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Early thymocyte development is regulated by modulation of E2A protein activity.

Engel I, Johns C, Bain G, Rivera RR, Murre C - J. Exp. Med. (2001)

Bottom Line: E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage.However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway.Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California at San Diego, La Jolla, CA 92093, USA.

ABSTRACT
The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

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E47 deficiency promotes the aberrant development of RAG-1  thymocytes. (A) Dot plots depicting surface CD4 and CD8 expression, as determined by flow cytometry, on thymocytes from 1-mo-old mice with RAG-1+/− E47+/−, RAG-1+/− E47−/−, RAG-1−/− E47−/−, and RAG-1−/− E4−/− genotypes. The RAG-1+/− E47−/− mouse used for this experiment was from a separate litter and analyzed separately from the other three mice. Numbers indicate the percentage of viable cells found in each quadrant. (B) Flow cytometric analysis of surface CD44 and CD25 expression on CD3−CD4−CD8− thymocytes from a 3.5-wk-old RAG-1−/− E47−/− mouse and three littermates. Numbers indicate the percentage of viable DN cells found in each quadrant. (C) Dot plots depicting surface CD4 and CD8 expression on thymocytes from 1-mo-old RAG-1−/− littermates that were either wild-type or heterozygous for E47, illustrating the appearance of DP thymocytes in some RAG-1−/− E47+/− mice. The percentage of DP thymocytes observed in RAG-1−/− E47+/− mice with significant numbers of DP thymocytes has ranged from ∼2–85% (data not shown). (D) Scatter plot indicating the number of thymocytes obtained from six RAG-1−/− E47−/− mice as compared with RAG-1−/− E47 wt or +/− littermates. Thymus cellularity in E47−/− mice typically ranges from 1.5–5 × 107 cells (data not shown). (E) Histograms of surface CD95 (Fas antigen) expression in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 4-wk-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47+/− littermate. (Dotted line) DP thymocytes from a RAG-1−/− E47+/− littermate. (F) Histograms of BCL-2 expression levels in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 1-mo-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47 wild-type littermate. (Dotted line) DP thymocytes from a RAG-1+/− E47+/+ littermate. All data presented are representative of analyses of at least three individual RAG-1−/− E47−/− mice and littermate controls.
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Figure 4: E47 deficiency promotes the aberrant development of RAG-1 thymocytes. (A) Dot plots depicting surface CD4 and CD8 expression, as determined by flow cytometry, on thymocytes from 1-mo-old mice with RAG-1+/− E47+/−, RAG-1+/− E47−/−, RAG-1−/− E47−/−, and RAG-1−/− E4−/− genotypes. The RAG-1+/− E47−/− mouse used for this experiment was from a separate litter and analyzed separately from the other three mice. Numbers indicate the percentage of viable cells found in each quadrant. (B) Flow cytometric analysis of surface CD44 and CD25 expression on CD3−CD4−CD8− thymocytes from a 3.5-wk-old RAG-1−/− E47−/− mouse and three littermates. Numbers indicate the percentage of viable DN cells found in each quadrant. (C) Dot plots depicting surface CD4 and CD8 expression on thymocytes from 1-mo-old RAG-1−/− littermates that were either wild-type or heterozygous for E47, illustrating the appearance of DP thymocytes in some RAG-1−/− E47+/− mice. The percentage of DP thymocytes observed in RAG-1−/− E47+/− mice with significant numbers of DP thymocytes has ranged from ∼2–85% (data not shown). (D) Scatter plot indicating the number of thymocytes obtained from six RAG-1−/− E47−/− mice as compared with RAG-1−/− E47 wt or +/− littermates. Thymus cellularity in E47−/− mice typically ranges from 1.5–5 × 107 cells (data not shown). (E) Histograms of surface CD95 (Fas antigen) expression in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 4-wk-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47+/− littermate. (Dotted line) DP thymocytes from a RAG-1−/− E47+/− littermate. (F) Histograms of BCL-2 expression levels in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 1-mo-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47 wild-type littermate. (Dotted line) DP thymocytes from a RAG-1+/− E47+/+ littermate. All data presented are representative of analyses of at least three individual RAG-1−/− E47−/− mice and littermate controls.

Mentions: The observation that E47 DNA binding was inhibited upon cross-linking of the CD3 complex in DN thymocytes raised the possibility that E2A proteins play a role in blocking the DN to DP transition in the absence of β selection. Therefore, we generated mice deficient for both E47 and RAG-1 and analyzed thymocytes from these mice for the expression of CD4 and CD8 by flow cytometry. As demonstrated previously, E47-deficient mice showed significant decreases in the proportion of DP thymocytes compared with the wild-type control littermates (Fig. 4 A, reference 8). DP thymocytes were absent from RAG-1−/− E47+/+ mice, due to the block in TCR gene rearrangement (Fig. 4 A, reference 1). However, the overwhelming majority of the thymocytes from the doubly deficient mice expressed high levels of both CD4 and CD8 (Fig. 4 A). The DP thymocytes isolated from the RAG-1−/− E47−/− mice expressed high levels of CD95 and low levels of BCL-2, similar to that of wild-type DP thymocytes (Fig. 4e and Fig. f, and references 30 and 31). DP thymocytes from mice deficient in both RAG-1 and E47 were also similar in size and cell-cycle status to that of wild-type DP thymocytes (data not shown). Strikingly, we also observed significant numbers of DP thymocytes in 6 out of 11 mice analyzed with a RAG1−/− E47+/− genotype (Fig. 4 C and data not shown). Thus a wild-type gene dosage of E47 is required to ensure that all RAG-deficient thymocytes arrest at the β selection checkpoint.


Early thymocyte development is regulated by modulation of E2A protein activity.

Engel I, Johns C, Bain G, Rivera RR, Murre C - J. Exp. Med. (2001)

E47 deficiency promotes the aberrant development of RAG-1  thymocytes. (A) Dot plots depicting surface CD4 and CD8 expression, as determined by flow cytometry, on thymocytes from 1-mo-old mice with RAG-1+/− E47+/−, RAG-1+/− E47−/−, RAG-1−/− E47−/−, and RAG-1−/− E4−/− genotypes. The RAG-1+/− E47−/− mouse used for this experiment was from a separate litter and analyzed separately from the other three mice. Numbers indicate the percentage of viable cells found in each quadrant. (B) Flow cytometric analysis of surface CD44 and CD25 expression on CD3−CD4−CD8− thymocytes from a 3.5-wk-old RAG-1−/− E47−/− mouse and three littermates. Numbers indicate the percentage of viable DN cells found in each quadrant. (C) Dot plots depicting surface CD4 and CD8 expression on thymocytes from 1-mo-old RAG-1−/− littermates that were either wild-type or heterozygous for E47, illustrating the appearance of DP thymocytes in some RAG-1−/− E47+/− mice. The percentage of DP thymocytes observed in RAG-1−/− E47+/− mice with significant numbers of DP thymocytes has ranged from ∼2–85% (data not shown). (D) Scatter plot indicating the number of thymocytes obtained from six RAG-1−/− E47−/− mice as compared with RAG-1−/− E47 wt or +/− littermates. Thymus cellularity in E47−/− mice typically ranges from 1.5–5 × 107 cells (data not shown). (E) Histograms of surface CD95 (Fas antigen) expression in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 4-wk-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47+/− littermate. (Dotted line) DP thymocytes from a RAG-1−/− E47+/− littermate. (F) Histograms of BCL-2 expression levels in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 1-mo-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47 wild-type littermate. (Dotted line) DP thymocytes from a RAG-1+/− E47+/+ littermate. All data presented are representative of analyses of at least three individual RAG-1−/− E47−/− mice and littermate controls.
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Figure 4: E47 deficiency promotes the aberrant development of RAG-1 thymocytes. (A) Dot plots depicting surface CD4 and CD8 expression, as determined by flow cytometry, on thymocytes from 1-mo-old mice with RAG-1+/− E47+/−, RAG-1+/− E47−/−, RAG-1−/− E47−/−, and RAG-1−/− E4−/− genotypes. The RAG-1+/− E47−/− mouse used for this experiment was from a separate litter and analyzed separately from the other three mice. Numbers indicate the percentage of viable cells found in each quadrant. (B) Flow cytometric analysis of surface CD44 and CD25 expression on CD3−CD4−CD8− thymocytes from a 3.5-wk-old RAG-1−/− E47−/− mouse and three littermates. Numbers indicate the percentage of viable DN cells found in each quadrant. (C) Dot plots depicting surface CD4 and CD8 expression on thymocytes from 1-mo-old RAG-1−/− littermates that were either wild-type or heterozygous for E47, illustrating the appearance of DP thymocytes in some RAG-1−/− E47+/− mice. The percentage of DP thymocytes observed in RAG-1−/− E47+/− mice with significant numbers of DP thymocytes has ranged from ∼2–85% (data not shown). (D) Scatter plot indicating the number of thymocytes obtained from six RAG-1−/− E47−/− mice as compared with RAG-1−/− E47 wt or +/− littermates. Thymus cellularity in E47−/− mice typically ranges from 1.5–5 × 107 cells (data not shown). (E) Histograms of surface CD95 (Fas antigen) expression in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 4-wk-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47+/− littermate. (Dotted line) DP thymocytes from a RAG-1−/− E47+/− littermate. (F) Histograms of BCL-2 expression levels in selected thymocyte populations as measured by flow cytometry. (Thick line) DP thymocytes from a 1-mo-old E47−/− RAG-1−/− mouse. (Thin line) DN thymocytes from a RAG-1−/− E47 wild-type littermate. (Dotted line) DP thymocytes from a RAG-1+/− E47+/+ littermate. All data presented are representative of analyses of at least three individual RAG-1−/− E47−/− mice and littermate controls.
Mentions: The observation that E47 DNA binding was inhibited upon cross-linking of the CD3 complex in DN thymocytes raised the possibility that E2A proteins play a role in blocking the DN to DP transition in the absence of β selection. Therefore, we generated mice deficient for both E47 and RAG-1 and analyzed thymocytes from these mice for the expression of CD4 and CD8 by flow cytometry. As demonstrated previously, E47-deficient mice showed significant decreases in the proportion of DP thymocytes compared with the wild-type control littermates (Fig. 4 A, reference 8). DP thymocytes were absent from RAG-1−/− E47+/+ mice, due to the block in TCR gene rearrangement (Fig. 4 A, reference 1). However, the overwhelming majority of the thymocytes from the doubly deficient mice expressed high levels of both CD4 and CD8 (Fig. 4 A). The DP thymocytes isolated from the RAG-1−/− E47−/− mice expressed high levels of CD95 and low levels of BCL-2, similar to that of wild-type DP thymocytes (Fig. 4e and Fig. f, and references 30 and 31). DP thymocytes from mice deficient in both RAG-1 and E47 were also similar in size and cell-cycle status to that of wild-type DP thymocytes (data not shown). Strikingly, we also observed significant numbers of DP thymocytes in 6 out of 11 mice analyzed with a RAG1−/− E47+/− genotype (Fig. 4 C and data not shown). Thus a wild-type gene dosage of E47 is required to ensure that all RAG-deficient thymocytes arrest at the β selection checkpoint.

Bottom Line: E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage.However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway.Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California at San Diego, La Jolla, CA 92093, USA.

ABSTRACT
The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

Show MeSH
Related in: MedlinePlus