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1.
FIG. 8.

FIG. 8. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Model depicting USF-mediated regulation of β-globin gene expression. The expression of USF increases during the differentiation of erythroid cells. USF regulates the recruitment of transcription complexes to the β-globin gene locus by interacting with E-boxes located in LCR element HS2 and in the adult β-globin gene promoter. Through the LCR, USF regulates the expression of the embryonic genes. USF further regulates the expression of the globin genes indirectly by enhancing the expression of erythroid cell-specific transcription factors with which it cooperates in mediating the recruitment of transcription complexes to the globin gene locus.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
2.
FIG. 2.

FIG. 2. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Analysis of transgenic mouse embryos at different stages of development. Male embryos were isolated at the indicated time points of development from A-USF transgenic females (F1 females from line II) mated with wild-type (WT) males. Embryos were placed in a culture dish with PBS either in the presence or absence of the yolk sac (YS) and photographed using a Leica MZ16F4 instrument and the Qcapture program. Embryos were genotyped for sex and determined to be wild-type or transgenic (A-USF).

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
3.
FIG. 5.

FIG. 5. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

μChIP analysis of RNA Pol II and USF1 association with LCR element HS2 and the GAPDH gene in the yolk sac of wild-type and A-USF transgenic embryos. Embryos (10.5 dpc) were taken from an A-USF transgenic female (mated to a wild-type male). Yolk sacs were isolated and subjected to μChIP analysis. (A) μChIP was performed with antibodies against negative control IgG and USF1. DNA was analyzed by qPCR using primers specific for LCR element HS2 as well as for the control GAPDH gene, as indicated. (B) μChIP was performed with antibodies against the negative control IgG and RNA Pol II. The DNA was analyzed by qPCR using primers specific for LCR element HS2 as well as for the control GAPDH gene, as indicated. Data were normalized to those for IgG and are represented as means ± standard errors of the means of three independent μChIP experiments with qPCRs performed in triplicate.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
4.
FIG. 4.

FIG. 4. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Generation and analysis of transient transgenic mouse embryos expressing A-USF. Fertilized oocytes were injected with the A-USF expression construct and implanted into the uterus of a pseudopregnant foster mother. Embryos (11.5 dpc) were isolated and subjected to DNA (embryo) and RNA (yolk sac) extraction. (A) cDNA from the embryos was analyzed by RT-PCR using primers specific for the A-USF transgene to verify A-USF expression. All four embryos, two transgenic (TG IV and TG V) and two wild-type (WT 7 and WT 8) embryos, were taken from the same litter. (B) RNA was subjected to qRT-PCR performed in triplicate for the analysis of ɛγ-globin, βH1-globin, βmin-globin, Hba α1, EKLF, Band3, and USF1 gene expression. Data were analyzed and are represented as described in the legend to Fig. 3A.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
5.
FIG. 7.

FIG. 7. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Transgenic (TG) A-USF embryos reveal a reduction in the number of CD71-positive and Ter-119-positive erythroid cells. Yolk sac cells from 10.5-dpc male embryos were isolated and subjected to flow cytometry against CD71 or Ter-119. Hatched areas indicate unstained yolk sac cells analyzed separately. Solid lines represent the analysis of cells from A-USF-expressing transgenic embryos, while gray-shaded areas represent cells from wild-type embryos. (A) FACS analysis using antibodies against CD71. (B) Number of CD71-positive cells in the 10.5-dpc yolk sac of wild-type (WT) and A-USF-expressing (A-USF) embryos. (C) FACS analysis using antibodies against Ter-119. (D) βH1-globin gene expression in Ter-119+ embryonic yolk sac cells. Yolk sac cells from 10.5-dpc embryos were sorted using Ter-119 antibodies, and a subset of Ter-119+ cells was collected and subjected to RNA extraction. Data were analyzed as described in the legend to Fig. 3A and are represented as the means ± standard errors of the means of two qRT-PCRs performed in duplicate.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
6.
FIG. 3.

FIG. 3. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Effects of A-USF expression on the expression of erythroid genes and erythroid cell-specific transcription factors. RNA was extracted from 10.5- or 11.5-dpc embryos, reverse transcribed, and subjected to qRT-PCR performed in triplicate. (A) qRT-PCR analysis of ɛγ-globin, βH1-globin, βmin-globin, Hba α1, USF1 (left; 10.5 dpc), and βmaj-globin (right; 11.5 dpc) gene expression in A-USF transgenic (TG II/1 to II/4) and wild-type (WT 1 to 4) mouse embryos. Two sets of four embryos, each containing two TG and two WT animals, were examined. GAPDH was used as an internal control, and sample data were normalized to those for a respective WT littermate. Data are represented as means ± standard errors of the means of at least three PCRs on each sample. (B) qRT-PCR analysis of EKLF, GATA-1, Tal-1, p45, Band3, HoxB4, and USF1 gene expression in the yolk sac of the transgenic (TG II/1 and II/2) and wild-type (WT 1 and 2) 10.5-dpc embryos examined in panel A (left). Data are presented as described for panel A.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
7.
FIG. 6.

FIG. 6. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Interaction of USF with regulatory elements of genes encoding hematopoietic-specific transcription factors. ChIP analysis of the interaction of USF1 and USF2 with regulatory elements of the EKLF, GATA-1, Tal-1, and NF-E2 (p45) genes as well as with the Necdin promoter serving as a negative control. (A) The diagrams at the top indicate the position of E-boxes with respect to the transcription start site of each individual gene, with arrows indicating the location of primers used to amplify each region. ChIP was performed on uninduced or induced MEL cells. Cells were induced to differentiate for 3 days in the presence of 1.5% DMSO. Uninduced and induced cells were incubated with 1% formaldehyde. After being quenched with 125 mM glycine, the cells were lysed and chromatin was fragmented by sonication prior to precipitation with antibodies specific for IgG, USF1, or USF2. The isolated DNA was analyzed by qPCR with primers specific for the EKLF, GATA-1, Tal-1, NF-E2 (p45), and Necdin gene promoters, as indicated. Results are represented as means ± standard errors of the means of three independent experiments, with each PCR performed in duplicate. (B) ChIP performed on 16.5-dpc liver cells, examining the interaction of USF2 with the regions examined in panel A. Results are represented as means ± standard errors of the means of two independent experiments with PCRs performed in duplicate.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.
8.
FIG. 1.

FIG. 1. From: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor .

Generation and analysis of mice expressing A-USF. (A) DNA construct pITRp543f2A-USF4 used to generate transgenic mice expressing dominant-negative USF (A-USF). The A-USF coding region is under the control of the human β-globin gene promoter (β-P) and 3′ enhancer (3′E) as well as human LCR elements HS2 and HS3 plus flanking DNA. The DNA construct is flanked on either site by insulator elements derived from the chicken β-globin gene locus (cHS4). (B) SYBR green stain of the RT-PCR analysis of A-USF expression in transgenic (founders I and III and line II F1 littermates 1 to 3 [II/1 to II/3]) and wild-type (WT) mice. RNA was isolated from the spleens of phenylhydrazine-treated mice, reverse transcribed, subjected to PCR analysis with primers specific to the A-USF coding region, and electrophoresed in 5% Tris-borate-EDTA polyacrylamide gels. (C) Western blot analysis of A-USF expression in transgenic or wild-type mice. Protein was isolated from the spleen or liver of phenylhydrazine-treated mice and subjected to Western blot analysis using an antibody against USF1, which also detects A-USF. (D) qRT-PCR analysis of βmaj-globin gene expression in spleens of A-USF transgenic line II F1 littermates, A-USF founder mouse I, and wild-type mice. Data from the three line II F1 littermates were combined and are designated II/1/2/3. GAPDH was used as a loading control, and results from samples were normalized to those of the wild type. (E) ChIP analysis of RNA Pol II and USF2 interactions with the βmaj-globin gene promoter control mice (WT) and transgenic mice (A-USF). Spleens taken from two phenylhydrazine-treated F1 females (derived from line II) or wild-type mice were homogenized and subjected to ChIP analysis using antibodies against IgG, RNA Pol II, or USF2. Error bars reflect standard deviations from two independent experiments.

Shermi Y. Liang, et al. Mol Cell Biol. 2009 November;29(21):5900-5910.

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