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

<p>Abstract</p> <p>Background</p> <p>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 <it>Runx1 </it>lack definitive hematopoiesis and die in mid-gestation. Expression of <it>Runx1 </it>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 <it>Runx1 </it>biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of <it>Runx1 </it>during development.</p> <p>Results</p> <p>We employed mice bearing a hypomorphic <it>Runx1 </it>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.</p> <p>Conclusion</p> <p>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.</p

Absence of Runx3 expression in normal gastrointestinal epithelium calls into question its tumour suppressor function

The Runx3 transcription factor regulates cell fate decisions during embryonic development and in adults. It was previously reported that Runx3 is strongly expressed in embryonic and adult gastrointestinal tract (GIT) epithelium (Ep) and that its loss causes gastric cancer. More than 280 publications have based their research on these findings and concluded that Runx3 is indeed a tumour suppressor (TS). In stark contrast, using various measures, we found that Runx3 expression is undetectable in GIT Ep. Employing a variety of biochemical and genetic techniques, including analysis of Runx3-GFP and R26LacZ/Runx3Cre or R26tdTomato/Runx3Cre reporter strains, we readily detected Runx3 in GIT-embedded leukocytes, dorsal root ganglia, skeletal elements and hair follicles. However, none of these approaches revealed detectable Runx3 levels in GIT Ep. Moreover, our analysis of the original Runx3LacZ/LacZ mice used in the previously reported study failed to reproduce the GIT expression of Runx3. The lack of evidence for Runx3 expression in normal GIT Ep creates a serious challenge to the published data and undermines the notion that Runx3 is a TS involved in cancer pathogenesis

Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis-3

<p><b>Copyright information:</b></p><p>Taken from "Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis"</p><p>http://www.biomedcentral.com/1471-213X/7/84</p><p>BMC Developmental Biology 2007;7():84-84.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1941738.</p><p></p>lots of E15.5 to E18.5 WT and P2thymocytes indicating the percentages of cells in each quadrant. Histograms in (B) show the average ± S.E. of TCRβ/TCRγδ proportions of WT and P2thymocytes in at least three experiments at each developmental stage. The difference between WT and P2was 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 P2compared to WT, at E18.5 the distribution of P2thymocytes resumed normal pattern. The mean fluorescence intensity (MFI) (B right) of E15.5 and E16.5 TCRβP2thymocytes was significantly higher than WT (p < 0.005; by Student's t test). (C) Expression of TCRβ/TCRγδ in P2thymocytes is transcriptionally upregulated. RT-PCR analysis of RNA derived from WT and P2E16.5 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 P2thymocytes compared to WT. (D and E) Enhanced apoptosis of P2thymocytes is associated with elevated expression of TCRβ/TCRγδ. (D) Histograms demonstrating the proportion of apoptotic cells among TCRβor TCRgδthymocytes. 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 TCRγδwhere all cells are apoptotic). Most of TCRβ/TCRγδ positive P2thymocytes (red), are at the M1 (Annexin V) apoptotic subset. (E) Proportion of apoptotic thymocytes among TCRβ/TCRγδ positive E15.5, E16.5 and E18.5 thymocytes. Barrepresent the average ± S.E. of at least three independent experiments for each time point using different mice. The differences between WT and P2in number of TCRβapoptotic thymocytes were significant at P < 0.01 (*) by Student's t test

Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis-4

<p><b>Copyright information:</b></p><p>Taken from "Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis"</p><p>http://www.biomedcentral.com/1471-213X/7/84</p><p>BMC Developmental Biology 2007;7():84-84.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1941738.</p><p></p>fferent 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 P2thymocytes 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

Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis-2

<p><b>Copyright information:</b></p><p>Taken from "Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis"</p><p>http://www.biomedcentral.com/1471-213X/7/84</p><p>BMC Developmental Biology 2007;7():84-84.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1941738.</p><p></p>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 P2thymi. (B) P2thymocytes display enhanced apoptosis throughout embryonic thymopoiesis. Proportion of non-viable (apoptotic+necrotic) WT and P2cells gradually decreased during embryonic development. Nevertheless, at any given time point, P2thymui contained a higher proportion of non-viable cells compared to WT littermates. After birth the proportion of non-viable thymocytes in P2became similar to WT. At least five mice of each genotype were analyzed. The differences between WT and P2apoptotic thymocytes were significant at P < 0.001 (*) and P < 0.01 (#) by Student's t test. (C) P2thymocytes retain normal proliferation capacity. Following FACS analysis an equal number (1 × 10) of E15.5 WT or P2Annexin V negative thymocytes were incubated with TPA (20 ng/ml) and ConA (5 μg/ml) for 48 h. H-thymidine was present during the last 18 h of incubation. Differences between WT and P2were 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 P2thymocytes. WT or P2E15.5 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) per lobe were sufficient to populate all available thymic lobes and therefore used 1000 FL cells per lobe. Thymocytes accumulation in FTOC populated with P2FL HPC is reduced compared to WT (left panel WT-black; P2-white). Proportion of apoptotic thymocytes (Annexin V) derived from FTOC populated with P2FL cells (red) was considerably higher compared to WT (light blue) (right panel)

Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis-0

<p><b>Copyright information:</b></p><p>Taken from "Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis"</p><p>http://www.biomedcentral.com/1471-213X/7/84</p><p>BMC Developmental Biology 2007;7():84-84.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1941738.</p><p></p>P2neonates. P2-mediated transcription in P2tissues diminished, whereas P1-mediated transcription was largely unaffected. (B) IHC analysis of glandular stomach of E16.5 WT (left) and P2(right) embryos. Runx1 expression is detected in epithelial cells of WT embryo, but missing in P2littermate (10× magnification). (C) Reduced size of thymic lobes in P1.5 P2mice (right) as compared to WT littermate (left). (D) RT-PCR analysis of P1- and P2-mediated transcription in thymus of WT embryos, neonates and young mice. (E) Analysis of expression by hybridization of E15.5 WT (left) and P2(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(10× magnification). (co) = cortex; (me) = medulla. (F) Western blot analysis of proteins extracted from thymus of E15.5–E17.5 WT and P2embryos. Runx1 proteins were not detected in E15.5 P2thymus, but gradually accumulated in E16.5 and E17.5 thymi. (G) Northern blot analysis of RNA from thymi of WT and P2newborn mice. Whereas P2-mediated transcription (the 4 Kb and 8 Kb transcripts) [30, 31], in P2thymocytes was markedly attenuated, P1-mediated transcription (the 2 Kb and 6 Kb transcripts) was apparently unaffected. (H) Reduced thymus cellularity in P2embryos and neonates. Five mice of each genotype were analyzed. The difference between the number of thymocytes from WT and P2thymus was significant at P < 0.0001 (*) and P < 0.05 (#) by Student's t test

Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis-8

<p><b>Copyright information:</b></p><p>Taken from "Developmentally regulated promoter-switch transcriptionally controls function during embryonic hematopoiesis"</p><p>http://www.biomedcentral.com/1471-213X/7/84</p><p>BMC Developmental Biology 2007;7():84-84.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1941738.</p><p></p>P2neonates. P2-mediated transcription in P2tissues diminished, whereas P1-mediated transcription was largely unaffected. (B) IHC analysis of glandular stomach of E16.5 WT (left) and P2(right) embryos. Runx1 expression is detected in epithelial cells of WT embryo, but missing in P2littermate (10× magnification). (C) Reduced size of thymic lobes in P1.5 P2mice (right) as compared to WT littermate (left). (D) RT-PCR analysis of P1- and P2-mediated transcription in thymus of WT embryos, neonates and young mice. (E) Analysis of expression by hybridization of E15.5 WT (left) and P2(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(10× magnification). (co) = cortex; (me) = medulla. (F) Western blot analysis of proteins extracted from thymus of E15.5–E17.5 WT and P2embryos. Runx1 proteins were not detected in E15.5 P2thymus, but gradually accumulated in E16.5 and E17.5 thymi. (G) Northern blot analysis of RNA from thymi of WT and P2newborn mice. Whereas P2-mediated transcription (the 4 Kb and 8 Kb transcripts) [30, 31], in P2thymocytes was markedly attenuated, P1-mediated transcription (the 2 Kb and 6 Kb transcripts) was apparently unaffected. (H) Reduced thymus cellularity in P2embryos and neonates. Five mice of each genotype were analyzed. The difference between the number of thymocytes from WT and P2thymus was significant at P < 0.0001 (*) and P < 0.05 (#) by Student's t test