Input:
BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1
Background
Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.
Results
The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.
Conclusion
In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.

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Transcription of a gene or DNA template into RNA.


Input:
Background
Members of the transforming growth factor beta (TGF-beta) superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis [1]. These cytokines can be divided into three subgroups: TGF-beta, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitute the largest family. BMPs are 30-38 kDa hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation [2,3]. Several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues [1]. TGF-beta has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date. [4]. In contrast, the effect of BMPs in the immune system has not been widely investigated. In that respect, BMP- 2, -4 and -7 have been found to control differentiation of hematopoietic stem cells [5] and early T cell development [6,7]. BMP-6 has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells [8]. Furthermore, BMP-2, -4, 6 and -7 had an antiproliferative and a proapoptotic effect on multiple myeloma cells [9-11]. In addition, by gene expression profiling, BMP-6 significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large B cell lymphomas (DLBCL) [12].
BMP-6, like the other BMP members, signals through ligation and heterodimerzation of BMP type I [activin-like-kinase (ALK)] and type II serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating Smad proteins. BMP-6 can signal through the ligation of the type I receptors Act-RIA, BMP-RIA, and BMP-RIB and the type II receptors BMP-RII, Act-RIIA and Act-RIIB, which lead to the phosphorylation of the receptor Smads (Smad-1, Smad-5, and Smad-8). The R- Smads then form complexes with the co-Smad (Smad4) and are translocated into the nucleus where they exert gene regulation [1,13].
Given the reported role of BMP-6 in B-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human B cells. We studied the effects of BMP-6 on proliferation and apoptosis on resting and stimulated B cells. Furthermore, the expression of BMP receptors and BMP-6 induced activation of the Smad signalling pathway with subsequent regulation of the target genes Id1-Id4, were resolved. Finally, we investigated whether B cells also were capable of producing BMP-6.

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Input:
BMP-6 inhibits anti-IgM induced proliferation of human B cells
The effects of BMP-6 on normal and neoplastic hematopoietic cells prompted us to investigate the effects of BMP-6 on normal human B cells. All experiments in this study were performed under serum-free conditions as FCS has been shown to interfere with BMP-signalling [14](own observations). To study the effect of BMP-6 on proliferation, B-cells from healthy volunteers were stimulated with anti-IgM and/or CD40L in the presence or absence of BMP-6 for three days. We found that BMP-6 led to a 35% mean reduction of anti-IgM- induced DNA synthesis (n = 8; p </= 0.0002, Figure 1A). Similar results were obtained for B cells treated with anti-IgM and CD40L (26% mean reduction, n = 6; p </= 0.023). The BMP-6-induced inhibition of proliferation was dose-dependent in both peripheral B cells (Figure 1B) and the Burkitt lymphoma cell line Ramos (40% reduction of DNA synthesis, Figure 1C). The BMP-6 effects could be reversed by addition of the extracellular inhibitor Noggin (Figure 1D). Similarly, a combination of the soluble BMP receptors BMP-RIB-Fc and BMP-RII-Fc also neutralized the effects of BMP-6 (data not shown). Next, we wanted to test whether BMP-6 had different effect on naive and memory B cells. Naive (CD19+CD27-) and memory (CD19+CD27+) B cells were isolated from peripheral blood by cell sorting of immunobead-isolated CD19+ B cells [15], and tested for their capacity to proliferate in the presence of BMP-6. However, BMP-6 inhibited anti-IgM induced DNA synthesis in the two subpopulations to a similar extent, with a mean reduction of DNA-synthesis of 45% (n = 5; p </= 0,004) for naive B cells and 48% (n = 5; p </= 0,001) for memory B cells (Figure 1E).

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Input:
BMP-6 induces cell death in human memory B cells and Ramos cells
Next, we wanted to establish whether BMP-6 also could affect the viability of normal B cells. Cell viability was determined by propidium iodide (PI) staining after culture with or without BMP-6 for 48 hours. Interestingly, BMP-6 showed a small, but reproducible mean increase of cell death from 17 to 23% (n = 5; p </= 0,003) in anti-IgM stimulated CD27+ memory B cells. Furthermore, Ramos cells showed a mean increase in cell death from 20 to 50% (n = 3, p < 0,001, figure 3) after BMP-6 treatment. In contrast, cell death of total CD19+ cells (n = 6; p </= 0,32; data not shown) or CD27-IgG- naive B cells was not significantly affected (n = 5; p </= 0,65, figure 2).

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 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.

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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.


Input:
Human B cells express BMP-6 receptors
Detailed knowledge regarding expression of different BMP receptors in B cells is currently not available. To further elucidate the role of BMPs in human B cells, we performed western blot analysis for type I and type II BMP receptors. This analysis revealed that the type I receptors Act-RIA, BMP-RIB and the type II receptors BMP-RII and Act-RIIb are expressed on resting human B-cells (Figure 4). Ramos cells expressed the type I receptors Act-RIA, weakly BMP-RIB and the type II receptor BMP-RII, but more weakly than normal B cells (Figure 4). HL60 cells were used for comparison and weakly expressed Act-RIA and BMP-RII.
Taken together, these data show that normal human B cells and Ramos cells express a set of BMP receptors, previously shown to bind BMP-6 [16].

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

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Expression or production of a gene product.

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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:
BMP-6 induces phosphorylation of Smad1/5/8
Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor. Type I receptors can signal via several pathways. We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17]. B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points. Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis. Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad. The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5). A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6). Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells. However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).

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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:
BMP-6 induces upregulation of Id1
Next, we wanted to explore whether the BMP-6 induced phosphorylation of Smad 1/5/8 also could induce transcriptional changes of target genes. In this regard, the inhibitors of DNA binding proteins (Ids) are considered to be some of the major target genes for Smad-signalling [17]. Thus, B cells were pre-incubated over night in X-VIVO 15, and then cultured in medium alone or in the presence of BMP-6 for various time points before preparation of total RNA. The amount of Id1-Id4 mRNA was quantified by real-time RT-PCR. Interestingly, we observed a specific four-fold upregulation of Id1 mRNA in BMP-6-treated B cells (Figure 7). The up-regulation of Id1 mRNA was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of BMP-6 and returned to baseline after 24 hours. In contrast, no significant changes were observed for Id2 and Id3 mRNA, whereas Id4-transcripts were not detectable (Figure 7, data not shown).
Western blot analysis revealed that the BMP-6-induced upregulation of Id1 mRNA also was correlated with upregulation of Id1 protein as well. The increase in Id1 protein level was detectable after one hour and increased until 24 hours after BMP-6 addition, showing a 16-fold upregulation compared with t0 (p </= 0.020, n = 4) (Figure 8 and 9). In line with the mRNA data, no consistent change in the amounts of Id2 and Id3 protein could be observed (Figure 8 and 9). We were able to block the Id1 specific band with a blocking peptide (data not shown). Taken together, these data suggest that Id1 could be a possible target gene for mediating the effects of BMP-6 in human B cells, whereas Id2 and Id3 not seem to be involved.

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Expression or production of a gene product.

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Transcription of a gene or DNA template into RNA.


Input:
BMP-6 production in B cells
The fact that BMP-6 has been reported to act as an autocrine stimulator in chondrocytes [18] and ovarium [19], prompted us to investigate whether normal human B cells could produce BMP-6 upon stimulation. Ramos cells, which have been described to express BMP-6 mRNA endogenously [20], and the T cell line Jurkat, served as positive and negative controls, respectively. Endogenous BMP-6 mRNA levels in normal B cells were quantified by real-time RT-PCR after stimulation with anti-IgM for different time points. Interestingly, the up-regulation of BMP-6 mRNA was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-IgM. The level of BMP-6 mRNA was back to baseline after 24 hours upon stimulation (Figure 10). Furthermore, both FCS and human AB-serum induced significant upregulation of BMP-6 mRNA (Figure 11). Interestingly, in a separate study we have found that normal human T cells do not express BMP-6 mRNA after activation (Sivertsen et al, manuscript in preparation). Next, we wanted to detect BMP-6 protein in normal B-cells and tested various commercially available antibodies. However, in our hands these anti-BMP-6 antibodies did only recognize the recombinant BMP-6 protein and not the native protein.

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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.

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Expression or production of a gene product.

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Addition of a phosphate group to a biological molecule.

Regulation that increases, promotes, or enables its theme.

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Regulation whose positive or negative direction is not specified.

Transcription of a gene or DNA template into RNA.


Input:
Discussion
Recent studies have demonstrated an important role for BMP superfamily members in hematopoietic stem cells, early thymocytes [6,7] and B-cell malignancies [8,11,12], but a role for BMPs in normal human B cells has previously not been reported. The present study demonstrated a significant antiproliferative effect of BMP-6 in peripheral blood CD19+ B cells. Additionally, BMP-6 induced cell death in CD27+ memory B cells as well as in a Burkitt lymphoma cell line (Ramos). Importantly, BMP-6 induced a rapid and marked increase in Smad-1/5/8 phosphorylation. Furthermore, the BMP-6 induced Smad phosphorylation was followed by a selective upregulation of Id1 mRNA and subsequent Id1 protein.
In the present study, the demonstrated antiproliferative effect of BMP-6 in anti-IgM treated B cells was significant and dose-dependent. Importantly, the anti-proliferative effect of BMP-6 could be completely neutralized by the use of a natural inhibitor, Noggin. This is in line with others, showing that Noggin can function as a BMP-6 antagonist [21,22]. In addition, the combination of soluble BMP-RIB-Fc and BMP-RII-Fc fusion proteins also neutralized the anti-proliferative effect of BMP-6 in human B cells. Interestingly, as for other TGF family members, bifunctional effects have also been demonstrated for BMPs. Whereas several of the BMPs have been shown to promote proliferation in various cell types including condrocytes [23], liver [24] and granulosa cells [25], antiproliferative effects and induction of apoptosis has been reported for B and T lineage cells. Similar effects as demonstrated for BMP-6 on human B cells in the present study, were demonstrated for BMP-2, 4, 6 and -7 in human myeloma cells [9-11]. Other members of the BMP-family have also been reported to induce apoptosis, including in mouse B lineage cells [26]. Additionally, BMP-4 inhibits thymocyte proliferation [6]. Taken together, these data suggest that the role of BMPs in the regulation of proliferation and apoptosis is highly cell type dependent.
To examine how BMP-6 exerts its functional effects in B cells, we analysed BMP receptor expression by western blot analysis. Human peripheral B cells were found to express the BMP type I receptors Act-RIA and BMP-RIB, and the type II-receptors BMP-RII and Act-RIIb, which signal after binding of several BMPs, including BMP-6 [16,13]. To further explore BMP-6 induced signalling, activation of several pathways is possible. The major signalling pathway known to date, is activation of R-Smads [13,27]. In that respect, BMPs have been shown to exert antiproliferative effects in B lineage cells via phosphorylation of R-Smad [11,28]. Furthermore, BMP-2 has been shown to induce activation of STAT3 in myeloma cells [9]. However, phosphorylation of R-Smad was not investigated in that study. BMP-2 has also been shown to induce phosphorylation of p38 [29]. Thus, phosphorylation of p38, STAT3 and Smad1/5/8 represent important BMP-signalling pathways that mediated the effects of BMPs and even cross-talk between these pathways has been reported [29,30]. In the present study, we were not able to detect BMP-6-induced changes in the phosphorylation status of STAT3 or p38 in human peripheral B cells. Instead, a rapid and marked phosphorylation of Smad1/5/8 was revealed. In a parallel study, we have found that other BMPs also induced phosphorylation of Smad1/5/8 in peripheral B cells (data not shown). We are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various BMPs in human B cells.
Upregulation of Id1 via Smad1/5/8 phosphorylation is a known mechanism for BMP-6 signalling in other cell systems [31,32] and regulation of Id-proteins is thought to be an important mechanism for Smad-signalling [17]. In the present study, real-time RT-PCR experiments revealed a specific four-fold upregulation for Id1 in BMP-6-treated B cells, while the amount of Id2-Id4 remained unchanged. In agreement with this, western blot analysis demonstrated an upregulation of Id1 protein, while the amount of Id2 and Id3 protein levels remained unchanged. Previously, Id1 has been considered not to be expressed in later developmental stages than pro-B cells [33,34], and its constitutive expression has been reported to impair mouse B cell development [35]. Therefore, our demonstration of the time-dependent upregulation of Id1 mRNA and protein in mature normal human B cells is of particular interest. In that respect, it is noteworthy that TGF-beta signalling in early and mature B cells induces both Id2 and Id3 expression [36,37], but not Id1 (data not shown). Interestingly, these results show that various members of the TGF-beta family regulate Id proteins differently. Id2 and Id3 are considered to be the Id proteins mainly expressed in mature B cells [38]. The present study also found Id2 and Id3 protein in B cells to be more highly expressed than Id1 in resting B cells. However, BMP-6 did not induce significant changes in the protein expression of Id2 and Id3. It is believed that Id proteins block differentiation and promote proliferation in various cell types [39,33]. Id proteins act as dominant-negative inhibitors of E-proteins and Pax5 function by forming dimers with these proteins, making them unable to bind DNA. It has been proposed that the balance among E-proteins, Pax5 and Id proteins might have an important role in activated B cells [38]. In that respect, E-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development [40]. The antiproliferative and death inducing effect of BMP-6 in B cells with concomitant upregulation of Id1 protein is therefore in line with the view that Id proteins are required for the induction of growth arrest and apoptosis in B-lymphocyte progenitors by TGF-beta [40].
Furthermore, Id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis [32]. It is well established that various members of the Id family are overexpressed in a range of human tumours and generally, Id1 appears to be the family member most widely overexpressed in a variety of human malignancies [41], including multiple myeloma [42,32]. Additionally, our findings that BMP-6 activates intracellular signalling pathways in human B cells might be of potential pathophysiological significance in lymphoma and inflammation. High BMP-6 mRNA expression in DLBCL has been shown to correlate to unfavourable outcome [12]. In this respect, it is of interest that targeted expression of Id1 to B-lymphocytes resulted in aberrant B cell development, massive apoptosis, and subsequent development of B cell lymphomas [35]. Moreover, BMP-6 has been suggested to play a role in rheumatoid arthritis (RA) [43,44] and elevated levels of Id1 and Id3 have been found in the synovia of RA-patients [45]. Altogether, these results point to an important role for Id proteins in the regulation of normal B cell homeostasis and in diseases, where B cells are involved. It will therefore be important to further elucidate the role of Id-1 in human B cells by selective over expression or inhibition of Id-1 gene expression.
Given the role of BMP-6 in mature human B cells demonstrated here, identification of BMP-6 producing cells in vivo with possibility of interaction with naive and memory B cells might contribute to the understanding of mature B cell biology. High BMP-6 mRNA expression in DLBCL has been detected by gene expression profiling [12]. Furthermore, production of BMP-6 transcripts in normal activated B cells was detected in the same study. Of note, an autocrine BMP-6 loop has been reported by others in chondrocytes and in the ovarium [46,19,18]. Therefore, we wanted to explore the possibility for an autocrine BMP-6 loop in human B cells. We analysed the expression BMP-6 mRNA in peripheral blood B cells by real-time PCR, and report here the upregulation of endogenous BMP-6 transcripts after stimulation with FCS, human AB-serum and, most importantly, anti-IgM. However, our attempts to study BMP-6 protein levels were unsuccessful due to problems with unspecific binding of the anti-BMP-6 antibodies tested, and lack of specific staining in control cells known to express BMP-6 mRNA. In contrast, the recombinant protein was readily detected. In that respect, few investigators have detected BMP-6 protein in humans, especially in non-pathogenic tissue. The possibility of BMP-6 production in human B-cells is in line with a recent work that reported the production of BMP-6 in mouse B cells, infiltrating the bone marrow of mice with inflammatory arthritis [43]. In this study, a role for BMPs in the inflammatoric process of arthritis was suggested. The upregulation of the BMP-6-transcripts after IgM-crosslinking is of pathophysiologic interest [12]. A loss of TGF-beta-responsiveness has been suggested to be a critical contribution to malignant transformation [47,48] and similar oncogenic mechanisms have been postulated for BMPs. Lines of evidence suggest [49] that at early stages of carcinogenesis, BMP-6 is not a tumour promoter, but suppresses benign and malignant tumour outgrowth. These findings are in good agreement with previous findings for other TGF-beta family members, including TGF-beta1 and BMP-4 [50], indicating that cellular context of the BMP target cell might define the various observed effects. In contrast to the upregulation of BMP-6 transcript in B cells, we were not able to detect BMP-6 transcripts in human peripheral blood CD4+ or CD8+ T cells (resting or stimulated with anti-CD3 and anti-CD28; data not shown), consistent with the findings in T cell lines [20] and T cells in mice [43]. Other potential BMP-6 sources for mature B cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system. One well recognized source for BMP-6 production is the human bone and bone marrow stroma [51,8]. Furthermore, it is noteworthy that human umbilical vein endothelial cells (HUVEC) highly express BMP-6 mRNA [52], and vascular endothelium has been reported to produce BMP-6 [53]. These studies might imply a role for BMP-6 in transendothelial migration of B cells. BMP-6 mRNA has been demonstrated in murine macrophage cell lines, but not in humans [54]. In accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the BMP-6 transcript [20]. To our knowledge, there is currently no report about BMP-6 production of human dendritic cells.

Smad

Noggin

Noggin

BMP-RIB

BMP-RII

Act-RIA

BMP-RIB

BMP-RII

Act-RIIb

TGF-beta

TGF-beta

TGF-beta

TGF-beta

TGF-beta

binding

Act-RIA

binding

BMP-RIB

binding

BMP-RII

binding

Act-RIIb

forming dimers

bind

express

Act-RIA

express

BMP-RIB

express

BMP-RII

express

Act-RIIb

amount

amount

amount

amount

expressed

expression

expression

expression

expressed

expressed

expressed

expressed

expressed

expression

expression

expression

levels

levels

expression

expression

producing

detected

production

production

production

produce

production

antagonist

Noggin

inhibitors

forming dimers

inhibition

expression

phosphorylation

phosphorylation

phosphorylation

phosphorylation

Smad

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

induced

increase

increase

phosphorylation

induced

phosphorylation

followed

phosphorylation

upregulation

upregulation

upregulation

induce

activation

activation

induced

changes

induced

phosphorylation

Upregulation

phosphorylation

upregulation

remained unchanged

amount

upregulation

remained unchanged

amount

upregulation

induces

expression

induces

induce

changes

making

forming dimers

bind

upregulation

overexpressed

High

mRNA expression

elevated

levels

over

expression

High

mRNA expression

upregulation

upregulation

upregulation

highly

express

changes

phosphorylation

mechanism

Upregulation

changes

expression

autocrine

autocrine

mRNA expression

mRNA expression

production

expression

express

detect

express

demonstrated

negative

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:
Conclusion
In conclusion, our results show that BMP-6 induces activation of intracellular Smad signalling in mature human B-cells with consecutive production of Id1 protein. Furthermore, we report that BMP-6 has an antiproliferative effect in B cells stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induces cell death in activated memory B cells and Ramos cells. Taken together, these results provide a rationale to further examine the role of BMP-6 signalling in normal B cell biology as well as in pathologic conditions like B cell malignancies and autoimmune disorders.

production

consecutive

production

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
If not specified, all cells were cultured in X-VIVO 15trade (BioWhittaker, Verviers, Belgium) serum-free medium at 37degreesC and 5% CO2 in air.
Peripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee. Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55]. This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.
The following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15trade when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).

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:
Growth factors/supplements
The following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 mug/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 mug/ml), rhu BMPR-II/Fc Chimera (5 mug/ml), and recombinant mouse Noggin (5 mug/ml) were purchased from R&D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 mug/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).

BMP-RIB

BMPR-II

Noggin

CD40 ligand

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:
Antibodies used for flow cytometric analysis and immunoblot analysis
Antibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R & D Systems (Abingdon, UK). Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).
Characterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA). Expression levels of Id1-3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA). As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis. Anti-beta-actin was from Santa-Cruz. From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC. The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).

Act-RIA

BMP-RIB

BMPR-II

Act-RIIA

Act-RIIb

beta-actin

Expression levels

Expression levels

blocked

Expression levels

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 sorting
Highly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4degreesC, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.

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:
Western-blot analysis
B cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, beta-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol). Total protein (30-100 mug) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher &Schuell GmbH, Dassel, Germany). Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT). After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT. Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK). Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to beta-actin using Image Quant 5.5 software (Molecular Dynamics).

beta-actin

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:
Analysis of BMP-6 messenger RNA (mRNA) expression
Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. Total RNA was isolated using Absolutely RNAtrade RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions. Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements. Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan(R) Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A). To measure mRNA expression of BMP6, Id1-Id4 and PGK1 PCR were carried out with TaqMan(R) universal master mix. Primers and probes were provided by Assay-on-Demand (Applied Biosystems). PCR reactions were carried out in a final volume of 25 mul (BMP-6) or 20 mul (ID1). The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA. The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems). The expression was then normalized to the expression level of PGK1. PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56]. Expression levels in B cells were then related to the expression levels in Ramos cells.

expression

expression

expression

expression

have low expression

expression

expression

expression

expression

mRNA expression

mRNA expression

mRNA expression

mRNA 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:
Cell proliferation
For estimation of DNA synthesis, CD19+ cells (7.5 x 104 cells/0.2 ml) or Ramos cells (1 x 104 cells/0.2 ml) were cultured in triplicate in microtiter wells. The cells were pulsed with 3.7 x 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation. The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).

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:
Determination of cell death
Cell death was measured by vital dye exclusion test by staining cells with 5 mug/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice. At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.

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:
Statistical analysis
The statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered significant.

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 regulate basal transcription of the human APOBEC3G gene
APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.

promoter

GC-box

Luciferase

luciferase

specificity protein (Sp) 1

binding

GC-box

specificity protein (Sp) 1

binding

GC-box

expressed

inducible

promoter

regulate

transcription

regulate

transcription

regulation

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:
INTRODUCTION
The recently discovered APOBEC3 family of cytidine deaminases is considered to play an important role in antiviral intrinsic immunity (1,2). In primates, the seven paralogs APOBEC3A, B, C, DE, F, G, H (A3A-H) have been described (3), and they appear to fulfill individual functions. Human APOBEC3G (A3G), the most prominent member of the APOBEC3 family has been identified as the cellular restriction factor that is responsible for inhibition of virion infectivity factor (Vif)-deleted human immunodeficiency virus-1 (HIV-1) replication in non-permissive cells (4). A3G is packaged into HIV-1Deltavif particles and causes C-to-U deaminations on the single-stranded viral DNA during reverse transcription (5-8). This leads to degradation of the uracile-containing DNA by cellular repair mechanisms or to hypermutation of the viral genome (5,6). As a result, only a marginal fraction of the A3G-containing HIV-1 particles is able to complete the replication cycle. In addition to the inhibition of HIV-1, A3G restricts replication of other lentiviruses, gammaretroviruses, deltaretroviruses, spumaviruses, long-terminal-repeat (LTR)-retrotransposons, orthohepadnaviruses and avihepadnaviruses (9-21). Interestingly, deamination seems not to be the only A3G-mediated antiviral mechanism; in the case of hepatitis B virus (HBV) and human T cell leukemia virus type 1 (HTLV-1), A3G was shown to restrict virus replication by deamination-independent mechanisms (12, 13, 19, 22-25). Another member of the APOBEC3 family, APOBEC3F (A3F), appears to have similar activities like A3G (26,27). A3F is also packaged into HIV-1Deltavif particles and induces similar C-to-U deaminations, although the proteins differ in their target sequences specificity (26,28). Furthermore, A3F proteins were detected in many tissues that express A3G and are able to form heteromultimers with A3G (26,29,30). Both proteins localize to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of non-translating mRNAs (30,31).
A3G has been shown to be expressed in T cells, a relevant cell target for HIV-1 in vivo, but little is known about its regulation (4,29,32). There is a report describing that mitogenic stimulation of T cells upregulates A3G mRNA levels, but this was not analyzed on the transcriptional level (33). Since the A3G promoter has not been systematically analyzed so far, our aim was to clone the A3G promoter and characterize its regulation in T cells. In our study, we observed that A3G uses multiple transcriptional start sites (TSS). By generating a series of 5' deletions of the A3G promoter, we identified a 180 bp region that mediated basal transcription. In T cells, transcriptional activity of this core promoter was not inducible by mitogenic stimulation or interferon treatment, but was dependent on a GC-box which was recognized by Sp (specificity protein) 1 and Sp3 transcription factors.

mRNA processing (P) bodies

promoter

GC-box

B

C

DE

F

G

H

virion infectivity factor

Vif

vif

vif

interferon

Sp (specificity protein) 1

form heteromultimers

recognized

GC-box

Sp (specificity protein) 1

recognized

GC-box

express

express

expressed

mRNA levels

localize

mRNA processing (P) bodies

localize

mRNA processing (P) bodies

deleted

virion infectivity factor

upregulates

mRNA levels

regulation

regulation

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:
Cell culture
The human T cell lines A3.01 and PM1 (NIBSC, UK) and the human myeloid cell line U937 (NIBSC, UK) were grown in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. The human hepatic cell lines HepG2 and Huh7 (kindly provided by Dr Thomas Pietschmann, Department of Molecular Virology, University of Heidelberg) as well as HeLa cells were maintained in Dulbecco's high glucose modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. Cells lines were incubated at 37degreesC with 100% humidity in 5-7% CO2 and passaged using standard cell culture techniques.

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:
Plasmids
For cloning of an APOBEC3G promoter-driven reporter plasmid, genomic DNA was prepared from the T cell line PM1 using the DNeasy Kit (Qiagen). The DNA sequence ranging from positions -959 to +66 relative to the identified transcription start was amplified via PCR using the primers 3Gprom1025 (5'-TGTGAACGCGTTGCTGCAGGCCATCTGGATGTATATG-3') and 3Gpromreverse (5'-ACAGCAGATCTAGGGACCTCTGATAAAGACAGG-3'). PCR reactions were performed with Pwo DNA Polymerase (Roche) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 58degreesC for 60 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. The amplicon was ligated into the promoterless luciferase reporter plasmid pGL3-Basic (Promega) via MluI and BglII restriction sites, which were introduced by the primers. The resulting construct contained 1025 bp of the A3G promoter and was designated pGL3-APOprom1025. Reporter plasmids containing shorter fragments of the APOBEC3G promoter were constructed using pGL3-APOprom1025 as template and the following forward primers: for plasmid pGL3-APOprom502 (containing sequence -436/+66): 3Gprom502 (5'-TGTGAACGCGTTCCATAACATGGGGACAAGA-3'); for plasmid pGL3-APOprom225 (containing sequence -159/+66): 3Gprom225 (5'-TGTGAACGCGTCGAGGGCAGGATCCGGGAGT-3'); for plasmid pGL3-APOprom180 (containing sequence -114/+66): 3Gprom180 (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGG-3'); for plasmid pGL3-APOprom150 (containing sequence -84/+66): 3Gprom150 (5'-TGTGAACGCGTGCGGGACCACCAGGGGAGGGGCTT-3'); for plasmid pGL3-APOprom120 (containing sequence -54/+66): 3Gprom120 (5'-TGTGAACGCGTTGCTGGCTCAGCCTGGTGTG-3'); for plasmid pGL3-APOprom60 (containing sequence +7/+66): 3Gprom60 (5'-TGTGAACGCGTCCCTTTGCAATTGCCTTG-3'); each in combination with the reverse primer 3Gpromreverse (described above). PCR reactions were performed with Pfu Ultra Hotstart (Stratagene) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 45 s, 58degreesC for 45 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. As for pGL3-APOprom1025, MluI and BglII restriction sites were introduced via the primers and PCR products were ligated into pGL3-Basic (Promega) via these restriction sites. pGL3-APOprom180mut carries two point mutations (bold) and was generated using the primer 3GProm180mut (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGTTCGGGACCACCAG-3') in combination with primer 3Gpromreverse. This PCR was performed with an annealing temperature of 65degreesC. pGL3promE1 (containing nucleotides -114/-85) and pGL3promE2 (containing nucleotides -92/-63) were constructed by annealing the following single-stranded oligonucleotides: 114_85Plus (5'- CGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGGA-3') and 114_85Minus (5'- GATCTCCAGCTGGAGCCTCCTCTCCACCATCAAGAA-3') or 92-63Plus (5'-CGCGTCCAGCTGGGCGGGACCACCAGGGGAGGGGCA-3') and 92_63Minus (5'-GATCTGCCCCTCCCCTGGTGGTCCCGCCCAGCTGGA-3'). After annealing, the double-stranded oligonucleotides which contained the respective 30 bp of the APOBEC3G promoter and sticky ends compatible with MluI and BglII restriction sites were ligated into the pGL3-Promoter (Promega) vector. The sequences of all constructed plasmids were verified by sequence analysis. Nucleotide -219 of the cloned APOBEC3G promoter differs from the sequence in the database (GenBanktrade accession number DQ147772). An A-to-C substitution is present at this position. Numbering is relative to the major transcriptional start site we identified.
The reporter plasmids pGL3-Control and phRG-TK were purchased from Promega. pGL2-CVX contains two repeats of the IFN-responsive GAS (gamma activated sequence) elements (GATCTGGATTTAGAGTAATATGAAACTGAAAGTACTTCG) of the guanylate-binding protein (GBP) gene in front of a CMV minimal promoter and was kindly provided by Ute Pagelow and Mario Koster from the Helmholtz-Zentrum fur Infektionsforschung. Plasmid pNL4-3 (NIBSC, UK) contains the full-length HIV-1NL4-3 genome and has been described previously (34). pcDNA3.1Vif was generously provided by Nathaniel R. Landau from the Salk Institute, La Jolla. It was generated by amplifying the Vif gene from pNL4-3 and ligating it into the pcDNA3.1 vector via BamHI and XhoI restriction sites. A 3'-WPRE element was included into the XhoI site. pBS-kRSPA-TatHIV-1(NL4-3) was constructed by amplifying the two exons of Tat via PCR reaction using the molecular clone pNL4-3 as template and the following primer sets: exon1, 5UXho1HIV-Tat 1Plus (5'-GCATGCTCGAGATGGAGCCAGTAGATCCTAG-3') and HIV-Tat 1Minus (5'-TGCTTTGATAGAGAAGCTTGATG-3'); exon2, 15FHIV-Tat2Plus (5'-TTCTCTATCAAAGCAACCCACCTCCCAATCCCG-3') and 5USpe1HIV-Tat2Minus (5'-GACGTACTAGTCTATTCCTTCGGGCCTGTC-3'). XhoI and SpeI restriction sites were introduced via the primers. The sense-primer of exon2 starts with a 15-mer which is homologous to the 3' end of exon1 and necessary for fusion of both exons. PCRs were performed with Expand High Fidelity PCR System (Roche) using the following conditions: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 55degreesC for 45 s, 68degreesC for 45 s; one cycle 68degreesC for 7 min. For fusion of both exons, the following PCR conditions were applied: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 58degreesC for 45 s, 68degreesC for 60 s. After 10 cycles without primers, the sense-primer of exon1 and the antisense-primer of exon2 were added for the remaining cycles. The resulting amplicon was ligated into the pBS-kRSPA vector (35) via XhoI and SpeI restriction sites.

Pwo

luciferase

Pfu

guanylate-binding protein

GBP

Vif

Tat

Tat

Tat

Tat

Tat

Tat

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'-Rapid amplification of cDNA ends analysis (RACE)
Total RNA was isolated from A3.01 T cells using RNeasy mini kit (Qiagen). The transcriptional start sites of A3G were identified using the 5'/3' RACE Kit, 2nd Generation (Roche) according to the manufacturer's instructions. The following primers were used: RACE-APO3G1 (5'-TATCCCTTGTACACTTTGT-3') for cDNA synthesis, RACE-APO3G2 (5'-CATACTCCTGGTCACGAT-3') for the first PCR and RACE-APO3Gnest (5'-GAATACACCTGGCCTCGAA-3') for the nested PCR. Reaction products were analyzed by agarose gel electrophoresis, purified using QIAquick gel extraction kit (Qiagen), T/A-cloned into vector pCR4-TOPO (Invitrogen) 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 assay
For transient transfection of A3.01 and U937 cells, DMRIE-C transfection reagent (Life Technologies) was used (36). Cells were seeded in 6-well tissue culture plates (5 x 105 cells per well) in 1.5 ml Opti-MEM (Life Technologies) containing 0.5 microg firefly luciferase reporter plasmid and 3.5 microl DMRIE-C. After 4-5 h of incubation, 1.5 ml complete RPMI medium were added. HepG2 and Huh7 cell lines were transfected using LipofectAMINE Plus as recommended by the manufacturer (Life Technologies). Briefly, exponential growing cells (1.5 x 106) were transfected with 5 microl LipofectAMINE, 6 microl PLUS reagent and the required amount of plasmid DNA in a final volume of 1 ml Opti-MEM. Following 4 h of incubation, cells were washed in PBS and 3 ml of complete DMEM medium were added.
For cotransfection of reporter plasmids and siRNA into HeLa cells, HiPerfect transfection reagent (Qiagen) was used according to the manufacturer's protocol for cotransfection of adherent cells with siRNA and plasmid DNA.
Two days after transfection of the respective cell lines, cells were harvested in 100 microl (suspension cells) or 300 microl (adherent cells) of Passive Lysis Buffer (Promega) and luciferase assay was performed using the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. As an internal control, 50 ng (adherent cells) or 100 ng (suspension cells) of ph-RG-TK plasmid (Promega), which constitutively expresses renilla luciferase was cotransfected in every sample and firefly luciferase activities were normalized to renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown in the figures. For stimulation of cells, final concentrations of 20 ng/ml TPA (Sigma) or 30 ng/ml IFN-alpha or 30 ng/ml IFN-gamma (Tebu-Bio) were applied approximately 15 h before harvesting for luciferase assay.

Luciferase

luciferase

luciferase

luciferase

luciferase

luciferase

IFN-alpha

IFN-gamma

luciferase

expresses

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:
Electrophoretic mobility shift assay (EMSA)
For preparation of nuclear extracts, 5 x 106 A3.01 T cells were washed in cold PBS and resuspended in 500 microl buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After incubation for 15 min on ice, swollen cells were pressed 10 times through a syringe with a 26G needle and centrifuged at 5000 r.p.m. for 5 min. Pellets contained the nuclei and were washed in buffer A for two times and resuspended in 50 microl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA 1 mM DTT, 1 mM PMSF). After shaking for 30 min at 4degreesC and centrifugation for 10 min at 13 000 r.p.m., supernatants were used as nuclear extracts.
EMSA probes were generated by annealing the following complementary oligonucleotides: APO-Sp1/3, 5'- CCAGCTGGGCGGGACCACCAGGGGAGGGGC-3' and 5'-GCCCCTCCCCTGGTGGTCCCGCCCAGCTGG-3'; APO-Sp1/3mut, 5'- CCAGCTGTTCGGGACCACCAGGGGAGGGGC-3' and 5'- GCCCCTCCCCTGGTGGTCCCGAACAGCTGG-3' according to standard procedures. Nucleotides differing from the original promoter sequence are shown in bold type. A commercially available Sp1 probe (sc-2502, referred to as Sp1cons) was purchased from Santa Cruz Biotechnology. The double-stranded oligonucleotides were 5' end-labeled using T4 polynucleotide kinase (New England Biolabs) and [gamma-32P]ATP (3000 Ci/mmol, Amersham) and purified by using Nick G50 columns (Amersham).
For EMSA, 5 microg of nuclear proteins were preincubated on ice with 2 microg of poly(dI-dC) (Roche) as an unspecific competitor and 1 microg of bovine serum albumin in band shift buffer (50 mM Tris, 150 mM KCl, 5 mM EDTA, 2.5 mM dithiothreitol, 20% Ficoll) for 15 min. 32P-labeled oligonucleotides (50 000 c.p.m.) were added in a total volume of 20 microl, incubated on ice for 20 min and loaded onto 5% native polyacrylamide gels in 0.5xTris-borate-EDTA buffer. Upon fractionation, gels were dried and exposed for autoradiography. For competition experiments, 1- or 30-fold molar excess of the unlabeled APO-Sp1/3 or APO-Sp1/3mut oligonucleotides was added to the preincubation mixture. For supershift experiments, 2 microg Sp1 antibody (sc-59x, Santa Cruz Biotechnology) or Sp3 antibody (sc-644x, Santa Cruz Biotechnology) were added to the preincubation mixture and preincubation time was extended to 30 min.

T4 polynucleotide kinase

bovine serum albumin

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:
Chromatin immunoprecipitation (ChIP) assay
A3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37degreesC. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 x 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 microl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 x 107 cells and 2 microg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 microl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 microl of the resolved DNA in the first PCR, and 1 microl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5'-ccacggtggcctccgagggtga-3' and ChIP3Gminus: 5'-ctctccaccatcaagacagac-3' (1. PCR); ChIP3G2plus: 5'-tactctccctccctgtcccca-3' and ChIP3G nested minus: 5'-aggctgatgcctccgcag-3' (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 60degreesC for 60 s, 72degreesC for 2 min; one cycle 72degreesC for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5'-taagtaccacccagagatgag-3' and ChIP3Gneg_minus: 5'-catgatcttcatggtggcacg-3' for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55degreesC.

Micrococcal Nuclease

actin

Taq polymerase

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:
RNA interference and western blot analysis
Sp1 and Sp3 translation was silenced in HeLa cells using the siRNA duplexes Hs_SP1_1_HP and Hs_SP3_1_HP (Qiagen). A nonspecific siRNA (Qiagen) was used as control. HeLa cells were transfected with 150 or 300 ng siRNA per 6-well, using the HiPerfect transfection reagent (Qiagen) according to the manufacturer's protocol for reverse transfection of adherent cells in 6-well plates.
Forty-eight hours after transfection, HeLa cells were harvested for detection of Sp1 and Sp3 proteins. Cells were washed in PBS, lysed in RIPA (25 mM Tris pH 8.0, 137 mM NaCl, 1% Glycerol, 0.5% sodium deoxycholate, 1% NP-40, 2 mM EDTA pH 8, 0.1% SDS and protease inhibitors) and lysates were cleared by centrifugation. After boiling with Laemmli's buffer, samples were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Sp1 and Sp3 proteins were detected using alpha-Sp1(Pep2) antibody (sc-59, Santa Cruz) or alpha-Sp3(D-20) antibody (sc-644, Santa Cruz) followed by incubation with alpha-rabbit-HRP (Amersham Biosciences). For detection of tubulin, alpha-tubulin (B5-1-2, Sigma) and alpha-mouse-HRP (Amersham Biosciences) antibodies were used. Signals were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).

tubulin

tubulin

detection

detection

detected

detected

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:
Characterization of the transcriptional start sites of APOBEC3G by 5'-RACE
To identify the transcriptional start sites (TSS) of APOBEC3G (A3G) in A3.01 T cells, we performed 5'-rapid amplification of cDNA ends analysis (RACE) with A3G-specific primers (see Figure 1). Agarose gel electrophoresis resolved the nested PCR products into three bands of different electrophoretic mobility with a dominant middle band (Figure 2). For each band, the DNA was cloned and sequence analysis of six or seven individual transformants was performed. We observed that the transcriptional start sites of the A3G gene were located between 58 and 361 nt upstream of the ATG start codon (Figure 1). Although TSS were variable and most sites were only detected once among the 19 clones analyzed, one TSS was identified in six individual clones. This TSS was located 66 nt upstream of the start of the published A3G mRNA sequence (GenBanktrade accession number NM021822) and we defined this position as the major transcriptional start site of the A3G gene.

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.