The specific activation (and repression) of the erythroid transcriptome is the critical feature of erythropoiesis. While a great deal is known about the genetic and epigenetic control of the α- and β-globin loci, much less is known about the regulation of 40% of erythroid genes have alternative promoters, defined as two or more distinct promoter/first exon regions that are transcriptionally active and spliced to common exons downstream. The human ankyrin 1 gene (ANK1) contains three tissue specific alternative promoter/first exons. The first, known as ANK1B, is located 100 kb 5 of exon 2 and is active in neuronal and muscle cells. The ANK1A promoter is located 5 kb 5 of exon 2 and is expressed at low levels in many cell types and at higher levels in cerebellar cells. The ANK1E promoter/first exon is located between ANK1A and ANK1B 20 kb 5 of exon 2 and is activated only in erythroid cells. The goal of this project is to define the cis and trans regulation of the ANK1E promoter as a model for the additional 40% of erythroid genes that are expressed from alternative promoters. Mutations in the ANK1 gene are the most common cause of Hereditary Spherocytosis. We had previously identified a di-nucleotide deletion (-72/73) in the 5 transcribed but untranslated region of the human ANK1E gene in a family with ankyrin deficient Hereditary Spherocytosis. We showed that the presence of the deletion decreased promoter function both in vitro and in vivo, establishing it as a causative Hereditary Spherocytosis mutation. We hypothesized that the nucleotides surrounding the -72/73 di-nucleotide deletion would define an uncharacterized regulatory sequence. To test this hypothesis, we functionally tested a library of over 16,000 ANK-1 promoters with degenerate sequence between -78 and -70. We identified the wild type sequence (15%) and three additional sequences (10-40%). The novel sequences were shown to be functional in cell-free transcription, transient transfection and transgenic mouse assays. We concluded from these results that the di-nucleotide deletion at -72/73 disrupts a functional motif in the ANK-1 promoter. (Laflamme K, Owen AN, Devlin EE, Yang MQ, Wong C, Steiner LA, Garrett LJ, Elnitski L, Gallagher PG, Bodine DM. Functional analysis of a novel cis-acting regulatory region within the human ankyrin gene (ANK-1) promoter. Mol Cell Biol. 30: 3493-502, 2010.) Using the functional sequences, we derived a consensus sequence for the element in the ANK1E promoter. We examined 17,181 human promoters for the consensus sequence, which we called the TFIID localization sequence (DLS). We found that the DLS motif was present in 9% of human promoters. The combination of a DLS and an Sp1 binding site was sufficient to activate transcription in transient transfection assays. These results demonstrated that novel promoter elements can be identified on a genome-wide scale through studies of human dieease. (Yang MQ, Laflamme K, Gotea V, Joiner CH, Seidel NE, Wong C, Petrykowska HM, Lichtenberg J, Lee S, Welch L, Gallagher PG, Bodine DM, Elnitski L. Genome-wide detection of a TFIID localization element from an initial human disease mutation. Nucleic Acids Res. 39 (6): 2175-87, 2011.) In some Hereditary Spherocytosis kindreds, linked -108/-153 nucleotide substitutions have been found in the 5 upstream (untranscribed) region of the ANK1E promoter. In previous work we showed that in vivo, the ANK1E promoter and upstream region directed position-independent, uniform expression in transgenic mice, properties that have been associated with barrier insulators. To test the hypothesis that the ANK1E upstream region is a barrier, we performed both K562-based and transgenic mouse barrier assays and found that this region is indeed a barrier insulator both in vitro and in vivo. The barrier exhibits all of the properties of a barrier, including the prevention of gene silencing in vivo and the appropriate chromatin modifications. When the -108/-153 Hereditary Spherocytosis-associated mutations are introduced into the barrier region, barrier function was lost with a loss of the barrier-associated chromatin modifications. These data identified the upstream region of the ANK1E promoter as a barrier insulator and is the first demonstration of mutations in a barrier element causing a human disease. (Gallagher PG, Steiner LA, Liem RI, Owen AN, Cline AP, Seidel NE, Garrett LJ, Bodine DM. Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis. J Clin Invest. 120 (12): 4453-65, 2010.) After identifying the 5 ANK1E promoter barrier, we sought to locate a partner downstream. We found a second ANK1E barrier insulator located in an adjacent pair of DNase I hypersensitive sites located 5.6kb 3 of the ANK1E promoter. Like the 5 barrier, the 3 barrier effectively prevents transgene silencing in vitro and in vivo. The ANK1E 3 barrier also contains an NF-E2 dependent enhancer that is not required for barrier function. We demonstrated that a chromatin loop formed by the two barrier elements brings the 3 enhancer and NF-E2 into proximity with the 5 barrier region containing the ANK1E promoter. This is the first demonstration of a mechanism for determining which alternative promoter is active in a specific cell type. (Yocum AO, Steiner LA, Seidel NE, Cline AP, Rout ED, Lin JY, Wong C, Garrett LJ, Gallagher PG, Bodine DM. A Tissue Specific Chromatin Loop Activates the Erythroid Ankyrin-1 Promoter. Blood. In press.) This project is largely completed, and our future efforts will be on more genome-wide analyses of erythroid gene expression. However, our detailed understanding of the function of the ANK1E promoter will allow us to use this locus in addition to the globin loci to test hypotheses generated from these analyses (see below). Project 2. Erythropoiesis and megakaryopoiesis are both dependent on the interactions of transcription factors and other proteins with the primary DNA sequence. The goal of this project is to define the interactome of the transcription factors GATA1, NF-E2 and KLF1 in the chromatin of primary erythroblasts, megakaryocytes and their common progenitor, the Megakaryocyte/Erythroid Progenitor (MEP). In our initial studies, we used ChIP-Seq to study the interactome of KLF1 in primary mouse erythroid progenitor cells and more differentiated erythroblasts. In erythroid progenitor cells, we found that KLF1 was located at the periphery of the nucleus, occupied the promoter regions of genes, and acted as a transcriptional activator. In erythroblasts, we found that KLF1 was distributed throughout the nucleus, and erythroblast-specific KLF1 occupancy was predominantly in intragenic regions. We found very little overlap between KLF1 occupancy and the other transcription factors, leading to a model in which KLF1 directs programs that are independent of those regulated by the GATA factors or TAL1. (Pilon AM, Ajay SS, Kumar SA, Steiner LA, Cherukuri PF, Wincovitch S, Anderson SM;NISC Comparative Sequencing Center, Mullikin JC, Gallagher PG, Hardison RC, Margulies EH, Bodine DM. Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation. Blood. 118 (17): e139-48, 2011.) The limitation of our model is that the comparative analyses we have done used data collected from cell lines. To fully test our model we will perform sequential ChIPSeq on primary MEP, erythroblasts and megakaryocytes sorted from adult mouse bone marrow. Our transcription factor profiling of GATA1, NF-E2 and KLF1 in MEP, erythroblasts and megakaryocytes will allow investigators to mine an enormous amount of data to develop testable hypotheses about the genetic programs associated with erythroid and megakaryocyte specification and differentiation.
Showing the most recent 10 out of 24 publications
