SATB1 nuclear protein, expressed predominantly in T-cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with {epsilon}-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases {gamma}-globin and downregulates SATB1 family protein and {epsilon}-globin gene expression. Chromatin immunoprecipitation (ChIP) using anti-SATB1 antibody shows selective binding in vivo in the {beta}-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the {epsilon}-globin promoter. SATB1 overexpression increases {epsilon}-globin and decreases{gamma}-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the {epsilon}-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the {gamma}-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In co-transfection experiments, increase in {epsilon}-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can upregulate the {epsilon}-globin gene by interaction with specific sites in the {beta}-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation.

The human β globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5'-HBE1, HBG2, HBG1, HBD, HBB-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in 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, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the β-globin ACH, and HPFH.

The active elements of the beta -globin locus control region (LCR) are located within domains of unique chromatin structure. These nuclease hypersensitive sites (HSs) are characterized by high DNase I sensitivity, erythroid specificity, similar nucleosomal structure, and evolutionarily conserved clusters of cis-acting elements that are required for the formation and function of the core elements. To determine the requirements for HS core formation in the setting of nuclear chromatin, we constructed a series of artificial HS cores containing binding sites for GATA-1, NF-E2, and Sp1. In contrast to the results of previous in vitro experiments, we found that when constructs were stably integrated in mouse erythroleukemia cells the binding sites for NF-E2, GATA-1, or Sp1 alone or in any combination were unable to form core HS structures. We subsequently identified two new cis-acting elements from the LCR HS4 core that, when combined with the NF-E2, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-acting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo beta -globin LCR HS core formation is more complex than previously thought and that several factors are required for this process to occur.

Nuclear compartmentalization seems to have an important role in regulating metazoan genes1, 2. Although studies on immunoglobulin and other loci have shown a correlation between positioning at the nuclear lamina and gene repression, the functional consequences of this compartmentalization remain untested2, 3. We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM), and tested the consequences of such repositioning on gene activity in mouse fibroblasts. Here, using three-dimensional DNA-immunoFISH, we demonstrate repositioning of chromosomal regions to the nuclear lamina that is dependent on breakdown and reformation of the nuclear envelope during mitosis. Moreover, tethering leads to the accumulation of lamin and INM proteins, but not to association with pericentromeric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Finally, we use targeted adenine methylation (DamID) to show that, as is the case for our model system, inactive immunoglobulin loci at the nuclear periphery are contacted by INM and lamina proteins. We propose that these molecular interactions may be used to compartmentalize and to limit the accessibility of immunoglobulin loci to transcription and recombination factors.

We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine beta-globin locus during erythroid differentiation. Murine fetal liver cells were separated into distinct erythroid maturation stages by fluorescence-activated cell sorting, and the nuclear position of the locus was determined at each stage. We find that the beta-globin locus progressively moves away from the nuclear periphery with increasing maturation. Contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, betamajor-globin expression begins at the nuclear periphery prior to relocalization. However, relocation of the locus to the nuclear interior with maturation is accompanied by an increase in betamajor-globin transcription. The distribution of nuclear polymerase II (Pol II) foci also changes with erythroid differentiation: Transcription factories decrease in number and contract toward the nuclear interior. Moreover, both efficient relocalization of the beta-globin locus from the periphery and its association with hyperphosphorylated Pol II transcription factories require the locus control region (LCR). These results suggest that the LCR-dependent association of the beta-globin locus with transcriptionally engaged Pol II foci provides the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.

Rapid prenatal diagnosis of Beta-thalassaemia