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

doi:10.1186/1471-213X-7-84

. 2007 Jul 12:7:84.

doi: 10.1186/1471-213X-7-84.

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

Amir Pozner¹, Joseph Lotem, Cuiying Xiao, Dalia Goldenberg, Ori Brenner, Varda Negreanu, Ditsa Levanon, Yoram Groner

Affiliations

PMID: 17626615
PMCID: PMC1941738
DOI: 10.1186/1471-213X-7-84

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

Amir Pozner et al. BMC Dev Biol. 2007.

. 2007 Jul 12:7:84.

doi: 10.1186/1471-213X-7-84.

Authors

Amir Pozner¹, Joseph Lotem, Cuiying Xiao, Dalia Goldenberg, Ori Brenner, Varda Negreanu, Ditsa Levanon, Yoram Groner

Affiliation

¹ Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. apozner@genetics.utah.edu <apozner@genetics.utah.edu>

PMID: 17626615
PMCID: PMC1941738
DOI: 10.1186/1471-213X-7-84

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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Figures

**Figure 1**
**Attenuated *Runx1* P2 expression in P2^neo/neoembryos impaired thymus development**. (A) RT-PCR analysis of RNA from kidney and stomach of E16.5 WT and P2^neo/neoembryos, and from tongue of P1.5 WT and P2^neo/neoneonates. P2-mediated transcription in P2^neo/neotissues diminished, whereas P1-mediated transcription was largely unaffected. (B) IHC analysis of glandular stomach of E16.5 WT (left) and P2^neo/neo(right) embryos. Runx1 expression is detected in epithelial cells of WT embryo, but missing in P2^neo/neolittermate (10× magnification). (C) Reduced size of thymic lobes in P1.5 P2^neo/neomice (right) as compared to WT littermate (left). (D) RT-PCR analysis of *Runx1* P1- and P2-mediated transcription in thymus of WT embryos, neonates and young mice. (E) Analysis of *Runx1* expression by *In-situ* hybridization of E15.5 WT (left) and P2^neo/neo(right) thymic sections, using the P2-5'UTR probe. P2-derived transcripts are clearly visible in the cortex of E16.5 WT, but not of P2^neo/neo(10× magnification). (co) = cortex; (me) = medulla. (F) Western blot analysis of proteins extracted from thymus of E15.5–E17.5 WT and P2^neo/neoembryos. Runx1 proteins were not detected in E15.5 P2^neo/neothymus, but gradually accumulated in E16.5 and E17.5 thymi. (G) Northern blot analysis of RNA from thymi of WT and P2^neo/neonewborn mice. Whereas P2-mediated transcription (the 4 Kb and 8 Kb transcripts) [30, 31], in P2^neo/neothymocytes was markedly attenuated, P1-mediated transcription (the 2 Kb and 6 Kb transcripts) was apparently unaffected. (H) Reduced thymus cellularity in P2^neo/neoembryos and neonates. Five mice of each genotype were analyzed. The difference between the number of thymocytes from WT and P2^neo/neothymus was significant at P < 0.0001 (*) and P < 0.05 (#) by Student's t test.

**Figure 2**
**Histological and IHC analysis of thymus development in WT and P2^neo/neomice**. (A, C, E, G, I, K) Transverse sections of WT (left panels) and P2^neo/neo(right panels) thymic lobes stained with hematoxylin and eosin (HE). co = cortex, me = medulla, scb = sub-cortical band, cy = cyst. (B, D, F, H, J, L) Immunostaining of Runx1 in sections of WT (left panels) and P2^neo/neo(right panels) thymic lobes. Runx1 is detected in thymocytes, but not in thymic epithelium. A reduced number of Runx1 expressing thymocytes (that are abnormally distributed) is seen in P2^neo/neothymic lobes.

**Figure 3**
**P2^neo/neothymocytes display enhanced apoptosis, but have normal cell proliferation capacity**. (A) FACS analysis of E14.5 and E17.5 WT (left panels) and P2^neo/neo(right panels) thymocytes stained with Annexin V and PI. FACS dot plots and percentages of cells in each quadrant are indicated. Note the >2 fold increase in the proportion of apoptotic cells (Annexin V⁺/PI^-) and of necrotic cells (Annexin V⁺/PI⁺) in P2^neo/neothymi. (B) P2^neo/neothymocytes display enhanced apoptosis throughout embryonic thymopoiesis. Proportion of non-viable (apoptotic+necrotic) WT and P2^neo/neocells gradually decreased during embryonic development. Nevertheless, at any given time point, P2^neo/neothymui contained a higher proportion of non-viable cells compared to WT littermates. After birth the proportion of non-viable thymocytes in P2^neo/neobecame similar to WT. At least five mice of each genotype were analyzed. The differences between WT and P2^neo/neoapoptotic thymocytes were significant at P < 0.001 (*) and P < 0.01 (#) by Student's t test. (C) P2^neo/neothymocytes retain normal proliferation capacity. Following FACS analysis an equal number (1 × 10⁴) of E15.5 WT or P2^neo/neoAnnexin V negative thymocytes were incubated with TPA (20 ng/ml) and ConA (5 μg/ml) for 48 h. ³H-thymidine was present during the last 18 h of incubation. Differences between WT and P2^neo/neowere statistically insignificant by Student's t test. Proliferation capability of thymocytes was further examined by immunostaining of E15.5 thymic lobes for the proliferation-associated antigen recognized by Ki-67 antibodies (Right panels). (D) Enhanced embryonal apoptosis is a cell-autonomous property of P2^neo/neothymocytes. WT or P2^neo/neoE15.5 FL HPC were differentiated under saturating conditions in E14.5 WT FTOC for 14 or 16 days. We predetermined that ~100 FL cells (either WT or P2^neo/neo) per lobe were sufficient to populate all available thymic lobes and therefore used 1000 FL cells per lobe. Thymocytes accumulation in FTOC populated with P2^neo/neoFL HPC is reduced compared to WT (left panel WT-black; P2^neo/neo-white). Proportion of apoptotic thymocytes (Annexin V⁺) derived from FTOC populated with P2^neo/neoFL cells (red) was considerably higher compared to WT (light blue) (right panel).

**Figure 4**
**The propensity of P2^neo/neothymocytes to undergo apoptosis is associated with elevated expression of TCRβ and TCRγδ**. (A and B) Expression of TCRβ and TCRγδ on WT and P2^neo/neoembryonal thymocytes. (A) Shown are FACS analysis dot plots of E15.5 to E18.5 WT and P2^neo/neothymocytes indicating the percentages of cells in each quadrant. Histograms in (B) show the average ± S.E. of TCRβ/TCRγδ proportions of WT and P2^neo/neothymocytes in at least three experiments at each developmental stage. The difference between WT and P2^neo/neowas significant at P < 0.005 (*) and P < 0.05 (#) by Student's t test. While at E15.5 to E17.5 the proportion of TCRβ/TCRγδ expressing thymocytes was much higher in P2^neo/neocompared to WT, at E18.5 the distribution of P2^neo/neothymocytes resumed normal pattern. The mean fluorescence intensity (MFI) (B right) of E15.5 and E16.5 TCRβ⁺P2^neo/neothymocytes was significantly higher than WT (p < 0.005; by Student's t test). (C) Expression of TCRβ/TCRγδ in P2^neo/neothymocytes is transcriptionally upregulated. RT-PCR analysis of RNA derived from WT and P2^neo/neoE16.5 thymocytes using primers specific for the pre-TCRα and the constant regions of TCRβ and TCRγ transcripts. Increased number of PCR cycles shows elevated steady-state levels of mRNAs in P2^neo/neothymocytes compared to WT. (D and E) Enhanced apoptosis of P2^neo/neothymocytes is associated with elevated expression of TCRβ/TCRγδ. (D) Histograms demonstrating the proportion of apoptotic cells among TCRβ⁺or TCRgδ⁺thymocytes. TCRβ/TCRγδ positive WT thymocytes (blue) are divided into two main populations, apoptotic (M1) and non-apoptotic, according to level of Annexin V (except for E15.5 TCRγδ⁺where all cells are apoptotic). Most of TCRβ/TCRγδ positive P2^neo/neothymocytes (red), are at the M1 (Annexin V^high) apoptotic subset. (E) Proportion of apoptotic thymocytes among TCRβ/TCRγδ positive E15.5, E16.5 and E18.5 thymocytes. Bars represent the average ± S.E. of at least three independent experiments for each time point using different mice. The differences between WT and P2^neo/neoin number of TCRβ⁺apoptotic thymocytes were significant at P < 0.01 (*) by Student's t test.

**Figure 5**
**P2-mediated expression of *Runx1* is required for embryonal thymopoiesis**. (A) Distribution of CD44/CD25 expressing thymocytes of E15.5 WT and P2^neo/neoembryos. 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 P2^neo/neothymocytes 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.

**Figure 6**
**P2-mediated embryonal expression of *Runx1* is required for development of FL HPC**. (A) RT-PCR analysis of RNA from E13.5 FL cells of WT and P2^neo/neoembryos. P2-mediated transcription in P2^neo/neowas largely attenuated, whereas P1-mediated transcription was apparently unaffected. (B) Colony forming activity of FL hematopoietic stem/progenitor cells. Colonies (>30 cells in size) were scored on day 7 of incubation. Shown are average numbers of colonies per FL ± S.E. of at least three different WT or P2^neo/neoembryos at E12.5 and E13.5. The difference between WT and P2^neo/neocolony number is significant (*) at P < 0.001 by Student's t test. (C and D) Altered expression of surface antigens on P2^neo/neoFL HPC. (C) Expression profiles of Gr-1, CD18, CD11b, CD11a, CD44 and c-Kit, in FL HPC from E17.5 WT and P2^neo/neoembryos. Representative data of at least three different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. Note the 2-fold decrease in the proportions of Gr-1⁺/CD18⁺and CD11a⁺/CD11b⁺(left panels) as well as CD44⁺/c-Kit⁺(right panel), FL cells in P2^neo/neocompared to WT. (D) Proportion of CD34⁺FL HPC. Profiles of WT and P2^neo/neoFL HPC, filled (blue) and unfilled (red) histograms, respectively, at E14.5, E16.5 and E18.5. Shown are representative results of at least three different experiments. Numbers at the upper right corners are percentages of CD34⁺cells in WT (upper) and P2^neo/neo(lower). Note the marked decrease in the proportions of P2^neo/neoCD34⁺HPC. (E) Analysis of committed FL T-cell precursors (TCPs). FACS analysis showing the distribution of B220⁺/c-Kit⁺/CD19^-FL HPC of E15.5 WT and P2^neo/neoembryos. Gated CD19^-precursors (R1) were analyzed for c-Kit and B220 expression. Note the 4-fold decrease in the proportions of c-Kit⁺/B220^low/D19^-TCPs (R2) in P2^neo/neoFL HPC. Data shown on the right are average ± S.E. of four independent experiments using four different mice. The difference between the percentages of TCPs from WT and P2^neo/neoFL HPC is significant at P < 0.005 by Student's t test. (F) Reduced capacity of P2^neo/neoFL HPC to colonize FTOC. Isolated E15.5 FL HPC from WT or P2^neo/neoembryos were set to differentiate, under limiting dilution conditions, for 16 days in E14.5 WT FTOC. Shown are percentage of unsuccessfully populated lobes per cultured cells and the calculated frequencies of three different independent experiments.

**Figure 7**
*Runx1* P2^neoallele 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 *Runx1*^lz;*Runx1*^P2neo. (B) *Runx1*^lz;*Runx1*^P2neodie 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 *Runx1*^lz;*Runx1*^P2neoE11.5 embryo stained for β-galactosidase activity. Hemorrhages are seen in the 4^thventricle, 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 *Runx1*^lz;*Runx1*^P2neo(right view) E12.5 fetal liver. Note the absence of definitive hematopoietic precursors (deep purple cells) in *Runx1*^lz;*Runx1*^P2neoFL 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.

**Figure 8**
**Rescue of P2^neo/neodependent thymic defect by T-cell specific removal of the *neo* cassette**. (A) Specific excision of the *neo* cassette in P2/Lck-Cre mice. Southern blot analysis using a probe spanning the region indicated by the red bar assessed the level of *neo* excision in thymocytes, stomach epithelium and splenocytes of 4 week-old P2/Lck-Cre mice. The positions of *Xba*I cleavage sites are shown (X). The probe hybridized to a 3.4 Kb- and a 5.4 Kb *Xba*I genomic fragments derived from WT and P2^neo/neoallele, respectively. Insertion/excision of the *neo* cassette by Lck-Cre eliminated the middle *Xba*I site (shown in WT as bold blue) generating the 4.6 Kb fragment derived from the P2/Lck-Cre allele. (B) Regeneration of thymus cellularity in P2/Lck-Cre mice. At day-1.5 the number of thymocytes in P2/Lck-Cre thymic lobes was near normal compared to WT littermates. Data shown are average ± S.E. of five independent experiments using five mice of each genotype (WT, P2^neo/neoand P2/Lck-Cre). The difference between WT and P2^neo/neois significant at P < 0.001 by Student's t test. (C) Recovery of thymopoiesis and thymic organogenesis in P2/Lck-Cre embryos. Histological analysis and *Runx1* expression in thymic lobes derived from E15.5 and E16.5 WT, P2^neo/neoand P2/Lck-Cre embryos. Shown are H&E stained transverse sections immunostained for Runx1. (D) Runx1 expression was not rescued in P2/Lck-Cre epithelial cells. IHC analysis of Runx1 expression in esophagi derived from E16.5 WT, P2^neo/neoand P2/Lck-Cre embryos. Transverse sections showing Runx1 expression in epithelial cells lining the lumen of WT (arrow), but not of P2^neo/neoor P2/Lck-Cre esophagus. Several other tissues including stomach and nasal cavity were similarly analyzed and revealed no expression of Runx1 in epithelia of P2^neo/neoor P2/Lck-Cre mice (not shown).

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References

1. Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene. 2004;23:4308–4314. doi: 10.1038/sj.onc.1207130. - DOI - PubMed
1. de Bruijn MF, Speck NA. Core-binding factors in hematopoiesis and immune function. Oncogene. 2004;23:4238–4248. doi: 10.1038/sj.onc.1207763. - DOI - PubMed
1. Levanon D, Groner Y. Structure and regulated expression of mammalian RUNX genes. Oncogene. 2004;23:4211–4219. doi: 10.1038/sj.onc.1207670. - DOI - PubMed
1. Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A. 1998;95:11590–11595. doi: 10.1073/pnas.95.20.11590. - DOI - PMC - PubMed
1. Durst KL, Hiebert SW. Role of RUNX family members in transcriptional repression and gene silencing. Oncogene. 2004;23:4220–4224. doi: 10.1038/sj.onc.1207122. - DOI - PubMed

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[1] Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene. 2004;23:4308–4314. doi: 10.1038/sj.onc.1207130. - DOI - PubMed

[2] Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene. 2004;23:4308–4314. doi: 10.1038/sj.onc.1207130. - DOI - PubMed

[3] de Bruijn MF, Speck NA. Core-binding factors in hematopoiesis and immune function. Oncogene. 2004;23:4238–4248. doi: 10.1038/sj.onc.1207763. - DOI - PubMed

[4] de Bruijn MF, Speck NA. Core-binding factors in hematopoiesis and immune function. Oncogene. 2004;23:4238–4248. doi: 10.1038/sj.onc.1207763. - DOI - PubMed

[5] Levanon D, Groner Y. Structure and regulated expression of mammalian RUNX genes. Oncogene. 2004;23:4211–4219. doi: 10.1038/sj.onc.1207670. - DOI - PubMed

[6] Levanon D, Groner Y. Structure and regulated expression of mammalian RUNX genes. Oncogene. 2004;23:4211–4219. doi: 10.1038/sj.onc.1207670. - DOI - PubMed

[7] Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A. 1998;95:11590–11595. doi: 10.1073/pnas.95.20.11590. - DOI - PMC - PubMed

[8] Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A. 1998;95:11590–11595. doi: 10.1073/pnas.95.20.11590. - DOI - PMC - PubMed

[9] Durst KL, Hiebert SW. Role of RUNX family members in transcriptional repression and gene silencing. Oncogene. 2004;23:4220–4224. doi: 10.1038/sj.onc.1207122. - DOI - PubMed

[10] Durst KL, Hiebert SW. Role of RUNX family members in transcriptional repression and gene silencing. Oncogene. 2004;23:4220–4224. doi: 10.1038/sj.onc.1207122. - DOI - PubMed

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

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

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