bind

RNA polymerase II

form heteromultimers

binding

core promoter

binding

core promoter

expression

detected

expression

expression

expression

expressed

expressed

overexpression

overexpression

expression

expression

expressed

express

expression

expression

block

transcription

silencing

silencing

reduced

transcription

reduced

transcriptional

enhance

transcriptional

upregulation

enhanced

transcription

increased

enhanced

inducible

promoter

IFN-alpha

inducible

promoter

IFN-gamma

upregulated

IFN-alpha

expression

upregulated

IFN-gamma

expression

enhance

the described interferon-responsive elements

transcription

enhanced

expression

IFN-alpha

Targeting

Vif

proteasomal degradation

from

transcription

promoter

active

promoter

play an important role

bind

serve as activators,

promoter

serve as activators,

promoter

mediating

transcription

mediating

transcription

active

promoter

inducible

promoter

crucial

GC-box

binding

proteasomal degradation

regulation

promoter

affected

IFN-alpha

60 bp fragment

affected

IFN-gamma

60 bp fragment

influence

IFN-alpha

expression

influence

IFN-gamma

expression

alters

Vif

controlled

controlled

transcription

promoter

unaffected

transcription

Vif

regulation

transcription

regulation

transcription

controlled

transcription

180 bp promoter

regulation

regulation

controlled

promoter with multiple transcriptional start sites

controlled

expression

transcription

transcriptional

transcription

transcription

transcription

transcription

transcription

transcription

transcription

transcription

transcriptional

transcription

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: Sequence of the A3G promoter and the downstream region. The first 1000 bp upstream of the major TSS are shown in lower case, the first 800 bp of the transcribed sequence are shown in upper case. Introns are removed, but their positions are indicated. Arrows refer to transcriptional start sites and their observed frequency is given by the numbers above. The primer binding sites for 5'-RACE analysis and cloning of the luciferase reporter constructs are underlined and the names of the primers are annotated. Gray and black arrowheads define the regions designated E1 and E2 which were cloned into vector pGL3-Promoter and used as EMSA probes. The ATG start codon and the identified Sp1/Sp3 transcription factor binding site are shown in bold.

transcriptional start sites

luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: 5'-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5'-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: Luciferase activities of A3G promoter constructs in different cell lines. (A) A3G promoter 5' deletion constructs of different sizes were cloned into pGL3-Basic luciferase reporter plasmids. Numbering is relative to the major TSS. A putative Sp1/Sp3 consensus site (gray square) is depicted. (B) A3.01 T cells were transiently transfected with the A3G promoter deletion constructs. Numbers on the x-axis refer to the length of the A3G promoter fragments in bp. (C) A3G promoter plasmids were transfected into U937, HepG2 and Huh7 cell lines. Numbers in the legends refer to the length of the A3G promoter fragments in bp. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.

promoter

Luciferase

luciferase

luciferase

luciferase

luciferase

coexpressed

luciferase

coexpressed

luciferase

activities

Luciferase

promoter

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: A3G promoter activities after coexpression of HIV-1 proteins or treatment with TPA or interferons. (A) A3.01 T cells were cotransfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter and 1 microg of Vif expression plasmid, 1 microg Tat expression plasmid or increasing amounts of HIV-1NL4-3 (0.1, 0.5 and 1 microg). After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (B) A3.01 T cells were transiently transfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter or with empty vector. Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 20 ng/ml TPA. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities and the values for the empty vectors (untreated and TPA-stimulated) were set as 1. (C) A3.01 T cells were transiently transfected with the A3G promoter constructs or with the interferon-responsive reporter plasmid pGL2-CVX (GAS). Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 30 ng/ml IFN-alpha or IFN-gamma. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (D) HepG2 cells were used for transfection. The experiment was performed as described in (C). Mean values (+/-SD) of representative experiments performed in triplicate are shown.

Vif

Tat

luciferase

luciferase

luciferase

luciferase

luciferase

luciferase

luciferase

interferon

luciferase

IFN-alpha

IFN-gamma

luciferase

luciferase

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: A GC-box mediates transcriptional activity of the 180 bp core promoter. (A) A3.01 T cells were transfected with reporter plasmid pGL3-Basic containing 180, 150 or 120 bp of the A3G promoter. The two G-to-T substitutions introduced into the GC-box of the 180 bp fragment (180mut) are specified. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown. (B) pGL3-Promoter reporter plasmids containing the regions E1 or E2 (see Figure 1) upstream of the SV40 promoter were transfected into A3.01 T cells. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.

luciferase

luciferase

luciferase

luciferase

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

coexpressed

luciferase

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: Sp1 and Sp3 bind to the GC-box present in the A3G promoter. (A) Nuclear extracts of A3.01 T cells were incubated with a 32P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein-DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). (B) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody-protein-oligo complexes (lanes 3, 4, 7, 8, 11, 12). (C) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.

GC-box

actin

bind

GC-box

bind

GC-box

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input: Silencing of Sp1 and Sp3 reduces A3G promoter activity. (A) HeLa cells were transfected with 150 and 300 ng of unspecific, Sp1-specific or Sp3-specific siRNA. After 48 h, cells were harvested and Sp1 and Sp3 proteins were detected by western blot analysis. As loading control, protein levels of tubulin are shown. (B) HeLa cells were cotransfected with reporter plasmid pGL3-Basic containing 180 or 150 bp of the A3G promoter and siRNA. Hundred nanogram of unspecific siRNA (control), Sp1-specific siRNA (Sp1), Sp3-specific siRNA (Sp3) or a mixture of 50 ng Sp1-specific plus 50 ng Sp3-specific siRNA (Sp1+Sp3) were used. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.

tubulin

luciferase

luciferase

coexpressed

luciferase

coexpressed

luciferase

Silencing

Silencing

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFkappaB activity in vertebrate B-cells
Abstract
To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569-1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFkappaB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology.

phosphorylation

required

regulate

regulate

phosphorylation

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Introduction
The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCgamma and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3-6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8-12].
PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13-16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFkappaB transcription factor and in regulating cell survival during oxidative stress [17,21-23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26-28].
To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFkappaB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.

binding

phosphorylation

contributes

binding

activation

activation

indispensable

phosphorylation

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Cell culture, transient transfections and cell stimulation
The generation, culture and activation of PKD1-/-, PKD3-/- and PKD1/3-/- knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29].

knockout

knockout

knockout

knockout

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
sIgM staining
DT40 B cells (2x106 cells per point) were resuspended in 200mul buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
All results shown are representative of at two to four independent experiments unless otherwise indicated.

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases
DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3-/- cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).
Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3-/- B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.

serine 82

express

express

expression

expression

expressed

expression

expression

expression

Loss

phosphorylation

lack

expression

double knockout

double knockout

defective

phosphorylation

lacking

lacking

knockout

knockout

abolished

induced

lacked

lacked

lacked

deficient

phosphorylation

phosphorylation

serine 82

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

increased

phosphorylation

increase

phosphorylation

induced

phosphorylation

induced

expression

sufficient to restore

induced

regulation

restore

expression

induced

induced

phosphorylation

regulation

phosphorylation

regulation

phosphorylation

regulate

phosphorylation

regulate

phosphorylation

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases
PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3-/-: Flag-PKD3+ve) or absence (PKD1/3-/-) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3-/- cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3-/- B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3-/- cells was slightly slower than that of wild type DT40 cells (12.7+/-2.8h versus 10.2+/-0.4h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3-/- cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.
PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30-32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.

re-expression

re-expression

expression

expression

loss

expression

A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Antigen receptor regulated signalling pathways in PKD-null DT40 B cells
To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3-/- (and in PKD1/3-/-:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3-/- cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3-/- B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33-35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3-/--Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3-/- (and PKD1/3-/--Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.
To search for other potential PKD targets that may show defective regulation in PKD1/3-/- DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3-/- cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3-/- DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.
BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFkappaB transcription factor. Thus, PKD-mediated activation of NFkappaB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFkappaB activation [38]. In B cells, NFkappaB is known to be regulated via DAG and PKCbeta [39,40] but whether PKDs are key intermediaries for NFkappaB regulation has not been explored.
The data (Fig. 4A) show that NFkappaB transcriptional activity was strongly induced in both wild-type and PKD1/3-/- DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFkappaB transcriptional activity was abolished in PKCbeta-/- DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCbeta-/- cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFkappaB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFkappaB.

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Input:
Discussion
Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27. It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFkappaB transcription factors [41]. Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression. We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFkappaB regulation in lymphocytes. We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42]. However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFkappaB transcription factors. These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFkappaB activation [20,23]. Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages. Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.

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Input:
GATA3-Driven Th2 Responses Inhibit TGF-beta1-Induced FOXP3 Expression and the Formation of Regulatory T Cells
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Author Summary
Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells. The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check. The current study focuses on the role of two key transcription factors-FOXP3 and GATA3-in controlling the commitment of these cells. We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells. We show that IL-4-induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter. We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.
It is shown that Th2 responses prevent the generation of inducible Tregs. This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter. This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.

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Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Introduction
Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently. Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively. A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations. The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa. Specifically interleukin 4 (IL-4)-induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2]. GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3]. Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5]. A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4-dominated responses can break antigen-specific immune tolerance [7]. Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.
The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens. Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery. These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines. Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions. These induced Treg (iTreg) cells were reported to express FOXP3 [10]. The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14]. TGF-beta has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-betaRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell-dependent multiorgan inflammatory disease [16]. Although the effect of TGF-beta on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.
The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively. We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo. We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.

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A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

Presence or movement of a biological participant at or to a location.

The location at which the event or its theme is situated.

The destination location to which a participant moves.

Regulation that decreases, suppresses, or prevents its theme.

The site on the cause that participates in a regulatory event.

The participant or event responsible for causing or regulating the event.

The site on the theme where the event occurs.

The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
FOXP3 Induction in T Cell Subsets
It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).
Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.

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A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

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The participant or event responsible for causing or regulating the event.

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The primary participant or event affected by the event.

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Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
FOXP3 and GATA3 Kinetics in Differentiating Cells
The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-gamma, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.

IFN-gamma

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A generic non-protein physical entity used as an event site or location argument.

A gene or gene-product mention, represented by the GENIA Protein label.

Association of two or more biological participants.

The site on the theme where the event occurs; Site2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme2 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme3 distinguishes an additional filler of the same role.

The primary participant or event affected by the event; Theme4 distinguishes an additional filler of the same role.

Expression or production of a gene product.

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The destination location to which a participant moves.

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The primary participant or event affected by the event.

Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

Degradation or catabolic processing of a protein.

Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.