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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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Runx1 P2neo allele failed to rescue the embryonal lethality phenotype of Runx1-/- mice. (A) Schematic representation of Runx1 locus in the two Runx1 mutant strains used to generate the compound mutant strain Runx1lz;Runx1P2neo. (B) Runx1lz;Runx1P2neo die at E11.5 to E12.5 due to hemorrhages in the CNS and/or lack of FL hematopoiesis. Left: Lateral view of a whole mount compound Runx1lz;Runx1P2neo E11.5 embryo stained for β-galactosidase activity. Hemorrhages are seen in the 4th ventricle, the ventral metencephalon and spinal cord. Right: Hematoxylin and eosin (H&E) staining of a section through the spinal cord exhibits focal hemorrhage (arrow head). (C) H&E staining of WT (left view) and Runx1lz;Runx1P2neo (right view) E12.5 fetal liver. Note the absence of definitive hematopoietic precursors (deep purple cells) in Runx1lz;Runx1P2neo FL as compared to WT. The data demonstrate that activity of a single Runx1 P1 was not sufficient to rescue the embryonal lethal phenotype of Runx1-/- mice.
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Figure 7: Runx1 P2neo allele failed to rescue the embryonal lethality phenotype of Runx1-/- mice. (A) Schematic representation of Runx1 locus in the two Runx1 mutant strains used to generate the compound mutant strain Runx1lz;Runx1P2neo. (B) Runx1lz;Runx1P2neo die at E11.5 to E12.5 due to hemorrhages in the CNS and/or lack of FL hematopoiesis. Left: Lateral view of a whole mount compound Runx1lz;Runx1P2neo E11.5 embryo stained for β-galactosidase activity. Hemorrhages are seen in the 4th ventricle, the ventral metencephalon and spinal cord. Right: Hematoxylin and eosin (H&E) staining of a section through the spinal cord exhibits focal hemorrhage (arrow head). (C) H&E staining of WT (left view) and Runx1lz;Runx1P2neo (right view) E12.5 fetal liver. Note the absence of definitive hematopoietic precursors (deep purple cells) in Runx1lz;Runx1P2neo FL as compared to WT. The data demonstrate that activity of a single Runx1 P1 was not sufficient to rescue the embryonal lethal phenotype of Runx1-/- mice.

Mentions: Homozygous disruption of Runx1 results in a complete absence of FL hematopoiesis and the mice die between E11.5 and E12.5 of hemorrhages in the central nervous system [19,20]. P2neo/neo mice were born and showed no sign of hemorrhages, indicating that expression levels of Runx1 in homozygous P2neo/neo during early embryogenesis were sufficient to rescue the Runx1-/- phenotype. This occurrence raised the question of whether Runx1 levels provided by activity of a single P1 will also be sufficient to rescue the phenotype. To address this issue we generated a compound mutant strain Runx1lz; Runx1P2neo by mating Runx1lz/+ mice [16] with Runx1P2neo/+. These mice had one allele expressing a fused Runx1-LacZ protein and one P2neo allele expressing WT P1 (Fig. 7A). Compound mutant Runx1lz;Runx1P2neo embryos died between E11.5 and E12.5 and suffered from hemorrhages in the nervous system (Fig. 7B). The most extensive bleedings was seen in the 4th ventricle, the ventral metencephalon and spinal cord. Moreover, the liver of E12.5 compound mutant embryos was depleted of hematopoietic cells (Fig. 7C), indicating lack of definitive hematopoiesis, similar to the previously reported Runx1-/- phenotype [16,19,20]. These results indicated, that during midgestation, angiogenesis and definitive hematopoiesis are highly sensitive to the dosage of Runx1 as activity of a single P1 was not sufficient to rescue the Runx1-/- phenotype. This conclusion is consistent with the previously reported data which was based on transplantation assays [17,18,55]. This observation may also pertain to the unique role of P2 activity during midgestation angiogenesis and/or early hematopoiesis.


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)

Runx1 P2neo allele failed to rescue the embryonal lethality phenotype of Runx1-/- mice. (A) Schematic representation of Runx1 locus in the two Runx1 mutant strains used to generate the compound mutant strain Runx1lz;Runx1P2neo. (B) Runx1lz;Runx1P2neo die at E11.5 to E12.5 due to hemorrhages in the CNS and/or lack of FL hematopoiesis. Left: Lateral view of a whole mount compound Runx1lz;Runx1P2neo E11.5 embryo stained for β-galactosidase activity. Hemorrhages are seen in the 4th ventricle, the ventral metencephalon and spinal cord. Right: Hematoxylin and eosin (H&E) staining of a section through the spinal cord exhibits focal hemorrhage (arrow head). (C) H&E staining of WT (left view) and Runx1lz;Runx1P2neo (right view) E12.5 fetal liver. Note the absence of definitive hematopoietic precursors (deep purple cells) in Runx1lz;Runx1P2neo FL as compared to WT. The data demonstrate that activity of a single Runx1 P1 was not sufficient to rescue the embryonal lethal phenotype of Runx1-/- mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Runx1 P2neo allele failed to rescue the embryonal lethality phenotype of Runx1-/- mice. (A) Schematic representation of Runx1 locus in the two Runx1 mutant strains used to generate the compound mutant strain Runx1lz;Runx1P2neo. (B) Runx1lz;Runx1P2neo die at E11.5 to E12.5 due to hemorrhages in the CNS and/or lack of FL hematopoiesis. Left: Lateral view of a whole mount compound Runx1lz;Runx1P2neo E11.5 embryo stained for β-galactosidase activity. Hemorrhages are seen in the 4th ventricle, the ventral metencephalon and spinal cord. Right: Hematoxylin and eosin (H&E) staining of a section through the spinal cord exhibits focal hemorrhage (arrow head). (C) H&E staining of WT (left view) and Runx1lz;Runx1P2neo (right view) E12.5 fetal liver. Note the absence of definitive hematopoietic precursors (deep purple cells) in Runx1lz;Runx1P2neo FL as compared to WT. The data demonstrate that activity of a single Runx1 P1 was not sufficient to rescue the embryonal lethal phenotype of Runx1-/- mice.
Mentions: Homozygous disruption of Runx1 results in a complete absence of FL hematopoiesis and the mice die between E11.5 and E12.5 of hemorrhages in the central nervous system [19,20]. P2neo/neo mice were born and showed no sign of hemorrhages, indicating that expression levels of Runx1 in homozygous P2neo/neo during early embryogenesis were sufficient to rescue the Runx1-/- phenotype. This occurrence raised the question of whether Runx1 levels provided by activity of a single P1 will also be sufficient to rescue the phenotype. To address this issue we generated a compound mutant strain Runx1lz; Runx1P2neo by mating Runx1lz/+ mice [16] with Runx1P2neo/+. These mice had one allele expressing a fused Runx1-LacZ protein and one P2neo allele expressing WT P1 (Fig. 7A). Compound mutant Runx1lz;Runx1P2neo embryos died between E11.5 and E12.5 and suffered from hemorrhages in the nervous system (Fig. 7B). The most extensive bleedings was seen in the 4th ventricle, the ventral metencephalon and spinal cord. Moreover, the liver of E12.5 compound mutant embryos was depleted of hematopoietic cells (Fig. 7C), indicating lack of definitive hematopoiesis, similar to the previously reported Runx1-/- phenotype [16,19,20]. These results indicated, that during midgestation, angiogenesis and definitive hematopoiesis are highly sensitive to the dosage of Runx1 as activity of a single P1 was not sufficient to rescue the Runx1-/- phenotype. This conclusion is consistent with the previously reported data which was based on transplantation assays [17,18,55]. This observation may also pertain to the unique role of P2 activity during midgestation angiogenesis and/or early hematopoiesis.

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