Disease relevance of Hbb-ar

• Induction of differentiation down a nonerythroid pathway causes a progressive loss of hypersensitivity in the LCR [1].
• Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives [2].
• These results suggest that HS1234 may function as a locus control region (LCR), deregulating c-myc expression in t(15;12) plasmacytomas, as well as potentially contributing to aspects of normal IgH chain expression [3].
• In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal [4].
• We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice [5].

High impact information on Hbb-ar

• The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication [6].
• We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction [6].
• Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced-a phenomenon known as position effect variegation (PEV) [7].
• The long-distance influence of LCR elements on chromatin structure and gene expression is remarkable and likely to be encountered in the analysis of other developmentally regulated, multigene loci [8].
• The five human beta-type-globin genes, epsilon, Ggamma, Agamma, delta and beta, are close together and are regulated by a locus control region (LCR) located at the 5' end of the locus [9].

Biological context of Hbb-ar

• Structure and function of the murine beta-globin locus control region 5' HS-3 [10].
• Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells [10].
• Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice [11].
• Mice derived from these ES cells (delta HS2 delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome [11].
• Replacement of 5'HS2 with a selectable marker gene (delta HS2 + neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis [11].

Anatomical context of Hbb-ar

• Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells [10].
• Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells [12].
• Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific [13].
• However, each site is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult red blood cells [14].
• In the T cell receptor (TCR) alpha/delta locus, an LCR might regulate the differential tissue and developmental expression and the rearrangement of TCR alpha and delta genes [15].

Associations of Hbb-ar with chemical compounds

• To define the array of nuclear factors interacting with beta-LCR HS-3, we have performed in vivo dimethyl sulfate footprinting of the active HS-3 core in erythroid cells by a modified procedure that permits assessment of protein-DNA contacts at adenine, as well as guanine, residues [16].
• Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7] [17].
• Furthermore, chromatin immunoprecipitation experiments revealed that Bach1 bound to the MARE of HS2 increased by the treatment of MEL cells with SA, and this was cancelled by hemin [18].
• Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15 [19].
• To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels [20].

Other interactions of Hbb-ar

• The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone [10].
• The results obtained support a general role of EKLF in beta-globin gene activation and are in agreement with models involving an advantage of the LCR proximal respect to distal gene [21].
• Activation of beta-major globin gene transcription is associated with recruitment of NF-E2 to the beta-globin LCR and gene promoter [22].
• We show that LCR hypersensitive sites characteristic of erythroid cells are present in three independent multilineage progenitors [FDCP (factor-dependent cell, Paterson)-mix A4, B6SUtA, and LyD9] under conditions of self-renewal [1].
• The LCR is composed of a series of 5 DNaseI hypersensitive sites (5'HSs) that form in the nucleus of erythroid precursors [23].

Analytical, diagnostic and therapeutic context of Hbb-ar

• Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver [24].
• Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects [25].
• To address these questions, we established a transgenic reporter model of TCRalpha LCR function that allows for analysis of LCR activity in individual cells by the use of flow cytometry [26].
• Our findings suggest that sequences in the distal promoter play a role in single copy transgene activation and that larger LCR and promoter elements are most suitable for gene therapy applications [27].
• In gel retardation assays, a short fragment from HS4 formed complexes with nuclear extracts from both erythroid and nonerythroid cells, and a core protected sequence 5'GACTGGC3' was revealed by DNAse I protection and methylation interference studies [28].

References