Morphological analysis of ln2 and ln2-Ik^null fetal livers. (A) Wright-Giemsa staining of ln2 (left panel) or ln2-Ik^null (right panel) cells. (B) Detailed counting of the different cellular elements in the two genetic backgrounds. The percentages represent the averages from three independent experiments with the corresponding standard errors of the mean.

Recruitment of chromatin modifying and remodeling activities to the human β-globin locus in ln2 and ln2-Ik^null 12.5-dpc fetal liver cells. ChIP analysis of ln2 and ln2-Ik^null cells was performed. Analysis and quantification of the immunoprecipitated samples were as described in Fig. 2C and D. *, P ≤ 0.05 (Student t test). The antibodies used are indicated at the top of each panel. AcH3, antiacetylated (K9 and K14) histone H3 antibodies. Black bars indicate ln2 cells; gray bars indicate ln2-Ik^null cells.

Binding of Ikaros to the exon 1-intron 1 junction of huγ-globin genes. EMSA of exon 1-intron 1 junction of huγ-globin genes (Huγ probe) was performed with 15 μg of NE from MEL cells. An Ikaros-specific retarded band (Ik) is competed out by a 100-fold molar excess of cold double-stranded oligonucleotide (Huγ comp, lane 3) but not by an oligonucleotide containing a mutated (TTGGAA instead of TGGGAA) Ikaros consensus binding site (Huγ comp^mut, lane 4). Supershift experiments were carried out with antibodies (Ik Ab) raised to the C (C, lane 5) and N (N, lane 6) termini of the Ikaros protein. The antibody-shifted complexes are indicated by an arrow; an asterisk indicates free, labeled probe.

Model of hypothetical Ikaros-dependent repressosome nucleation leading to huγ-gene repression at the time of γ- to β-globin switching. A hypothetical model of huγ-globin gene repression mediated by the Ikaros/GATA-1/FOG-1/Mi-2/HDAC1 repressosome (for simplicity, only one of the two huγ genes is depicted). Repressosome nucleation at the huγ promoters requires the presence of Ikaros. In ln2 cells, chromatin conformation at the huγ-region limits transcriptional activator recruitment to huγ promoters. Thus, huγ genes are progressively and efficiently silenced. However, in ln2-Ik^null cells (ln2-Ik^null), the repressosome is formed less efficiently, and chromatin at the huγ promoters maintains an accessible conformation, which sustains recruitment of transcriptional activators and coactivators hence, higher huγ-gene expression. Repressosome nucleation, by reducing chromatin accessibility, progressively decreases the frequency of productive interactions between βLCR and huγ promoters. At the same time, several transactivators (such as EKLF) and chromatin-modifying activities (such as that of the SWI/SNF related complex, E-RC1), gathered to the huβ promoter, contribute to chromatin activation and facilitate βLCR/huβ over βLCR/huγ long-range interactions.

Ikaros and GATA-1 recruitment to the GATA-2 IG promoter in ln2 and ln2-Ik^null 12.5-dpc fetal liver cells. (A) Schematic overview of GATA-2 IG promoter region. In boldface and underlined are the GATA-1 and Ikaros DNA consensus binding sites; in boldface italics is the beginning of exon IG (29). (B) ChIP analysis of ln2 and ln2-Ik^null cells with Ikaros and GATA-1 specific antibodies. Analysis and quantification of immunoprecipitated samples are as described in Fig. 2C and D. *, P ≤ 0.05 (Student t test). (C) Representative example of semiquantitative RT-PCR performed on equal amounts of RNA purified from ln2 or ln2-Ik^null 12.5- dpc fetal liver cells. Top panel, mouse GATA-2 cDNA; bottom panel, mouse actin cDNA, used as a control. Band intensities were quantified with the MultiGauge 2.0 program, and the relative levels of GATA-2 gene expression were quantified according to the formula depicted underneath the panels.

Physical proximity between βLCR and globin gene promoters in ln2 and ln2-Ik^null 12.5-dpc fetal liver cells. 3C was applied on HCHO-fixed ln2 or ln2-Ik^null 12.5-dpc fetal liver cells. Nuclei were digested with EcoRI, and genomic DNA was ligated and subjected to qPCR with SYBR green. βLCR HS2-HS4 (HS2-4) EcoRI fragment was used as a “fixed” fragment, and specific primer sets were designed in order to amplify the genomic regions corresponding to the ɛ gene (ɛ), inter-ɛ-γ region (iɛ-γ), ^Gγ^Aγ genes (^Gγ^Aγ), ^Aγ gene (^Aγ), ψβ region (ψβ), inter-γ-δ region (iγ-δ), δ gene (δ), inter-δ-β region (iδ-β), and β gene region (β). Relative cross-linking frequencies (y axis) of the fixed fragment with globin fragments were defined using naked DNA encompassing the whole huβ-globin as a control and normalized to endogenous mouse actin. A value of 1 was attributed to the highest cross-linking frequency obtained with ln2 samples. Error bars represent standard deviations. The x axis indicates the position across the locus.

Ikaros-GATA-1 interaction. (A, D, and E) ChIP analysis of ln2 and ln2-Ik^null cells. Analysis and quantification of chromatin immunoprecipitated samples are as described for Fig. 2C and D. *, P ≤ 0.05 (Student t test). (B) GATA-1 gene expression. Representative examples of qRT-PCR carried out on ln2 (blue circles) and ln2-Ik^null (green squares) 12.5-dpc fetal liver cells. GATA-1 (left panel) expression levels in ln2 relative to ln2-Ik^null cells were calculated according to the method of Pfaffl (42) (see also Materials and Methods) using mouse actin (right panel) as an internal control, and they are expressed as the ln2/ln2-Ik^null ratio. y axis, derivative of SYBR green fluorescence. (C) EMSA of huγ-promoter AvaI-ApaII fragment (25) (ApaI-AvaII probe) was carried out with 5 μg of NE from ln2 (Ik^WT NE) or ln2-Ik^null (Ik^null NE) fetal liver cells. Lane 1, no NE; lanes 3, 5, 7, and 9, competition with a 100-fold molar excess of cold ApaI-AvaII oligonucleotide. Arrow A, Oct-1-specific retarded band; arrow B, GATA-1-specific retarded band; *, free, labeled probe. (F and G) Representative examples of protein IP on whole-cell extracts prepared from ln2 12.5-dpc fetal liver cells (F) or pOZ-FH-N or Ikaros-FH K562-infected cells (G). The antibodies used for IP or WB assays are indicated at the top and the bottom of the panels, respectively. Ikaros (Ikaros and Ik-FH)- and GATA-1-specific bands are indicated on both sides of the panels. Filled circles represent contaminating immunoglobulin heavy chain band. A higher-molecular-weight Ikaros-1-specific band is indicated by an asterisk. Ig, isotype-matched immunoglobulin control; Mock, pOZ-FH-N K562-infected cells; Ik, Ikaros-pOZ-FH-N K562-infected cells; NE, wild-type K562 NE.

Ikaros recruitment to the human β-globin locus in ln2 and ln2-Ik^null 12.5-dpc fetal liver cells. (A) Map of the huβ-globin locus. The locations of the βLCR HSs are indicated by arrows; genes are indicated as black boxes and Pyr region as a white box. Black arrowheads mark GATA-1 binding sites; asterisks indicate potential Ikaros binding sites according to the TGGGAA hexanucleotide consensus sequence. Amplicons for ChIP analysis are represented by gray lines depicted underneath each genomic region. (B) RT-PCR performed on equal amounts of RNA purified from ln2 (lane 1) or ln2-Ik^null (lane 2) 12.5-dpc fetal liver cells. Th, thymus control (lane 3); Neg, negative control (lane 4). Ikaros cDNA spliced variants are indicated on the left side of the panel. (C and D) Ikaros ChIP. Immunoprecipitated and unbound (input) chromatin samples were used as templates in qPCR analyses with primers specific for amylase 2.1y (Amy) promoter or the huβ-globin regions βLCR HS3 and HS2, the huγ promoters (Huγ), the proximal (Pyr) and distal (Pyr3′) Pyr regions, the proximal (Huβ5′) and distal (Huβ) huβ-promoter regions, and the huβ-gene region (Huβg). Quantification was carried out according to the 2^−ΔΔCT method, using mouse kidney-specific Thp protein promoter as an internal control, since this gene is not expressed in erythroid cells. Mouse Amy/Thp control is included to confirm that no enrichment was observed at regulatory regions of nonhematopoietic genes. Enrichment levels are represented by bars, with their corresponding standard deviations. A value of 1 indicates no enrichment. *, P ≤ 0.05 (Student t test). All data shown are the results of at least four independent ChIP experiments, with qPCR from each ChIP performed in triplicate and averaged (standard deviation). (C) HCHO-fixed cells. (D) EGS-fixed cells.