6 research outputs found
Chromatin dynamics and the regulation of B-glohin gene expression
The human B-globin locus is frequently used as a model system to study mechanisms
controlling tissue-specific and developmentally regulated gene expression. Much of the recent
progress in understanding the regulation of B-globin gene expression has come from a better
knowledge of the process of transcription. Proper transcriptional regulation of the human B-globin
genes occurs, at least in part, through specific interactions of regulatory traus-acting
proteins to defined cis-regulatory sequences that include promoters, enhancers, silencers, and
elements of the locus control region
The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene
The beta-globin locus control region (LCR) is contained on a 20 kb DNA
fragment and is characterized by the presence of five DNaseI
hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active
6.5 kb version of the LCR, called the muLCR, has been described.
Expression of the beta-like globin genes is absolutely dependent on the
presence of the LCR. The developmental expression pattern of the genes in
the cluster is achieved through competition of the promoters for the
activating function of the LCR. Transgenic mice experiments suggest that
subtle changes in the transcription factor environment lead to the
successive silencing of the embryonic epsilon-globin and fetal
gamma-globin promoters, resulting in the almost exclusive transcription of
the beta-globin gene in adult erythropoiesis. In this paper, we have asked
the question whether the LCR and its individual hypersensitive sites
5'HS1-4 can activate a basic promoter in the absence of any other globin
sequences. We have employed a minimal promoter derived from the mouse
Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The
results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic
expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at
any stage of development. Expression of the muLCR and 5'HS3 transgenes is
repressed during fetal stages of development. The transgenes are in an
inactive chromatin conformation and the lacZ gene is not transcribed, as
shown by in situ hybridization. These data are compatible with the
hypothesis that the LCR requires the presence of an active promoter to
adopt an open chromatin conformation and with models proposing progressive
heterochromatization during embryogenesis. The results suggest that the
presence of a beta-globin gene is required for LCR function as conditions
become more stringent during development
The role of EKLF in human β-globin gene competition.
We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKL
The human β-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial β-galactosidase gene.
The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5'HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at any stage of development. Expression of the muLCR and 5'HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development
Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo.
The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3
Tumor stroma-derived Wnt5a induces differentiation of basal cell carcinoma of ptch-mutant mice via caMKII
Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptchr flox/floxERT2 +/- knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca 2+ signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling