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. 2009 Nov 27;394(2):197-208.
doi: 10.1016/j.jmb.2009.09.046. Epub 2009 Sep 23.

The higher structure of chromatin in the LCR of the beta-globin locus changes during development

Xiangdong Fang  1 , Wenxuan Yin, Ping Xiang, Hemei Han, George Stamatoyannopoulos, Qiliang Li
Affiliations

The higher structure of chromatin in the LCR of the beta-globin locus changes during development

Xiangdong Fang et al. J Mol Biol. .

Abstract

The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

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Figures

Fig. 1
Fig. 1
Occurrences of histones H2A, H2B, H3, and H4 in the human transgenic and the mouse endogenous β-globin loci. Bindings of histones H2A, H2B, H3, and H4 were measured by ChIP assay using the specific antibodies. (a) Comparison of histone H3 amounts at the HSs of the human β-globin LCR between adult and embryonic erythroblasts. The tested HSs are shown at the x-axis. (b) Comparison of histone H3 amounts at the four HS intervening regions of the human β-globin LCR between adult and embryonic erythroblasts. HS4/3 represents the region between HS4 and HS3, and so on. (c) Histone H2A, H2B, and H4 bindings at the HSs and the intervening regions of the human LCR in adult erythroblasts. The data columns in each group are arranged based on their genomic order from 5′ to 3′ related to the ε-globin gene. (d) Comparison of histone H3 amounts at the HSs of the mouse endogenous β-globin LCR between adult and embryonic erythroblasts. (e) Comparison of histone H3 amounts at the five HS intervening regions of the mouse endogenous β-globin LCR between adult and embryonic erythroblasts. (f) Histone H2A, H2B, and H4 bindings at the HSs and the intervening regions of the mouse endogenous LCR in adult erythroblasts. The data columns in each group are arranged based on their genomic order from 5′ to 3′ related to the εy-globin gene.
Fig. 2
Fig. 2
The profiles of histone H3 acetylation in the β-globin locus in embryonic and adult erythroblasts. The level of histone H3 at K9 and K14 acetylation in the β-globin locus was measured in embryonic and adult erythroblasts by ChIP assay. Enrichments were corrected by the mAire gene. The line drawing on the bottom shows the human β-globin locus. The globin genes are marked by their names. The ε, Gγ and Aγ genes are expressed in the yolk sac and early fetal liver of transgenic mice; the δ and β genes are expressed in fetal liver and adult spleen erythroblasts. The olfactoryol receptor genes are shown as ovals. The arrows indicate the DNase I hypersensitive site flanking the locus, which are constantly present in erythroid cells at all stages of development. The HSs at the promoters, which are associated with developmental stage-specific gene expression, are omitted. The LCR consists of HSs 1–5.
Fig. 3
Fig. 3
Comparison of MNase sensitivity of the mouse β-globin locus between embryonic and adult erythroblasts. Schematic illustration of the mouse β-globin locus is shown in the line drawing. The εy-and βH1-globin genes are expressed in embryonic erythroid cells, and the βmaj- and βmin-globin genes are expressed in definitive erythroid cells. The ovals represent the olfactory receptor genes. The arrows indicate DNase I hypersensitive sites. (a and b) The MNase digestion profiles of the GAPDH gene and 3′ HS1 of human Jurkat cells, which were added into the yolk sac and the spleen prior to the MNase treatment. The y-axis represents the portion of the remaining DNA after treatment over the untreated DNA at each enzyme concentration. The x-axis represents different MNase concentrations from zero (untreated) to 30 units. (c to k) The MNase digestion profiles at the gene promoters and various positions of the LCR of the endogenous mouse β-globin locus in embryonic and adult erythroid cells.
Fig. 4
Fig. 4
Developmental-stage-associated changes in the proximity between HSs of the LCR. 3C assay was employed to estimate the proximity between HS5 and HS1 and between HS 4 and 2 in embryonic and fetal erythroblasts. The asterisks indicate that the difference between two different stages is statistically significant (n=4).
Fig. 5
Fig. 5
Effects of the HS3 core and 2.3-kb HS3 deletions on the chromatin structure of the LCR. (a) Comparison of the effects of ΔHS3c and ΔHS3 on the proximity between 5HS5 and 5′HS1 and between5′HS4 and 5′HS2 (a) in the yolk sac and (b) in the fetal liver. The error bars represent standard deviation. (b) Southern blot hybridization to determine HSs 1, 2, 4, and 5 of the LCR in the yolk sac and fetal liver in transgenic mice. HSs 4 and 5 were detected in the MfeI-digested DNA (upper panels) and HSs 1 and 2 were detected in the NdeI-digested DNA (lower panels). FL, fetal liver; YS, yolk sac; wild, wild-type βYAC; ΔHS3c, the βYAC harboring a 234-bp deletion containing the HS3 core; ΔHS3, the βYAC harboring a 2.3-kb deletion of HS3. (c) Comparison of the effects of the HS3 core and 2.3-kb HS3 deletions on histone acetylation in embryonic erythroblasts. See details in the legend to Fig. 2. (d) Comparison of histone binding in the human LCR between the wild-type, ΔHS3 and ΔHS3c βYACs in adult erythroblasts. (a) Histone H3; (b) histone H4. The error bars represent standard deviation.
Fig. 6
Fig. 6
The putative 30-nm fiber of the LCR in a simplified form of the two-start zigzag model. The purple balls represent nucleosome cores, and the green line indicates linkers between nucleosomes. The drawings represent a segment of the LCR encompassing HSs 4, 3, and 2, which is ~7.5 kb in length, with the nucleosome spacing ~200 bp. A long 30-nm fiber has to bend in order to be packaged into the nucleus. The positions of HSs are indicated in blue. Note that in embryonic erythroid cells the HSs are located in the linkers as histones at HSs are depleted (b). In contrast, the array of nucleosomes is uninterrupted in the LCR in adult erythroid cells (a) as the regular level of histones is retained at HSs (see the text). The red spheres represent trans-activating factors recruited by HSs with the highest protein concentration at the center. When the distance between HSs is short and the chromatin fiber bends, the protein spheres will overlap. Assuming that enhancer activity of the LCR is a function of the concentration of trans-acting factors recruited by HSs, the overlapping will result in a synergistic effect on the enhancer activity of the LCR because the overall concentration of trans-activating factors in the LCR is greater than the sum of those recruited by each HS. The overlapping will be augmented in erythroid cells as HSs of the LCR are heavily acetylated, leading to higher chromatin flexibility (depicted as the bends at HSs in the figures). The overlapping will be further increased in the embryonic stage as the degree of acetylation per histone protein is higher in embryonic than adult erythroid cells [compare (a) with (b)]. This model predicts that the enhancer activity of the LCR is determined not only by trans-acting factors recruited by HSs, but also the spatial distance between HSs and the flexibility of chromatin. (c and d) Ten and one nucleosomes were presumably removed by the 2.3-kb HS3 and the HS3 core deletions, respectively. Both the deletions eliminate a protein sphere. However, the reduction of the final concentration of trans-acting factors in the 2.3-kb HS3-deleted LCR is partially compensated for by the decrease in the distance between HSs 2 and 4 as 10 nucleosomes were eliminated by the deletion (c and d). Therefore, the overall effect of the 2.3-kb deletion on enhancer activity of the LCR would be more moderate than that of the HS3 core deletion.
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