A tissue-specific chromatin loop activates the erythroid ankyrin-1 promoter.

The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the ∼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.

Exploiting pre-rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis.

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

Genome-wide detection of a TFIID localization element from an initial human disease mutation.

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17,181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.

Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis.

Defects of the ankyrin-1 gene are the most common cause in humans of hereditary spherocytosis, an inherited anemia that affects patients of all ethnic groups. In some kindreds, linked -108/-153 nucleotide substitutions have been found in the upstream region of the ankyrin gene promoter that is active in erythroid cells. In vivo, the ankyrin erythroid promoter and its upstream region direct position-independent, uniform expression, a property of barrier insulators. Using human erythroid cell lines and primary cells and transgenic mice, here we have demonstrated that a region upstream of the erythroid promoter is a barrier insulator in vivo in erythroid cells. The region exhibited both functional and structural characteristics of a barrier, including prevention of gene silencing in an in vivo functional assay, appropriate chromatin configuration, and occupancy by barrier-associated proteins. Fragments with the -108/-153 spherocytosis-associated mutations failed to function as barrier insulators in vivo and demonstrated perturbations in barrier-associated chromatin configuration. In transgenic mice, flanking a mutant -108/-153 ankyrin gene promoter with the well-characterized chicken HS4 barrier insulator restored position-independent, uniform expression at levels comparable to wild-type. These data indicate that an upstream region of the ankyrin-1 erythroid promoter acts as a barrier insulator and identify disruption of the barrier element as a potential pathogenetic mechanism of human disease.

Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes.

Gene therapy for hematopoietic diseases has been hampered by the low frequency of transduction of human hematopoietic stem cells (HSCs) with retroviral vectors pseudotyped with amphotropic envelopes. We hypothesized that transduction could be increased by the use of retroviral vectors pseudotyped with envelopes that recognize more abundant cellular receptors. The levels of mRNA encoding the receptors of the feline retroviruses, RD114 and feline leukemia virus type C (FeLV-C), were significantly higher than the level of gibbon ape leukemia virus (GaLV) receptor mRNA in cells enriched for human HSCs (Lin- CD34+ CD38-). We cotransduced human peripheral blood CD34+ cells with equivalent numbers of FeLV-C and GALV or RD114 and GALV-pseudotyped retroviruses for injection into fetal sheep. Analysis of DNA from peripheral blood and bone marrow from recipient sheep demonstrated that FeLV-C- or RD114-pseudotyped vectors were present at significantly higher levels than GALV-pseudotyped vectors. Analysis of individual myeloid colonies demonstrated that retrovirus vectors with FeLV-C and RD114 pseudotypes were present at 1.5 to 1.6 copies per cell and were preferentially integrated near known genes We conclude that the more efficient transduction of human HSCs with either FeLV-C- or RD114-pseudotyped retroviral particles may improve gene transfer in human clinical trials.

Hmgb3: an HMG-box family member expressed in primitive hematopoietic cells that inhibits myeloid and B-cell differentiation.

Hmgb3 is a member of a family of chromatin-binding proteins that can alter DNA structure to facilitate transcription factor binding. We identified the Hmgb3 cDNA in a subtractive hybridization screen for transcripts that are preferentially expressed in hematopoietic stem cells. We inserted an internal ribosomal entry site-green fluorescence protein cassette into the 3' untranslated region of the X-linked Hmgb3 locus to identify Hmgb3-expressing cells. In adult mice, Hmgb3 mRNA is detected in bone marrow cells, primitive Lin-, c-kit+, Sca-1+, IL-7Ralpha- cells, and Ter119+ erythroid cells. We observed that long-term repopulating ability is entirely contained in the subpopulation of Lin-, c-kitHI cells that express Hmgb3. Most common lymphoid and myeloid progenitors express Hmgb3. Introduction of a retrovirus containing the Hmgb3 cDNA into mouse bone marrow stem cells demonstrated that enforced expression of Hmgb3 inhibited B-cell and myeloid differentiation. We conclude that down-regulation of Hmgb3 protein levels is an important step for myeloid and B-cell differentiation.

Retroviral transduction of IL2RG into CD34(+) cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras.

X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain of cytokine receptors, gamma(c). Because bone marrow transplantation (BMT) for XSCID does not provide complete immune reconstitution for many patients and because of the natural selective advantage conferred on lymphoid progenitors by the expression of normal gamma(c), XSCID is a good candidate disease for therapeutic retroviral gene transfer to hematopoietic stem cells. We studied XSCID patients who have persistent defects in B-cell and/or combined B- and T-cell function despite having received T cell-depleted haploidentical BMT. We compared transduction of autologous B-cell lines and granulocyte colony-stimulating factor-mobilized peripheral CD34(+) cells from these patients using an MFGS retrovirus vector containing the gamma(c) gene IL2RG pseudotyped with amphotropic, gibbon ape leukemia virus, or RD114 envelopes. Transduced B-cell lines and peripheral CD34(+) cells demonstrated provirus integration and new cell-surface gamma(c) expression. The chimeric sheep model was exploited to test development of XSCID CD34(+) cells into mature myeloid and lymphoid lineages. Transduced and untransduced XSCID CD34(+) cells injected into developing sheep fetuses gave rise to myeloid cells. However, only transduced gamma progenitors from XSCID patients developed into T and B cells. These results suggest that gene transfer to autologous peripheral CD34(+) cells using MFGS-gc retrovirus may benefit XSCID patients with persistent T- and B-cell deficits despite prior BMT.