![Figure 1. In vitro and in vivo functional analysis of mouse Runx1 promoter fragments. (A) Schematic of mouse Runx1 locus showing the location of the P1 and P2 promoters, the +23 hematopoietic enhancer (black arrow) and exons (light blue indicates untranslated region [UTR]; dark blue, coding sequences). (B) Vista plots showing percentage identity in pairwise alignments of the mouse Runx1 P1 and P2 promoter fragments used in this study and their corresponding human (Homo sapiens), dog (Canis familiaris), opossum (Monodelphis domestica), chicken (Gallus gallus), and frog (Xenopus tropicalis) sequences. A 6-way RankVista25 alignment indicates the extent of multispecies conservation and the likelihood (−log10[P value]) this conservation arose by chance. Genomic sequences and alignments as in Nottingham et al.9 Pink denotes areas of more than 70% noncoding sequence conservation more than 100 bp; light blue, conserved UTRs. (C) Luciferase activity of mouse P1 and P2 promoter constructs in transiently transfected 416B myeloid progenitor cells. Constructs with the SV40 core promoter were used as controls. Data are the mean plus or minus SD of more than or equal to 5 independent transfections, using more than or equal to 2 separately prepared batches of test plasmids. (D) In vivo analysis of mouse P1 and P2 promoter fragments in F0 transgenic mouse embryos. Embryos were analyzed for LacZ expression as whole mounts and after cryosectioning as before.9 No reproducible Runx1-specific Xgal staining could be observed in F0 transgenic mouse embryos carrying the P1 LacZ (n = 10) or P2 LacZ (n = 20) transgenes. Nonspecific staining, or no staining, is presumably the result of random integration of the constructs at or near endogenous enhancers, or in heterochromatin, respectively. (E) Real-time PCR analysis of ChIP for SCL and Gata2 in mouse (CBAxC57BL/6)/F1 FL cells and 416B cells, respectively. ChIP for SCL shows strong enrichment for the +23, but no/low-level enrichment for the P1 and P2. Gata2 strongly binds the +23 and to a lesser extent the P1, but not the P2. Both the +23 and the P1 harbor a mouse-frog conserved Gata motif, whereas the putative Gata motif in the P2 is only conserved between mouse, human, and dog (not shown). For SCL, data are the mean plus or minus SD of 2 independent ChIP experiments with 2 real-time PCR assays per ChIP; for Gata2 one representative experiment is shown. No Ab indicates no antibody control. Runx1 +23 enhancer and P1 and P2 promoter primers and probes used for real-time PCR are listed in Table S2.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/21/10.1182_blood-2008-12-193003/6/zh89990936270001.jpeg?Expires=1724234062&Signature=l-Ss~tug7PkleI~t4IBN-pQwWbXgQuCl0YJJtbbSTl0iRmjkLH~YCYkK1V9wmE71xWcWqFVOvEbb42~v867Lg~Bnvwi28ZAnvHVsp0s~S5tXbifqUnVACFY6NC8KuWpcXiZ~vdBt0uKRwz5I5jFE4nb3AF2wRB1c9QTYfwWqIpb~g-POcbUBjv-K07-6SK5GRmmMVzcH5PageD7nkJX7gPTelAlwFT6B6YH4dk-6DO2~v4YHfbB~APjjSZxuwkhF13jCicA6DvoogFtmVeNyJPbIlIVO8PuIMGWytxRs1nHBSNqgwoglqX2qIdh6w-NUaRNs1~ox3EwAxSdR0i~gng__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In vitro and in vivo functional analysis of mouse Runx1 promoter fragments. (A) Schematic of mouse Runx1 locus showing the location of the P1 and P2 promoters, the +23 hematopoietic enhancer (black arrow) and exons (light blue indicates untranslated region [UTR]; dark blue, coding sequences). (B) Vista plots showing percentage identity in pairwise alignments of the mouse Runx1 P1 and P2 promoter fragments used in this study and their corresponding human (Homo sapiens), dog (Canis familiaris), opossum (Monodelphis domestica), chicken (Gallus gallus), and frog (Xenopus tropicalis) sequences. A 6-way RankVista25 alignment indicates the extent of multispecies conservation and the likelihood (−log10[P value]) this conservation arose by chance. Genomic sequences and alignments as in Nottingham et al.9 Pink denotes areas of more than 70% noncoding sequence conservation more than 100 bp; light blue, conserved UTRs. (C) Luciferase activity of mouse P1 and P2 promoter constructs in transiently transfected 416B myeloid progenitor cells. Constructs with the SV40 core promoter were used as controls. Data are the mean plus or minus SD of more than or equal to 5 independent transfections, using more than or equal to 2 separately prepared batches of test plasmids. (D) In vivo analysis of mouse P1 and P2 promoter fragments in F0 transgenic mouse embryos. Embryos were analyzed for LacZ expression as whole mounts and after cryosectioning as before.9 No reproducible Runx1-specific Xgal staining could be observed in F0 transgenic mouse embryos carrying the P1 LacZ (n = 10) or P2 LacZ (n = 20) transgenes. Nonspecific staining, or no staining, is presumably the result of random integration of the constructs at or near endogenous enhancers, or in heterochromatin, respectively. (E) Real-time PCR analysis of ChIP for SCL and Gata2 in mouse (CBAxC57BL/6)/F1 FL cells and 416B cells, respectively. ChIP for SCL shows strong enrichment for the +23, but no/low-level enrichment for the P1 and P2. Gata2 strongly binds the +23 and to a lesser extent the P1, but not the P2. Both the +23 and the P1 harbor a mouse-frog conserved Gata motif, whereas the putative Gata motif in the P2 is only conserved between mouse, human, and dog (not shown). For SCL, data are the mean plus or minus SD of 2 independent ChIP experiments with 2 real-time PCR assays per ChIP; for Gata2 one representative experiment is shown. No Ab indicates no antibody control. Runx1 +23 enhancer and P1 and P2 promoter primers and probes used for real-time PCR are listed in Table S2.
