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Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors.

Woodcroft MW, Nanan K, Thompson P, Tyryshkin K, Smith SP, Slany RK, LeBrun DP - PLoS ONE (2015)

Bottom Line: We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself.However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis.Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

ABSTRACT
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

No MeSH data available.


Related in: MedlinePlus

B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1.(A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.
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pone.0130495.g005: B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1.(A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.

Mentions: During B-lymphopoiesis, the progeny of HSCs undergo progressive restriction of their lineage potential through the multipotent progenitor (MPP), lymphoid-primed MPP and common lymphoid progenitor (CLP) stages [23]. Commitment to the B-lineage occurs subsequently at the pro-B-cells stage, which coincides with surface expression of CD19. The B-lymphopoietic transcription factor genes Ebf1 and Pax5 are subjected to Polycomb group- (PcG) mediated silencing in HSCs and MPPs but are transcriptionally induced when cells reach the CLP stage [11]. Accordingly, the Ebf1 and Pax5 promoters are associated with the PcG-mediated repressive mark H3K27me3 in early hematopoietic and non-lymphoid progenitors, whereas they acquire the activating H3K4me3 mark in CLPs. Evaluating the E2A-PBX1-expressing FLPs indicated low to absent expression of Ebf1 or Pax5 at either the protein or transcript levels (Fig 5A and 5C). Examining the chromatin status at the Ebf1 and Pax5 promoters by ChIP-qPCR demonstrated failure of either promoter to switch from the silenced H3K27me3- to the active H3K4me3-marked state in E2A-PBX1-expressing FLPs (Fig 5B and 5D). Therefore, B-lymphopoiesis is blocked prior to the CLP stage as a consequence of E2A-PBX1 expression.


Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors.

Woodcroft MW, Nanan K, Thompson P, Tyryshkin K, Smith SP, Slany RK, LeBrun DP - PLoS ONE (2015)

B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1.(A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.
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Related In: Results  -  Collection

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pone.0130495.g005: B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1.(A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.
Mentions: During B-lymphopoiesis, the progeny of HSCs undergo progressive restriction of their lineage potential through the multipotent progenitor (MPP), lymphoid-primed MPP and common lymphoid progenitor (CLP) stages [23]. Commitment to the B-lineage occurs subsequently at the pro-B-cells stage, which coincides with surface expression of CD19. The B-lymphopoietic transcription factor genes Ebf1 and Pax5 are subjected to Polycomb group- (PcG) mediated silencing in HSCs and MPPs but are transcriptionally induced when cells reach the CLP stage [11]. Accordingly, the Ebf1 and Pax5 promoters are associated with the PcG-mediated repressive mark H3K27me3 in early hematopoietic and non-lymphoid progenitors, whereas they acquire the activating H3K4me3 mark in CLPs. Evaluating the E2A-PBX1-expressing FLPs indicated low to absent expression of Ebf1 or Pax5 at either the protein or transcript levels (Fig 5A and 5C). Examining the chromatin status at the Ebf1 and Pax5 promoters by ChIP-qPCR demonstrated failure of either promoter to switch from the silenced H3K27me3- to the active H3K4me3-marked state in E2A-PBX1-expressing FLPs (Fig 5B and 5D). Therefore, B-lymphopoiesis is blocked prior to the CLP stage as a consequence of E2A-PBX1 expression.

Bottom Line: We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself.However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis.Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

ABSTRACT
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

No MeSH data available.


Related in: MedlinePlus