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Abstract 349 The human and mouse β-globin loci share a conserved structure in which the locus control region (LCR) and genes are flanked by three CTCF bound DNaseI hypersensitive sites (HSs); 3‘HS1 downstream, and 5‘HS5 and human and mouse orthologues HS-111 or HS-62 upstream. In mice HS-62 and 3‘HS1 delineate a DNase sensitive domain. During erythroid differentiation, high-level expression of the β-globin locus is associated with LCR-dependent re-localization of the locus from the nuclear periphery to the nucleoplasm, where it associates with foci of serine-phosphorylated PolII deemed transcription factories (TFs). To investigate the relationships among chromatin structure, nuclear localization and β-globin expression during human erythropoiesis, CD34 progenitor cells were differentiated and analyzed by ChIP-array, primary transcript FISH, immuno-FISH, and chromatin conformation capture, carbon copy (5C). Localization of the β-locus away from the nuclear periphery and to TFs, and detection of β-nascent transcripts are rare events at day 4 (proerythroblasts), whereas by day 15 (polychromatic erythroblasts), nearly all loci are centrally located, associated with TFs and actively expressing. Three megabase profiles of complementary active and repressive histone marks (H3 lysine 4 di-methylation (DiK4) and H3 lysine 27 tri-methylation) reveal that DiK4 is enriched in the LCR and adult genes in undifferentiated CD34 cells and nears maximal enrichment by day 4. Thus the chromatin landscape is set up prior to erythroid commitment and is increased at day 4, but shows little change with activation. These profiles also reveal a previously un-described 257kb domain spanning from HS-111 to +146 relative to the ε-gene cap, with CTCF bound at its boundaries. 5C analysis reveals a high linkage frequency between the LCR and β-gene at day 4, prior to β-gene activation. Thus proximity may be necessary, but is not sufficient for high-level expression. In addition, the LCR and adult genes have frequent contact with surrounding regions, but interactions are sharply demarcated by HS-111 and +146, linking the above histone modification domain and 5C structure. The flanking regions HS-111, 3‘HS1 and +146 associate with the LCR and genes in an active chromatin hub (ACH)-like structure. By combining 5C with the Integrated Modeling Platform, a high-resolution three-dimensional (3D) model of chromatin structure was generated and revealed that the CTCF containing flanking regions, HS-111, 5'HS5, 3‘HS1 and +146 are in proximity and anchor loops of the intervening regions. The LCR and β-gene lie in close proximity ( In summary, our results reveal that acquisition of DiK4 precedes erythroid commitment. Enrichment of this active histone modification may occur in the nuclear periphery and is associated with a compact structure in which the flanking HSs, LCR and β-gene are in close proximity. Notably, close association of the LCR and β-gene precedes association with a TF and is not sufficient for expression. This suggests that if an ACH structure is important for function, its role may be limited to the initiation, but not the maintenance of gene expression. Alternatively, this compact structure may reflect the mafK-mediated recruitment of co-repressors to the LCR, as we demonstrated previously. The exchange of these repressors for activators could lead to the observed relocation from the periphery to TFs where high-level expression occurs and provides an explanation for the large change in expression that occurs despite a subtle change in the proximity of the LCR and β-gene. Preliminary studies on cell lines harboring intact, and LCR deficient chromosomes suggest the LCR may effect boundary element formation, domains of histone modifications and structure of the region. Disclosures: No relevant conflicts of interest to declare.

The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.