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. 2012 Sep 7;5(1):16.
doi: 10.1186/1756-8935-5-16.

Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages

Sergey V Ulianov  1 , Alexey A Gavrilov, Sergey V Razin
Affiliations

Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages

Sergey V Ulianov et al. Epigenetics Chromatin. .

Abstract

Background: The β-globin gene domains of vertebrate animals constitute popular models for studying the regulation of eukaryotic gene transcription. It has previously been shown that in the mouse the developmental switching of globin gene expression correlates with the reconfiguration of an active chromatin hub (ACH), a complex of promoters of transcribed genes with distant regulatory elements. Although it is likely that observations made in the mouse β-globin gene domain are also relevant for this locus in other species, the validity of this supposition still lacks direct experimental evidence. Here, we have studied the spatial organization of the chicken β-globin gene domain. This domain is of particular interest because it represents the perfect example of the so-called 'strong' tissue-specific gene domain flanked by insulators, which delimit the area of preferential sensitivity to DNase I in erythroid cells.

Results: Using chromosome conformation capture (3C), we have compared the spatial configuration of the β-globin gene domain in chicken red blood cells (RBCs) expressing embryonic (3-day-old RBCs) and adult (9-day-old RBCs) β-globin genes. In contrast to observations made in the mouse model, we found that in the chicken, the early embryonic β-globin gene, Ε, did not interact with the locus control region in RBCs of embryonic lineage (3-day RBCs), where this gene is actively transcribed. In contrast to the mouse model, a strong interaction of the promoter of another embryonic β-globin gene, ρ, with the promoter of the adult β-globin gene, βA, was observed in RBCs from both 3-day and 9-day chicken embryos. Finally, we have demonstrated that insulators flanking the chicken β-globin gene domain from the upstream and from the downstream interact with each other, which places the area characterized by lineage-specific sensitivity to DNase I in a separate chromatin loop.

Conclusions: Taken together, our results strongly support the ACH model but show that within a domain of tissue-specific genes, the active status of a promoter does not necessarily correlate with the recruitment of this promoter to the ACH.

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Figures

Figure 1
Figure 1
Analysis of β-globin mRNA levels in chicken 3-day and 9-day embryonic red blood cells. The vertical axis shows the relative amounts of β-globin gene primary transcripts, as determined by RT-qPCR analysis of RNA samples prepared from the blood of 3- and 9-day old chicken embryos. (A) Normalization of data to the level of β-actin mRNA. (B) Normalization of data to the amount of cells used for RNA preparation. The amounts of β-globin gene transcripts detected in different RNA samples were normalized to the number of cells used for preparation of these RNA samples. The amount of the most abundant transcript (ρ-transcript in 3-day RBCs) was set as 100 relative units and the other data were normalized to this value. The error bars represent the S.E.M. for two independent experiments.
Figure 2
Figure 2
MboI and NlaIII q3C analysis of the chicken β-globin gene domain. (A) Map showing the positions of genes and known regulatory elements of the chicken β-globin gene domain. The open boxes indicate genes, the blue lines represent exons, and the arrows indicate the direction of transcription. The closed green boxes show HSs. Upstream insulator, downstream enhancer-blocking elements and the β/Ε enhancer are shown by orange ellipses. FOLR1 - gene encoding folate receptor, transcribed in earlier erythroid progenitors; HAS - tissue-specific erythroid DNase I-hypersensitive site; HS1-3 - locus control region (LCR) of β-globin gene domain; OR51M1 - open reading frame for odorant receptor. The map was made using nucleotide sequences NW_001471556 and L17432.1 [GenBank:NW_001471556, GenBank:L17432.1]. (B-G) The relative frequencies of crosslinking of the anchor fragments containing the HS2 (B), ρ-promoter (C), upstream portion of gene βA(D), β/Ε-enhancer (E), upstream portion of gene Ε(F) and Ε-promoter (G). (B-F) show results of MboI 3C analysis and (G) shows the results of NlaIII 3C analysis. The dark grey rectangle in each panel indicates the anchor DNA fragment, and the light grey rectangles indicate the test fragments. The white rectangles indicate the restriction fragments that were not analyzed. The borders between neighboring fragments are indicated by dark grey lines. The tailless arrows represent the primers used for q3C analysis. The positions of test fragments on the restriction map are plotted on the x-axis. The cross-linking frequencies are plotted on the y-axis; the highest cross-linking frequency observed was set as 100 relative units (the cross-linking frequency between the fragment containing the ρ-promoter and the fragment containing the βA gene in 3-day RBCs for MboI 3C analysis, and the cross-linking frequency between the fragment containing HS2 and the fragment containing HS1 in 3-day RBCs for NlaIII 3C analysis (data not shown)). The results of 3C analysis for 3-day RBCs, 9-day RBCs and CEF are shown by the blue, red and green lines, respectively. The error bars represent the S.E.M. for three (B-F) or two (G) independent experiments.
Figure 3
Figure 3
MboI q3C analysis of spatial interactions of β-globin boundary insulators. The relative frequencies of cross-linking of the anchor fragments containing the 5′-insulator (A) and 3′-insulator (B) of the chicken β-globin gene domain. All designations are as described in Figure 2.
Figure 4
Figure 4
β-globin active chromatin hubs in the nuclei of 3-day and 9-day red blood cells of chicken embryos. The filled disks designate ACH, and the blue ellipses show protein complexes assembled on the boundary insulators.
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