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Twist-2 controls myeloid lineage development and function.

Sharabi AB, Aldrich M, Sosic D, Olson EN, Friedman AD, Lee SH, Chen SY - PLoS Biol. (2008)

Bottom Line: Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells.In addition, Twist-2 was found to be essential for endotoxin tolerance.Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT
Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

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Twist-2 Inhibits the Transcriptional Activity of Runx1 and C/EBPα(A) Immunoassay of COS7 cells transiently transfected with indicated plasmids. After 48 h, cells were lysed in NTN buffer containing 0.5% NP-40 and 0.5% Triton X-100. Cell lysates were analyzed by immunoblotting (IB) with anti-FLAG or anti-myc antibodies (upper two panels). Cell lysates were also immunoprecipitated (IP) with anti-myc antibody followed by immunoblotting with anti-FLAG antibody (lower panel).(B and C) COS7 cells were transiently transfected with a Runx1-responsive luciferase reporter (CBF4_Luc, 1 μg), or Runx1 expression plasmid (Runx1, 1 μg), or indicated amounts of Twist expression constructs. pcDNA3.1 blank vector control was cotransfected so that each well received the same total amount of DNA (4 μg). After 24 h, luciferase activity in cell extracts was quantified via luciferase assay (B). Runx1 responsive luciferase reporter cotransfected with empty vector control or Twist-2 expression vector only (C). Data are presented as mean ± SD and are representative of three independent experiments.(D and E) Luciferase reporter assays were performed on cells transfected with indicated plasmids (1 μg each). Twist-2 vector cotransfection inhibited the activity of a C/EBPα-dependent luciferase reporter in COS7 cells ([D] left panel), but not a PU.1-dependent luciferase reporter ([D] right panel). A CREB-responsive luciferase reporter from the PEPCK promoter cotransfected with empty vector control or Twist-2 expression vector only (E). Data are presented as mean ± SD, and the results are representative of three independent experiments.
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pbio-0060316-g004: Twist-2 Inhibits the Transcriptional Activity of Runx1 and C/EBPα(A) Immunoassay of COS7 cells transiently transfected with indicated plasmids. After 48 h, cells were lysed in NTN buffer containing 0.5% NP-40 and 0.5% Triton X-100. Cell lysates were analyzed by immunoblotting (IB) with anti-FLAG or anti-myc antibodies (upper two panels). Cell lysates were also immunoprecipitated (IP) with anti-myc antibody followed by immunoblotting with anti-FLAG antibody (lower panel).(B and C) COS7 cells were transiently transfected with a Runx1-responsive luciferase reporter (CBF4_Luc, 1 μg), or Runx1 expression plasmid (Runx1, 1 μg), or indicated amounts of Twist expression constructs. pcDNA3.1 blank vector control was cotransfected so that each well received the same total amount of DNA (4 μg). After 24 h, luciferase activity in cell extracts was quantified via luciferase assay (B). Runx1 responsive luciferase reporter cotransfected with empty vector control or Twist-2 expression vector only (C). Data are presented as mean ± SD and are representative of three independent experiments.(D and E) Luciferase reporter assays were performed on cells transfected with indicated plasmids (1 μg each). Twist-2 vector cotransfection inhibited the activity of a C/EBPα-dependent luciferase reporter in COS7 cells ([D] left panel), but not a PU.1-dependent luciferase reporter ([D] right panel). A CREB-responsive luciferase reporter from the PEPCK promoter cotransfected with empty vector control or Twist-2 expression vector only (E). Data are presented as mean ± SD, and the results are representative of three independent experiments.

Mentions: Twist-2 forms homodimers or heterodimers with ubiquitously expressed members of the E2A bHLH family [31,45], and can bind E-box consensus sites present in target gene promoters or can directly bind transcriptions factors such as MEF2, Runx2, and NF-κB via its C-terminal domain [31,35,46]. The Twist family has been previously shown to bind the runt homology domain of the Runx2 family member and inhibit its transcriptional activity [46,47]. Given that Runx1 is a known regulator of hematopoiesis, this prompted us to examine whether Runx1 transcriptional activity may be regulated by Twist-2. In order to address this possibility, COS7 cells were transfected with constructs encoding FLAG-tagged Twist-2 and Myc-tagged Runx1. Upon immunoprecipitation of Runx1, we detected Flag-tagged Twist-2 by coimmunoprecipitation (Figure 4A). To investigate the functionality of this interaction, COS7 cells were transfected with a Runx-dependent luciferase reporter construct containing four core binding factor consensus sites. Interestingly, we observed a basal level of reporter activity in COS7 cells, which could be due to a low-level endogenous expression of Runx family members. Cotransfection of Twist-2 resulted in a significant dose-response inhibition of Runx1 reporter activity that was able to reduce the promoter activity to near basal levels (Figure 4B). Neither Twist-2 vector nor empty vector pcDNA alone inhibited the activity of the Runx1-responsive luciferase reporter (CBF4_Luc) (Figure 4C). In addition, neither Twist-2 vector nor empty vector pcDNA inhibited the activity of an unrelated CREB-responsive luciferase reporter (Figure 4E). These data suggest the specificity of Runx1 inhibition by Twist-2. We also observed a subtle increase in Runx1-specific DNA binding in GMP from Twist-2 KO mice as compared to WT controls using electrophoretic mobility shift assays (EMSA) (Figure S2). Since multiple transcription factors play critical roles in myeloid lineage development, we examined the effect of Twist-2 on the transcription factors PU.1 and C/EBPα, which are required for myeloid lineage development. We found that Twist-2 inhibited the transcriptional activity of C/EBPα, but did not significantly affect the transcriptional activity of PU.1 (Figure 4D). These data suggest that Twist-2 suppresses myeloid lineage differentiation and the proliferation of GMP, possibly via inhibition of Runx1 and C/EBPα.


Twist-2 controls myeloid lineage development and function.

Sharabi AB, Aldrich M, Sosic D, Olson EN, Friedman AD, Lee SH, Chen SY - PLoS Biol. (2008)

Twist-2 Inhibits the Transcriptional Activity of Runx1 and C/EBPα(A) Immunoassay of COS7 cells transiently transfected with indicated plasmids. After 48 h, cells were lysed in NTN buffer containing 0.5% NP-40 and 0.5% Triton X-100. Cell lysates were analyzed by immunoblotting (IB) with anti-FLAG or anti-myc antibodies (upper two panels). Cell lysates were also immunoprecipitated (IP) with anti-myc antibody followed by immunoblotting with anti-FLAG antibody (lower panel).(B and C) COS7 cells were transiently transfected with a Runx1-responsive luciferase reporter (CBF4_Luc, 1 μg), or Runx1 expression plasmid (Runx1, 1 μg), or indicated amounts of Twist expression constructs. pcDNA3.1 blank vector control was cotransfected so that each well received the same total amount of DNA (4 μg). After 24 h, luciferase activity in cell extracts was quantified via luciferase assay (B). Runx1 responsive luciferase reporter cotransfected with empty vector control or Twist-2 expression vector only (C). Data are presented as mean ± SD and are representative of three independent experiments.(D and E) Luciferase reporter assays were performed on cells transfected with indicated plasmids (1 μg each). Twist-2 vector cotransfection inhibited the activity of a C/EBPα-dependent luciferase reporter in COS7 cells ([D] left panel), but not a PU.1-dependent luciferase reporter ([D] right panel). A CREB-responsive luciferase reporter from the PEPCK promoter cotransfected with empty vector control or Twist-2 expression vector only (E). Data are presented as mean ± SD, and the results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2602725&req=5

pbio-0060316-g004: Twist-2 Inhibits the Transcriptional Activity of Runx1 and C/EBPα(A) Immunoassay of COS7 cells transiently transfected with indicated plasmids. After 48 h, cells were lysed in NTN buffer containing 0.5% NP-40 and 0.5% Triton X-100. Cell lysates were analyzed by immunoblotting (IB) with anti-FLAG or anti-myc antibodies (upper two panels). Cell lysates were also immunoprecipitated (IP) with anti-myc antibody followed by immunoblotting with anti-FLAG antibody (lower panel).(B and C) COS7 cells were transiently transfected with a Runx1-responsive luciferase reporter (CBF4_Luc, 1 μg), or Runx1 expression plasmid (Runx1, 1 μg), or indicated amounts of Twist expression constructs. pcDNA3.1 blank vector control was cotransfected so that each well received the same total amount of DNA (4 μg). After 24 h, luciferase activity in cell extracts was quantified via luciferase assay (B). Runx1 responsive luciferase reporter cotransfected with empty vector control or Twist-2 expression vector only (C). Data are presented as mean ± SD and are representative of three independent experiments.(D and E) Luciferase reporter assays were performed on cells transfected with indicated plasmids (1 μg each). Twist-2 vector cotransfection inhibited the activity of a C/EBPα-dependent luciferase reporter in COS7 cells ([D] left panel), but not a PU.1-dependent luciferase reporter ([D] right panel). A CREB-responsive luciferase reporter from the PEPCK promoter cotransfected with empty vector control or Twist-2 expression vector only (E). Data are presented as mean ± SD, and the results are representative of three independent experiments.
Mentions: Twist-2 forms homodimers or heterodimers with ubiquitously expressed members of the E2A bHLH family [31,45], and can bind E-box consensus sites present in target gene promoters or can directly bind transcriptions factors such as MEF2, Runx2, and NF-κB via its C-terminal domain [31,35,46]. The Twist family has been previously shown to bind the runt homology domain of the Runx2 family member and inhibit its transcriptional activity [46,47]. Given that Runx1 is a known regulator of hematopoiesis, this prompted us to examine whether Runx1 transcriptional activity may be regulated by Twist-2. In order to address this possibility, COS7 cells were transfected with constructs encoding FLAG-tagged Twist-2 and Myc-tagged Runx1. Upon immunoprecipitation of Runx1, we detected Flag-tagged Twist-2 by coimmunoprecipitation (Figure 4A). To investigate the functionality of this interaction, COS7 cells were transfected with a Runx-dependent luciferase reporter construct containing four core binding factor consensus sites. Interestingly, we observed a basal level of reporter activity in COS7 cells, which could be due to a low-level endogenous expression of Runx family members. Cotransfection of Twist-2 resulted in a significant dose-response inhibition of Runx1 reporter activity that was able to reduce the promoter activity to near basal levels (Figure 4B). Neither Twist-2 vector nor empty vector pcDNA alone inhibited the activity of the Runx1-responsive luciferase reporter (CBF4_Luc) (Figure 4C). In addition, neither Twist-2 vector nor empty vector pcDNA inhibited the activity of an unrelated CREB-responsive luciferase reporter (Figure 4E). These data suggest the specificity of Runx1 inhibition by Twist-2. We also observed a subtle increase in Runx1-specific DNA binding in GMP from Twist-2 KO mice as compared to WT controls using electrophoretic mobility shift assays (EMSA) (Figure S2). Since multiple transcription factors play critical roles in myeloid lineage development, we examined the effect of Twist-2 on the transcription factors PU.1 and C/EBPα, which are required for myeloid lineage development. We found that Twist-2 inhibited the transcriptional activity of C/EBPα, but did not significantly affect the transcriptional activity of PU.1 (Figure 4D). These data suggest that Twist-2 suppresses myeloid lineage differentiation and the proliferation of GMP, possibly via inhibition of Runx1 and C/EBPα.

Bottom Line: Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells.In addition, Twist-2 was found to be essential for endotoxin tolerance.Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT
Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

Show MeSH
Related in: MedlinePlus