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Molecular and Cellular Biology, January 2008, p. 575-586, Vol. 28, No. 2
0270-7306/08/$08.00+0     doi:10.1128/MCB.00943-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

GATA-1 Modulates the Chromatin Structure and Activity of the Chicken -Globin 3' Enhancer ^,

Martín Escamilla-Del-Arenal and Félix Recillas-Targa^*

Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, México D.F. 04510, México

Received 28 May 2007/ Returned for modification 31 August 2007/ Accepted 23 October 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken -globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken -globin enhancer plays a central role in its capacity to differentially regulate -globin gene expression during erythroid differentiation and development.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In recent years, the relevance of promoter elements has been outlined based on the varied promoter architectures required for regulatory specificity (28). A large amount of data has been generated describing how the basal transcriptional machinery is incorporated into promoters and how proximal elements are required for full and specific activity (17). However, a subset of genes requires long-distance regulatory elements for their developmental timing and tissue-specific gene expression (9, 39). Locus control regions (LCRs), enhancers, silencers, and insulators represent some of the elements exerting remote regulation (5, 10, 26). Such regulatory elements require characteristic chromatin structures, and the great majority of these regulatory elements and chromatin components are usually associated with nuclease hypersensitive sites (HSs) (6, 39). Despite progress in our understanding of promoters and long-distance regulatory elements, the mechanisms by which enhancers control gene expression are poorly understood, particularly in terms of how their own chromatin structure modulates their activity.

In early work, we identified a silencer-enhancer element located at the chicken 3' side of the -globin domain, around 400 bp downstream of the adult ^A gene (Fig. 1A) (16, 32, 33). We have adopted this enhancer element as a model system to investigate the differential regulation of chicken -globin gene expression during erythroid differentiation and development. Based on recent data, we have proposed that the 3'-side enhancer is able to modulate its own function through the binding of GATA-1 and YY1 (Fig. 1B) (33) and the associated chromatin remodeling machinery. Much less is known about the molecular features of the chicken -globin silencer. What we have found until now is that the silencer is located side by side with the enhancer and that such location may give particular regulatory properties to both elements. The silencer was functionally defined in transient transfections as capable of reducing the activity of the heterologous simian virus 40 promoter, a weak promoter in erythroid cells (31). In addition, we know that at least three unidentified nuclear factors bind in vitro to it (31).

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FIG. 1. Scheme of the chicken -globin gene domain, the silencer-enhancer, and the detailed distribution of nuclear factor binding sites. (A) The silencer-enhancer regulatory elements are located 397 bp downstream of the adult A globin gene. These elements are included in a 1,036-bp BamHI genomic DNA fragment. A and B probes used in Southern blotting are shown (Fig. 2 and 3; also see Fig. S1 and S2 in the supplemental material). (B) Detailed scheme of the silencer-enhancer DNA-binding sites and key restriction enzyme-cutting sequences. The silencer possesses at least three binding sites for nuclear factors defined by in vitro footprinting (SF1 to SF3) (31). The identity of these nuclear factors remains to be determined. The initial characterization of the enhancer described the presence of three GATA-1 binding sites named the core enhancer (16). The core enhancer GATA-1 sites are named G1, G2, and G3. Sequence alignment of the duck and chicken homologous sequences revealed a highly conserved 120-bp DNA fragment with binding sites for Sp1/EKLF, NF-E2, a fourth GATA-1 (G4), and YY1 (33).

To better understand the function of the chicken 3' silencer-enhancer elements, here we studied the chromatin organization of this element during chicken development and erythroid differentiation. We hypothesized that the silencer-enhancer activity could be regulated by the interplay between key transcription factors and the acquisition of differential conformation of its chromatin structure. DNase I accessibility and nucleosomal mapping revealed an erythrocyte-specific conformation, based on the generation of two characteristic HSs. In situ restriction assays confirmed a differential chromatin conformation of the silencer-enhancer elements during erythroid differentiation and development. High-resolution mapping showed that the HSs are located between the silencer-enhancer elements, colocalizing with two GATA-1 binding sites. Mutational analysis in vivo demonstrated that the two most 5' GATA-1 binding sites regulate the enhancer chromatin conformation and are responsible for its optimal and regulated activity. These experiments further showed that in an integrated context, the silencer element previously defined in transient assays is a functional part of the enhancer. The chromatin remodeling process that occurs over the enhancer is consistent with an enrichment of histone acetylation followed by the local formation of an apparent nucleosome-free region, coincident with the stage of maximal enhancer activity. Our results demonstrate that GATA-1 is mainly responsible for the erythrocyte-specific chromatin conformation and activity of the chicken -globin 3' enhancer.

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Cell culture. All cell lines are derived from chicken (Gallus gallus). HD24 cells, which are pluripotent erythroid-myeloid cells transformed by the E26 virus, were grown as previously reported (3, 4, 30). 6C2 cells, corresponding to erythroblastosis virus-transformed bone marrow cells arrested at the CFU-E stage, are considered to be a preerythroblast line (3, 4). 6C2 cells, as well as the avian erythroblastosis virus-transformed and temperature-sensitive erythroblast line LSCCHD3 (herein referred to as HD3), were grown as previously described (6, 33). The DT40 lymphoid cell line was grown in DMEM supplemented with 50 µM 2-mercaptoethanol, 10% (vol/vol) fetal calf serum, 5% (vol/vol) chicken serum, and 10% (vol/vol) tryptose phosphate broth (13).

Plasmid constructs. DNA subfragments containing the chicken -globin 3' silencer-enhancer was PCR amplified from 10-day-old embryonic erythrocyte (10d-RBC) genomic DNA of G. gallus (Alpes, Puebla, México) by use of the following primers: ASIL1-PacI (5'-CGCGCTTAATTAAGATCCCATGCACTCCTTACC-3'), A7-AscI (5'-CAAGTGGCGCGCCTGCAGCAGGTTGAGCAGACC-3'), and AE1-NheI (5'-CGCGCGCTAGCGCGCAGGGTGAAGCTGTGCTG-3'). All DNA fragments were cloned into pG^D3 (kindly provided by Héctor Rincón-Arano) containing the GFP reporter gene under control of the ^D promoter. Mutations of the three GATA-1 binding sites present in the core enhancer (Fig. 1B; also see Fig. 7 below; also see Fig. S5 in the supplemental material) were generated in the context of the 1,036-bp silencer-enhancer construct by use of the QuikChange site-directed mutagenesis kit (Stratagene) with the following primers: Mutgata01U (5'-GGCCCCCGGCTCTTTAAACGGCCAGCAGCAGGG-3'), Mutgata01D (5'-CCCTGCTGCTGGCCGTTTAAAGAGCCGGGGGCC-3'), Mutgata02U (5'-GGGGGCTGCAGGTGGCTTTAAAAGAGCTGACAGGC-3'), Mutgata02D (5'-GCCTGTCAGCTCTTTTAAAGCCACCTGCAGCCCCC-3'), Mutgata03U (5'-GGGACGTGGGCAGCATTAAGCCTCGGGTGGG-3'), and Mutgata03D (5'-CCCACCCGAGGCTTAATGCTGCCCACGTCCC-3'). All the DNA fragments were NaeI-EcoRI digested and subcloned into the SmaI-EcoRI sites of the pL1-HYTK-L2 vector, replacing the cytomegalovirus HYTK gene (14). All recombinant plasmids were sequenced employing the primer ASIL1-NheI (5'CGCGGATCCCATGCACTCCTTACCCCATG-3').

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FIG. 7. Mutational analysis of the core enhancer GATA-1 binding sites and their effect on gene expression and chromatin structure. The transcriptional activity of the construct containing the D promoter (D), D plus the enhancer (core enhancer and the 120-bp DNA fragment; shown as DE120), and the silencer-enhancer at the RMCE-HD3-13 integration site was tested by fluorescence flow cytometry. DNase I- and MNase-HSs assays are shown in the lower panel. In addition, mutations of the first and second GATA-1 binding sites at the core enhancer were analyzed (DSE120G1 and DSE120G2). Black circles show the positions of the two HSs. The dotted oval shows the positioning of the static nucleosome that is lost in the GATA-1 point mutation clones.

DNase I, MNase, and restriction endonuclease nuclear digestions. For DNase I digestion, 1 x 10⁹ cells were washed twice in cold phosphate-buffered saline and resuspended in 2.5 ml of NI buffer (15 mM Tris-HCl [pH 7.5], 300 mM sucrose, 10 mM HEPES, 60 mM KCl, 4 mM MgCl₂, 1 mM dithiothreitol, 5 mM NaCl), and 2.5 ml of lysis buffer (NI buffer plus 0.5% NP-40) was then added to the isolated nuclei and incubated for 1 min. Nucleus integrity was monitored during this incubation, and the nuclei were then pelleted and washed once with NI buffer and resuspended in 100 µl of NI buffer. Five microliters of the resuspended nuclei was mixed with stop buffer (10 mM Tris-HCl [pH 7.5], 20 mM EDTA, 0.5% sodium dodecyl sulfate), and optical densities (OD) were determined (A₂₆₀ x 200). The volume corresponding to 3,000 OD units was adjusted to 300 µl with NI buffer solution in a final concentration of 2 mM CaCl₂, and nuclei were digested with 0, 0.1, 0.5, 1, 3, 5, and 15 U of DNase (Worthington) for 5 min at 25°C. The reaction was stopped with 300 µl of stop buffer. Samples were treated with 150 µg of proteinase K at 55°C for 6 h, phenol-chloroform extracted twice, and incubated with 50 µg of RNase A at 37°C for 1 h. The samples were phenol-chloroform extracted once more and DNA was precipitated with isopropanol. For micrococcal nuclease (MNase) digestions, we followed the same protocol as for DNase I with two modifications. Nuclei were adjusted to 1 mM of CaCl₂ and digested with increasing concentrations of MNase (Worthington). For restriction endonuclease digestions, we followed the same protocol as for DNase I with minor modifications. Nuclei were resuspended in buffer A (10 mM Tris-HCl, 15 mM NaCl, 60 mM KCl, 0.1 mM EDTA, 5 mM MgCl₂, 5% [vol/vol] glycerol, 1 mM dithiothreitol). Nuclear concentrations were adjusted to 3,000 OD in 300 µl buffer A and digested with 200 U of the corresponding restriction enzyme for 15 min at 30°C.

Isolation of mono- and dinucleosomes. Mono- and dinucleosomes were purified as described previously (18, 27). Three independent aliquots of isolated nuclei were digested with increasing concentrations of MNase. All MNase digestions were incubated for 25 min at 25°C. The reactions were stopped by adding EDTA (pH 8.0) to a final concentration of 10 mM. The three digests were combined and mono- and dinucleosomes were separated on a 6 to 30% sucrose gradient (18). For the MNase sensitivity assay, duplex PCR was performed with DNA associated with mono- and dinucleosomes insolated by sucrose gradient. The normalization was done using the FR gene sequence (primer 5.613 in reference 27). Genomic DNA from 10d-RBCs was used to determine the difference in sensitivity enrichment (n-fold) between genomic DNA and MNase-digested input DNA.

ChIP. The chromatin immunoprecipitation (ChIP) assay was done as previously reported (35) employing 2 µg of acetylated H3 (acH3), acH4, and H3K4me2 (Upstate), -RNA polymerase II (Pol II), anti-GATA-1 (H-200), and -immunoglobulin G (Santa Cruz Biotechnology) antibodies. H4K20me3 antibody was kindly provided by Thomas Jenuwein and mouse antihistone, panmonoclonal antibody recognizing histones H1, H3, H4, H2A, and H2B from Chemicon International (MAB3422) was kindly provided by Mario Zurita (24). Immunoprecipitated DNA was analyzed by PCR using primers specific for the silencer-enhancer (SE120) region with the primers RealTSil-U (5'-TGTGGCCCTCATAGGACTTCC-3'), RealTSil-D (5'-GACCCCACAAGTGGCACATAG-3'), RealTSE-U (5'-GGATGAGCGCAGGGTGAAGCT-3'), and RealTSE-D (5'-GCTGCTGGCCGTGATAAGACG-3') and the enhancer (E4) RealTenh-U (5'-CAGGCTCTCCTCCAGCTCACG-3') and RealTenh-D (5'-TACCCACCCGAGGCTATCTGC-3'). For semiquantitative ChIP assay, duplex PCR was performed as described previously (35). Control primers for normalization were selected from the chicken β-globin-adjacent 16 kb of condensed chromatin and the FR gene HSA sequence corresponding to an open chromatin region in the chicken genome (see primers 10.35 and 5.613 in reference 27). For GATA-1 ChIP assays, normalization primers were selected around 3 kb downstream of the enhancer, the 3' noncoding region of the chicken -globin domain and the primer sequence area and were as follows: Ctrl3'F (5'-CCGTTTCAAACCAACCTACTGGACT-3') and Ctrl3'R (5'-GCTGTACGCTTCAGCTCAATATCAG-3') (16, 33).

RMCE. Recombinase-mediated cassette exchange (RMCE) was basically performed as described previously (14). The pL1-HYTK-L2 vector was stably transfected in HD3 cells, and three independent clones were isolated (RMCE-HD3-13, -14, and -20). Transgene integrity was confirmed by Southern blotting (clones kindly provided by Héctor Rinón-Arano). Newly generated plasmids were cotransfected with the pBS185 plasmid, which contains the Cre recombinase (cytomegalovirus Cre; Clontech). Three rounds of hygromycin selection, each for a period of 3 to 5 days, to allow cell recovery, were performed. Stably transfected cells were selected based on Southern blotting and PCR from a pool of 700 hygromycin-resistant individual colonies.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Erythrocyte-specific formation of DNase I-HSs defining the silencer-enhancer transition. To assess the contribution of the local chromatin structure to the activity of the 3' chicken -globin silencer-enhancer (Fig. 1), we initially characterized its chromatin organization during erythroid differentiation and development. We took advantage of transformed chicken erythroid cell lines representing different developmental stages: the most primitive are HD24 cells, representing a myeloid-erythroid stage, whereas the most mature are HD3 cells, corresponding to erythroblasts. To complete the differentiation spectrum, we isolated nuclei from 10d-RBCs as well as terminally differentiated erythrocytes (TD-RBCs) (Fig. 2). Based on the identification of DNase I-HSs by DNase I digestions, we observed the progressive appearance of a cluster of at least three DNase I-HSs located between the enhancer and the silencer (Fig. 2). The nonerythroid DT40 cell line showed no DNase I-HS formation. Consistent with this change in chromatin structure, we have previously demonstrated that the enhancer is inactive in HD24 and 6C2 cells, is modestly active in HD3 cells, and reaches the highest levels of trans-activation potential in 10d-RBCs (33). These results demonstrate a localized and progressive increment of DNase I sensitivity over the silencer-enhancer.

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FIG. 2. Chromatin structure over the chicken 3'-side -globin silencer-enhancer. Southern blots showing the presence of DNase I-HSs. Notice the appearance of three DNase I-HSs over the core enhancer and the transition zone between the silencer and the enhancer, from HD24 cells to TD-RBCs (vertical arrows). In 10d-RBCs, the appearance of the HSs is almost 1 order of magnitude higher than that seen for HD3 digested nuclei (compare lanes corresponding to 6 and 1.5 units of DNase I). DT40 cells show only the higher concentration of enzyme to evidence the absence of HSs. M, 100-bp molecular mass marker.

Chromatin structure of the silencer-enhancer based on differential positioning of a nucleosome. To further characterize the chromatin structure of these regulatory elements and determine nucleosome positions over the silencer-enhancer elements, we carried out MNase digestions (Fig. 3). We incorporated two additional controls: naked (genomic) DNA (see Fig. S1 in the supplemental material) and chicken preerythroblast 6C2 cells, considered to be differentiation intermediates between the HD24 and HD3 cell lines (Fig. 3) (30). Two prominent erythrocyte-specific MNase-HSs are clearly observed with hypersensitivity progressively increasing in intensity in a differentiation stage-specific manner (Fig. 3), as observed for the DNase I-HSs (Fig. 2). The 5' MNase-HS is located between the silencer and enhancer, and the most 3' MNase-HS, which is consistently more intense, appears to colocalize between the first and second GATA-1 binding sites (Fig. 3). The distance between these two MNase-HSs is apparently not sufficient to accommodate a nucleosome (data not shown). We found that additional bands appear for HD3 cells and 10d-RBCs, suggesting different nucleosome positions over the enhancer representative of an average of templates (Fig. 3). This chromatin remodeling affects sequences corresponding to the 120-bp DNA fragment previously shown to be responsible for modulation of the enhancer activity (Fig. 1B and 3) (33). For TD-RBCs, we observed a general decrease in MNase sensitivity consistent with the progressive silencing of the -globin gene expression (Fig. 3; TD-RBCs and data not shown).

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FIG. 3. Nucleosome positioning determined by MNase digestion. The patterns of MNase digestion of naked DNA and of DT40 nuclei are similar. Interestingly, at the earliest stages of erythroid differentiation there are two MNase-HSs, the increasing intensity of which reaches the highest level in 10d-RBCs (black arrows). These two MNase-HSs are found between the silencer and the enhancer, and the most 3' HS is more intense (large horizontal arrow). Starting with HD3 cells, when the enhancer begins to be active, there is the appearance of several bands (asterisks) 3' to the second MNase-HS over the 120-bp DNA fragment (open white box in the vertical scheme), suggestive of an increase in nucleosome mobility or nucleosome exclusion, consistent with a more open chromatin organization (asterisks). The open horizontal arrows indicate the digestion pattern created by the constant positioning of a nucleosome downstream to the enhancer.

Utilizing Southern hybridization with probe B, we then determined the nucleosome positioning pattern over the silencer element, revealing stable positioning of one nucleosome over the silencer (Fig. 3; also see Fig. S2 in the supplemental material). Notice that another well-positioned nucleosome is also observed downstream from the 120-bp DNA fragment (Fig. 3).

In conclusion, the DNase I and MNase assays support the fact that the silencer-enhancer chromatin structure is gradually remodeled in a stage-specific manner, reaching maximal chromatin accessibility when the enhancer is fully active (HD3 cells and 10d-RBCs) (see Fig. S3 in the supplemental material).

Evaluation of the chromatin template accessibility during erythroid differentiation and development. Chromatin remodeling in vivo can be detected by an increase in the restriction enzyme accessibility of nucleosome assembled DNA in preparations of isolated nuclei (1, 41). Restriction enzyme accessibility assays were used to determine if particular nucleosomes over the silencer-enhancer elements were subjected to chromatin remodeling (Fig. 4). Nuclei from cells representative of the distinct erythroid stages and convenient restriction sites were chosen, based on the predicted nucleosome positioning. We found that the silencer element is poorly accessible to DraIII in DT40, HD24, 6C2, and HD3 cells (Fig. 4A and data not shown). In contrast, there is a clear accessibility increase in 10d-RBCs, suggesting a change in the local chromatin structure (Fig. 4A and B). Based on these results, we propose that the silencer is remodeled until late stages of erythroid differentiation. In an analysis of the region between the silencer and enhancer through MspI and HhaI digestion and over the enhancer through PstI (P1 and P2) (Fig. 4B), we detected a gradual increase of enzyme accessibility consistent with the DNase I- and MNase-HS results (Fig. 4B). Interestingly, the second and most 3' MspI site (M2) located outside of the enhancer shows a constant and weak accessibility in 6C2 and HD3 cells and 10d-RBCs, suggesting that the chromatin remodeling is restricted solely to the enhancer sequence (Fig. 4 and data not shown).

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FIG. 4. Analysis of chromatin accessibility by in situ restriction enzyme assays in the course of erythroid differentiation and development. (A) Scheme representing the -globin domain with the location of restriction enzymes used. D, DraIII; M1 and M2, MspI; A1 and A2, AvaII; P1 and P2, PstI; H1 and H2, HpaII; and H, HhaI. Vertical arrows indicate the positions of the two MNase-HSs. In situ restriction enzyme accessibility assays were performed with isolated nuclei. For DT40 cells, the region is in a repressive conformation, as shown using all the restriction enzymes. It is important to outline the progressive accessibility observed over the course of erythroid differentiation. In all cases, genomic DNA was BamHI digested and Southern blotting performed with probes from the 5' and 3' sides of the silencer-enhancer (data not shown). (B) Evaluation of local chromatin accessibility during chicken development. Using DraIII (D), there is a decay in enzyme digestion, suggesting a more accessible and relaxed chromatin conformation over the silencer during early stages of development. In contrast, for MspI (M1), accessibility is continuous during development. In TD-RBCs, there is a notorious increment on nondigested material in both cases. The results were quantitated using a phosphorimager and are presented as the percentage of digestion relative to that seen for the input DNA.

To better understand the differential chromatin remodeling on the silencer-enhancer region in an in vivo context, we surveyed restriction enzyme accessibility during chicken development by incubating DraIII and MspI restriction enzymes on isolated nuclei from embryonic primary red blood cells (Fig. 4B). The silencer chromatin, which is digested by DraIII, is more accessible during embryonic developmental days 5 to 8 (Fig. 4B), despite the possible well-positioned nucleosome (Fig. 4A). In contrast, restricted accessibility is concomitant with the switch from embryonic to adult gene expression (Fig. 4B, 10-d to 15d-RBCs). When we analyzed the region between the silencer and enhancer through MspI digestion, we found a constant degree of accessibility coincident with the fact that adult genes are transcribed (Fig. 4B, M1 site). Similar results were obtained using the HpaII restriction enzyme (data not shown).

In summary, in situ restriction enzyme accessibility assays confirmed the progressive changes in chromatin conformation. These results are in agreement with our initial hypothesis that predicts that a fully active enhancer is subjected to extensive local chromatin remodeling.

Mapping of the erythrocyte-specific MNase I-HSs and in vivo differential binding of GATA-1. The appearance of two prominent erythrocyte-specific MNase-HSs prompted us to map more precisely their position in relation to the silencer-enhancer sequences. By Southern blotting and different restriction enzyme digestions, we determined that the 5' MNase HSs is located over the silencer and the most prominent 3' MNase-HS was found between the first and second GATA-1 binding sites of the core enhancer encompassing a DNA fragment of 100 ± 20 bp (Fig. 4A and data not shown). Furthermore, we determined the in vitro GATA-1 interaction in this central motif (data not shown). To demonstrate GATA-1 association at the enhancer in vivo, we performed a semiquantitative ChIP assay (Fig. 5). Interestingly, we found a gradual incorporation of GATA-1 to the enhancer, in which the highest amount of factor coincides with the peak of enhancer activity. These results suggest a possible correlation between GATA-1 association and the degree of enhancer chromatin remodeling and activity.

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FIG. 5. In vivo interaction of the transcriptional factor GATA-1 with the chicken 3' enhancer -globin. The histograms summarize the results of semiquantitative duplex PCR performed in triplicate from two independent ChIPs. A representative gel of 10d-RBC ChIP is shown. Enh represents the PCR amplification products covering the silencer-enhancer sequences and Ctrl3' represents the corresponding normalization product from a distal 3' noncoding sequence located 3 kb downstream of the enhancer (16). IgG, immunoglobulin G.

GATA-1 binding is critical for the enhancer chromatin structure conformation. Based on these observations and the known contribution of GATA-1 to enhancer activity, we determined by use of point mutations that the interactions of GATA-1 at the first and second GATA-1 binding sequences are critical for enhancer activity in transient transfection assays (data not shown). To determine whether GATA-1 binding sites in the core of the 3'-side chicken -globin enhancer contribute in vivo to the establishment of its chromatin organization, we used a RMCE strategy, which allows comparison of the activities of different constructs within the same genomic insertion site (13, 14). First, we generated three independent stable clones with a single copy of the pL1-HYTK-L2 vector. Clones were generated in HD3 cells and named RMCE-HD3-13, -14, and -20. We then constructed a new series of vectors, including an intact silencer-enhancer with and without mutated GATA-1 sites and the adult ^D globin gene promoter driving GFP gene expression (Fig. 6A). A portion of the pEGF-1 plasmid was left on the 3' side to allow hybridization of a specific probe (Fig. 6A). Inverted LoxP sequences (Cre recombinase target sites) were placed flanking the ^D promoter, the EGFP reporter gene, and the silencer-enhancer element to induce cassette exchange through Cre recombinase transient expression. Nonrecombinant clones were discarded by negative selection with ganciclovir (14). It is important to outline that such a system allowed us to eliminate the influence of selection marker genes on stably integrated constructs. Once we established the RMCE experimental system, we were able to asses the contribution of the GATA-1 sites of the core enhancer to the local chromatin structure, as well as the enhancer-mediated transcriptional activation.

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FIG. 6. Reconstitution of the chromatin structure at the silencer-enhancer in stably transfected HD3 cell lines. (A) Transgene flanked by opposing LoxP sequence-specific homologous recombination sites. The specific probes used for Southern blot hybridization are shown (GFP and fi-ori). Arrows represent the HS determined. (B) RMCE was used to direct the insertion of different constructs into the same integration RMCE-HD3-13 site (in the absence of a selectable marker). MNase- and DNase I-HSs assays were performed with individual clones, showing recapitulation of the endogenous chromatin structure over the silencer-enhancer. EGFP expression was evaluated by fluorescent flow cytometry. It is important to note that in the presence of the silencer (DSE120), there is a remarkable enhancement of gene expression and the reconstitution of the endogenous chromatin structure. Black dots indicate the positions of the two MNase-HSs described previously in the endogenous context.

With this experimental system, we were able to recapitulate the endogenous chromatin structure pattern of the 3'-side -globin silencer-enhancer elements (compare Fig. 6B with Fig. 3). Unexpectedly, and contrary to our previously published in vitro results (31), when the silencer is incorporated to the construct we found a robust enhancement of the GFP expression (Fig. 7, top; compare pL^DE120 with pL^DSE120). Based on these results, we incorporated in this study a construct lacking the silencer (Fig. 7, ^DE120). In the absence of the silencer, we observed a reduction in GFP gene expression along with changes in chromatin structure (Fig. 7; compare ^DE120 with ^DSE120). The strong 3' MNase-HS is diminished, whereas the DNase I-HS remains. We attribute the permanence of this DNase I-HS to the presence of GATA-1 binding sites. These data are consistently reproducible in the context of the two other integration sites (see Fig. S4 and S5 in the supplemental material). In contrast to our earlier results, the silencer is not behaving as a negative regulator in the integrated context. Instead, it seems to be an integral component of the enhancer.

Analysis of constructs harboring GATA-1 point mutations revealed that the first (G1) and second (G2) GATA-1 binding sites are critical for enhancer activity and, most importantly, that their mutation caused the complete loss of DNase I-HSs (Fig. 7; also see Fig. S5 in the supplemental material). In addition, no reporter gene expression was detected from either construct by fluorescence-activated cell sorting analysis (Fig. 7). With respect to the nucleosome positioning, the general chromatin structure over the region was completely altered, with the loss of the 3' MNase-HS, characteristic of all the erythroid cells tested. These observations are reproducible in the context of the RMCE-HD3-14 and -20 integration sites (see Fig. S4 and S5 in the supplemental material; also data not shown).

In summary, we have been able to recreate the native chromatin structure pattern of the silencer-enhancer in distinct integration sites in HD3 cells. Furthermore, the previously defined silencer seems to contribute to the overall enhancer activity, possibly through the stable positioning of a nucleosome (see Fig. S1 to S3 in the supplemental material). But most importantly, the first and second GATA-1 binding sequences are responsible for the acquisition of a chromatin structure permissive for enhancer activity.

Histone posttranslational modifications over the -globin 3'-side regulatory element. Covalent histone modifications, particularly histone acetylation and methylation, regulate chromatin structure and function in intimate association with transcription factors. We therefore performed a series of semiquantitative ChIP assays to address the dynamics of histone modification on the complete -globin 3' enhancer during erythroid differentiation (Fig. 8). Chromatin from DT40 and HD3 cells and 7d-RBCs was immunoprecipitated using specific antibodies against H3K4me2, acH3, and acH4 as representatives of open chromatin and H4K20me3 as a representative of a repressive modification (Fig. 8A). Furthermore, we also surveyed the region for the in vivo association of RNA Pol II and verified the presence of nucleosomes employing a panmonoclonal antibody which is able to recognize all five histones (Fig. 8A). Interestingly, for HD3 cells we found both activating and repressive marks (Fig. 8A). We also found RNA Pol II present predominantly on the 3' of the enhancer (Fig. 8A). For 7d-RBCs, in which embryonic gene expression switches to adult ^D and ^A gene expression (16), we found enrichment of open chromatin marks, in agreement with our previous observations. In addition, we observed a redistribution of RNA Pol II, which was equally present across the enhancer. We note that these data are still consistent with the recruitment of RNA Pol II and components of the preinitiation complex to distal regulatory elements (22, 39, 40). Unexpectedly, the general levels of immunoprecipitated chromatin were low in 7d-RBCs in comparison to what was seen for DT40 and HD3 cells, suggesting a diminishment or even absence of nucleosomes in the regions studied, as supported by the pan-histone monoclonal antibody ChIP. For DT40 nonerythroid cells, we observed the absence of open chromatin marks, a modest presence of RNA Pol II, enrichment of histone H4K20me3, and the presence of histones (Fig. 8A). Overall, these results are in agreement with the progressive changes in the chromatin structure of the enhancer and its associated activity.

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FIG. 8. Histone modification analysis by ChIP assays and identification of a potential nucleosome-free region over the enhancer. (A) The histograms summarize the results of quantitative duplex PCR performed in triplicate from two independent immunoprecipitations. We amplified the 5' part of the enhancer (enhancer 5') and the 3' part (enhancer 3') as well as the entire enhancer (see scheme in panel B). A representative gel of 7d-RBC ChIP is shown, with each PCR performed in triplicate. Exp represents the PCR amplification products of the entire enhancer and Ctrl represents the corresponding normalization primers. For normalization, the central part of the 16 kb of condensed chromatin (for open marks), located upstream to the chicken β-globin domain, and the FR gene HSA sequences (for repressive marks) were used. (B) Localization of the primers used as shown in panels A and C. (C) Purification of mono- and dinucleosomes by use of a sucrose gradient. Numbers represent the sucrose fractions. DNA associated with mono- and dinucleosomes was used as the template for PCR amplification to determine the degree of template protection as a measure of nucleosome depletion. The normalization was done as described for panel A. The difference (n-fold) between genomic DNA and MNase-digested input DNA was determined. (D) Duplex PCR was performed with DNA associated with mono- and dinucleosomes isolated by sucrose gradient treatment to determine the MNase sensitivity as a measure of nucleosome depletion. The normalization was done as described for panel A. The difference (n-fold) between genomic DNA and MNase-digested input DNA was determined. Data are presented as increase (n-fold) in MNase sensitivity with respect to that seen for DT40.

To further assess the observed nucleosome depletion, MNase-digested chromatin was purified and mono- and dinucleosomes were isolated by use of a sucrose gradient (Fig. 8C). Duplex PCR was used for normalization, with primers covering the entire enhancer and the chicken FR gene HSA (27) (Fig. 8B; also see Materials and Methods). These results were clearer when we analyzed individual parts of the enhancer with primers used in the ChIP analysis (Fig. 8A and C) with the chicken FR gene HSA as a control for duplex PCR normalization (27). The entire enhancer region shows a gradual increase in MNase sensitivity (Fig. 2 to 5), but higher levels of sensitivity are associated with the central region, for which we demonstrated a GATA-1-dependent chromatin remodeling (Fig. 8D; data are presented as enrichment [n-fold] of MNase sensitivity [see Materials and Methods]).

Together, the ChIP results support a progressive chromatin remodeling during erythroid differentiation. Furthermore, in 7d-RBCs, a developmental stage in which the enhancer is more active, there is an apparent nucleosomal depletion over the 3'-side -globin enhancer.

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The contribution of the local chromatin structure to the enhancer's method of action is not well understood. Our hypothesis predicts that characteristic chromatin structures over enhancers and LCRs are needed for their function and long-range interaction with promoters. In the present work, we assessed the role of the chromatin structure and GATA-1-dependent remodeling on the activity of the 3' -globin silencer-enhancer during erythroid differentiation. A systematic characterization of the chromatin structure of this approximately 1-kb element established the erythrocyte-specific appearance of two characteristic HSs located over the enhancer and the transition point between the silencer and the enhancer. In the later erythroid differentiation stages, the silencer and the enhancer acquire the most accessible (relaxed) chromatin conformation. We also observed chromatin remodeling over a 120-bp region previously proposed to be responsible for the modulation of the enhancer activity in the stages during which the enhancer is active and the adult -globin genes are expressed (33). Fine mapping of the two HSs showed that a GATA-1 binding site is located between them. These chromatin-based studies demonstrated that the first and second GATA-1 binding sequences are critical for the proper chromatin remodeling of the silencer-enhancer elements and for enhancer activity. Unexpectedly and in contrast to our previous transient transfection results, in the integrated context, the silencer contributes to the enhancer activity. Finally, at the stage in which the enhancer is most active, we observed the formation of an apparent nucleosome-free zone. Together, our results demonstrate that local chromatin structure plays essential role in enhancer activity and that GATA-1 is fundamental for the formation of a characteristic erythrocyte-specific chromatin conformation and for regulated enhancer function. GATA-1 and GATA-2 are expressed in megakaryocytic and erythroid lineages, playing crucial roles in cell maturation and differentiation (8, 29). GATA-1 possesses a fundamental function in the control of transcriptional programs during development (11, 12) and interacts with a plethora of transcription factors, such as YY1 (33) and FOG-1 (8). GATA-1, like several other transcriptional factors, is able to induce transcriptional activation or repression in erythroid cells (34). Moreover, it is well documented that GATA-1 interacts stably with chromatin at promoters and enhancers and functions at the level of chromatin structure (2, 20, 37) and local chromatin remodeling (6, 7, 25). Indeed, in a reconstituted experimental system, GATA-1 perturbs the nucleosome, generating an apparently nucleosome-free DNA-GATA-1 complex responsible for HS formation (7).

An interesting aspect of our results is the observation of a nucleosome positioned over the previously defined silencer element, which we now find contributes to enhancer activity (see Fig. S1 to S3 in the supplemental material). It is possible that the nucleosome over this region represents a signal for the regulated attraction of GATA-1 and associated chromatin remodeling of the -globin enhancer. We believe that GATA-1 binding to the first motif and the generation of an apparent nucleosome-free region depends, at least in part, on such nucleosome positioning. This does not seem so unlikely based on the growing list of examples in which specific and strategically located nucleosomes contribute to the formation of nucleosome-free zones, in particular, over promoter elements (15, 42). In the same way, when we deleted the silencer or mutated the GATA-1 binding site, we observed the delocalization of a putative nucleosome just 5' of the first HS. The delocalization of the nucleosome by GATA-1 point mutation supports a model in which GATA-1 is working as a nucleation center of chromatin remodeling activities.

There has been an increased interest in the existence of a genomic positioning code for nucleosomes (1, 21, 36). It has been proposed that the degree of nucleosome depletion correlates with the level of gene expression and that nucleosome-free regions found for active promoters could also be a novel feature of enhancers (19). Our data are consistent with these observations. There is also an apparent correlation between the maximal degree of nucleosome depletion, the highest 3'-side -globin enhancer activity, and local chromatin remodeling (Fig. 3; also see Fig. S3 in the supplemental material). Recent elegant observations from others describe GATA-1 and GATA-2 binding along the mouse -globin domain during hematopoiesis that, in collaboration with FOG-1, contributes to regulated long-range intrachromosomal interactions (2, 37, 38). Based on our results, we propose that such regulation is highly dependent on the local chromatin structure of the enhancer. A detailed survey of histone marks and transcription factors and cofactors has suggested that each HS in the human β-globin LCR has distinct structural and functional features (23). These results are accompanied by the demonstration of nucleosome exclusion, and an inverse correlation between GATA-1 binding and nucleosome loss seems to emerge (23). In the case of the chicken -globin enhancer, one possible scenario is that the enhancer is behaving like an LCR in terms of the diversity of its genomic organization, chromatin structure, and activity.

In conclusion, we propose a tripartite composition for the regulatory element located at the 3' end of the chicken -globin domain. This element possesses a modular organization and has an activity strictly regulated with erythroid differentiation and development. The central component is tightly dependent on GATA-1 trans-acting potential, chromatin remodeling, and nucleosome displacement properties. The second, initially called a silencer element, is associated with a well-positioned nucleosome, and the third component is the 120-bp DNA fragment previously demonstrated to modulate the enhancer activity (33). In summary, the GATA-1 binding sites of the -globin enhancer are fundamental for the generation of an active chromatin structure and the modulation of the enhancer function in an erythroid environment. At this point, the challenge is to integrate and define how these activities operate in vivo and in the context of the -globin differential gene expression and chromatin domain organization.

 
We thank Ann Dean, Gary Felsenfeld, David J. Clark, Rodolfo Ghirlando, Mayra Furlan-Magaril, Ernesto Soto-Reyes, and Paul Delgado-Olguín for helpful comments on the manuscript. We thank Georgina Guerrero for her excellent technical assistance and members of the Félix Recillas-Targa laboratory for stimulating scientific discussions and suggestions. We also thank the technical assistance of Héctor Rincón-Arano, Cristina Aranda, Estela García Conzález, and Itzel Escobedo Ávila. We thank L. Ongay, G. Codiz, and M. Mora from the Unidad de Biología Molecular from the Instituto de Fisiología Celular, UNAM, for DNA sequencing and fluorescence-activated cell sorting facility.

This work was supported by grants from the Dirección General de Asuntos del Personal Académico-UNAM (IN203200, IX230104, IN209403, and IN214407), Consejo Nacional de Ciencia y Tecnología, CONACyT (33863-N, 42653-Q, and 58767), and the Third World Academy of Sciences (TWAS, grant 01-055 RG/BIO/LA). M.E.-D.-A was a fellowship recipient from CONACyT.

 
* Corresponding author. Mailing address: Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, México D.F. 04510, México. Phone: (52 55) 56 22 56 74. Fax: (52 55) 56 22 56 30. E-mail: frecilla{at}ifc.unam.mx

Published ahead of print on 5 November 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, January 2008, p. 575-586, Vol. 28, No. 2
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