In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human A

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.The erythrocyte membrane is a multifunctional, complex structure that provides the red cell the deformability and stability required to withstand its travels through macro-and microcirculation. It plays critical roles in erythropoiesis, including responding to erythropoietin, importing iron required for hemoglobin synthesis, and regulating cellular metabolism. Qualitative and quantitative disorders of erythrocyte membrane proteins have been associated with inherited abnormalities of red cell shape, including hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis syndromes (65, 103). Despite biochemical and genetic linkage to specific erythrocyte membrane protein genes, e.g., ankyrin-1, ␣-or ␤-spectrin, and band 3, mutations are found in the coding exons and promoter regions of only ϳ75% of cases studied. This suggests that the disease-causing mutation is located in critical regulatory regions outside the promoters and exons in a quarter of cases.Most erythrocyte membrane protein genes are large, comprised of Ͼ25 exons. They encode numerous diverse and complex isoforms, frequently generated by alternate splicing, alternate promoter usage, or alternate polyadenylation (18). In many cases, alternate promoters direct combinations of exons encoding diverse tissue-specific, cell type-specific, developmental-stage-specific, and/or differentiation stage-specific isoforms (6, 12, 13, 19, 21-24, 44, 52, 62, 78, 86, 108, 112-114). As such, erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and fu...

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, ؊108T to C and ؊108T to C in cis with ؊153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human A ␥-globin gene expressed ␥-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant ؊108 promoter demonstrated variegated ␥-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked ؊108/؊153 mutations. Three transgenic lines had undetectable levels of A ␥-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of A ␥-globin mRNA than wild type. Two of four expressing lines showed variegated ␥-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy numberindependent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations. Hereditary spherocytosis (HS)1 is a common hemolytic anemia characterized by the presence of spherically shaped erythrocytes on peripheral blood smear. The principal cellular defect in HS is loss of erythrocyte membrane surface area relative to intracellular volume, accounting for the spherical shape as well as decreased deformability of the erythrocyte (1). The primary biochemical defects in HS reside in the proteins of the erythrocyte membrane, particularly those proteins involved in the interactions between the membrane skeleton and the lipid bilayer: ankyrin, ␣ and ␤ spectrin, band 3, and protein 4.2 (2, 3). In two thirds to three quarters of cases, HS is inherited in an autosomal dominant fashion (1-4). In the remaining patients, HS is inherited in a recessive fashion or is the result of a de novo mutation.Ankyrin-1 (ANK1, Mendelian Inheritance in Man 182900) deficiency is one of the most common abnormalities found in the erythrocyte membranes of HS patients (5, 6). First identified in preparations of erythrocyte membranes, ankyrin provides the primary linkage between the spectrin-actin-based erythrocyte membrane skeleton and the plasma membrane by attaching tetramers of spectrin to the cytoplasmic domain of band 3 (7, 8). Studies have revealed that abnormalities of the ankyrin gene, primarily frameshift or nonsense mutations, are the most common cause of typical, dominant HS (3, 9 -11).Ankyrin-1 is transcribed in erythroid cells from a compact, erythroid-specific promoter (12). One molecular mechanism that could lead to ankyrin deficiency is a mutation in the ankyrin erythroid promoter leading to decr...

Growth Factor Independence(Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

Post-translational histone modifications, acting alone or in a context-dependent manner, influence numerous cellular processes via their regulation of gene expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly understood histone modification. Some reports describe depletion of K27Me1 at promoters and transcription start sites (TSS), implying its depletion at TSS is necessary for active transcription, while others have associated enrichment of H3K27me1 at TSS with increased levels of mRNA expression. Tissue-and gene-specific patterns of H3K27me1 enrichment and their correlation with gene expression were determined via chromatin immunoprecipitation on chip microarray (ChIP-chip) and human mRNA expression array analyses. Results from erythroid cells were compared with those in neural and muscle cells. H3K27me1 enrichment varied depending on levels of cell-type specific gene expression, with highest enrichment over transcriptionally active genes. Over individual genes, the highest levels of H3K27me1 enrichment were found over the gene bodies of highly expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of actively transcribing genes, H3K27me1 was selectively depleted at the TSS of actively transcribed genes. There was markedly decreased to no H3K27me1 enrichment in genes with low expression. At some locations, H3K27 monomethylation was also found to be associated with chromatin signatures of gene enhancers.The dynamic state of chromatin is influenced by many factors including nucleosomal configuration (1). Nucleosomes are composed of repetitive histone octamer units wrapped by DNA. Histones are evolutionarily conserved proteins with a characteristic histone fold and flexible NH 2 -terminal tails. Post-translational modifications of histones (2) acting alone or in a context-dependent manner, influence various cellular processes such as transcription, replication, DNA repair, and cell cycle progression by regulating the intrinsic chromatin environment or modifying the binding of non-histone, chromatinmodifying protein complexes (3, 4). Although some histone modifications correlate with specific biologic activities, it is clear that the complexity of histone modifications, and their interactions are just beginning to be revealed (4 -7). Understanding the effect these modifications have on cellular processes will provide important insights into both normal and disease-associated processes (8).Methylation of position-specific lysine residues in NH 2 -terminal histone tails is a critical post translational modification that can be associated with either active or repressed chromatin (9). For example, modifications such as trimethylation of histone H3 lysine 4 and acetylation of histone H3 lysine 9 are generally associated with euchromatin and gene activation (2) while trimethylation of histone H3 lysine 9 is generally associated with hetero-chromatin and gene repression.In addition to the unmodified form, histone H3 K27 exists in three methylation states, monomethylated, dimeth...