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Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis.

Pozner A, Lotem J, Xiao C, Goldenberg D, Brenner O, Negreanu V, Levanon D, Groner Y - BMC Dev. Biol. (2007)

Bottom Line: Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors.The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1.The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. apozner@genetics.utah.edu <apozner@genetics.utah.edu>

ABSTRACT

Background: Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development.

Results: We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors.

Conclusion: The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

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P2-mediated expression of Runx1 is required for embryonal thymopoiesis. (A) Distribution of CD44/CD25 expressing thymocytes of E15.5 WT and P2neo/neo embryos. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. (B) Distribution of embryonic and neonatal CD4/CD8 thymocytes. WT and P2neo/neo thymocytes of E16.5 and E17.5 embryos and day 1.5 and 3.5 neonates were analyzed. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated.
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Figure 5: P2-mediated expression of Runx1 is required for embryonal thymopoiesis. (A) Distribution of CD44/CD25 expressing thymocytes of E15.5 WT and P2neo/neo embryos. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. (B) Distribution of embryonic and neonatal CD4/CD8 thymocytes. WT and P2neo/neo thymocytes of E16.5 and E17.5 embryos and day 1.5 and 3.5 neonates were analyzed. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated.

Mentions: We next employed flow cytometry to study thymocyte differentiation in P2neo/neo embryos and neonates. First we examined the CD4/CD8 double negative (DN) population at E15.5 when nearly 100% of thymocytes are DN. The DN population is divided into four sub-groups, DN1 to DN4, according to the expression of CD25 and CD44 surface markers. Compared to WT, P2neo/neo embryos displayed an increase in the proportion of DN3 (CD25+/CD44-) thymocytes and a corresponding reduction in DN4 cells (CD25-/CD44-) (Fig. 5A), resulting in a significant increase in the ratio DN3/DN4 among P2neo/neo thymocytes (WT DN3/DN4 = 0.65 ± 0.15 and P2neo/neo = 1.51 ± 0.31; P < 0.001). To examine P1/P2 usage in the DN sub-groups we used flow cytometry to sort E15.5 thymocytes based on surface expression of CD25 and CD44 (as shown in Fig. 5A), and levels of P1 and P2 transcripts was determined. The levels of P1 and P2 expression did not differ significantly between DN3 and DN4 subgroups and was similar to the level shown in Fig. 1D.


Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis.

Pozner A, Lotem J, Xiao C, Goldenberg D, Brenner O, Negreanu V, Levanon D, Groner Y - BMC Dev. Biol. (2007)

P2-mediated expression of Runx1 is required for embryonal thymopoiesis. (A) Distribution of CD44/CD25 expressing thymocytes of E15.5 WT and P2neo/neo embryos. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. (B) Distribution of embryonic and neonatal CD4/CD8 thymocytes. WT and P2neo/neo thymocytes of E16.5 and E17.5 embryos and day 1.5 and 3.5 neonates were analyzed. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941738&req=5

Figure 5: P2-mediated expression of Runx1 is required for embryonal thymopoiesis. (A) Distribution of CD44/CD25 expressing thymocytes of E15.5 WT and P2neo/neo embryos. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. (B) Distribution of embryonic and neonatal CD4/CD8 thymocytes. WT and P2neo/neo thymocytes of E16.5 and E17.5 embryos and day 1.5 and 3.5 neonates were analyzed. Representative data of five different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated.
Mentions: We next employed flow cytometry to study thymocyte differentiation in P2neo/neo embryos and neonates. First we examined the CD4/CD8 double negative (DN) population at E15.5 when nearly 100% of thymocytes are DN. The DN population is divided into four sub-groups, DN1 to DN4, according to the expression of CD25 and CD44 surface markers. Compared to WT, P2neo/neo embryos displayed an increase in the proportion of DN3 (CD25+/CD44-) thymocytes and a corresponding reduction in DN4 cells (CD25-/CD44-) (Fig. 5A), resulting in a significant increase in the ratio DN3/DN4 among P2neo/neo thymocytes (WT DN3/DN4 = 0.65 ± 0.15 and P2neo/neo = 1.51 ± 0.31; P < 0.001). To examine P1/P2 usage in the DN sub-groups we used flow cytometry to sort E15.5 thymocytes based on surface expression of CD25 and CD44 (as shown in Fig. 5A), and levels of P1 and P2 transcripts was determined. The levels of P1 and P2 expression did not differ significantly between DN3 and DN4 subgroups and was similar to the level shown in Fig. 1D.

Bottom Line: Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors.The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1.The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. apozner@genetics.utah.edu <apozner@genetics.utah.edu>

ABSTRACT

Background: Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development.

Results: We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors.

Conclusion: The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

Show MeSH
Related in: MedlinePlus