Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages
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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.
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.
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.
Keywordsβ-globin genes Active chromatin hub Insulator Chicken RBCs
Active chromatin hub
Chicken embryonic fibroblast
Site of hypersensitivity to DNase I
Locus control region
Red blood cell
real-time reverse transcriptase polymerase chain reaction
quantitative chromosome conformation capture assay.
Recent studies of the spatial configuration of the mouse and human β-globin gene domain suggest that in erythroid cells, all regulatory elements of the domain become assembled into a common activation complex (the ACH) where the promoters of different globin genes are recruited in a developmental stage-specific fashion[22, 23, 24].
In the present work, we have analyzed the spatial configuration of the chicken β-globin gene domain in circulating red blood cells (RBCs) both before (3-day RBCs) and after (9-day RBCs) the β-globin switching. Using the quantitative chromosome conformation capture assay (3C-qPCR)[25, 26], we have demonstrated that the switching of chicken β-globin gene expression correlates with prominent changes in the spatial configuration of the domain. However, the observed spectrum of changes does not perfectly fit the simplistic model that postulates that only the transcriptionally active genes are recruited to the ACH.
Measurement of β-globin mRNA levels in red blood cells of 3- and 9-day-old chicken embryos
To be able to compare the relative levels of transcription of different globin genes in 3-day and 9-day RBCs, the experimentally detected amounts of globin gene primary transcripts were normalized to the experimentally detected amount of β-actin mRNA (Figure1A). However, it is worth mentioning that the total amount of RNA routinely extracted from the same number of 3-day and 9-day RBCs was much higher in the case of 3-day RBCs (approximately 50 μg and 1.7 μg from 10 million 3-day and 9-day RBCs, respectively). Consequently, the amounts of mRNAs of housekeeping genes differ drastically in samples prepared from the same number of 3-day and 9-day RBCs. For this reason, normalization to the amount of cells (3-day and 9-day RBCs) used for the extraction of RNA (Figure1B) appears to be more appropriate for comparison of the absolute levels of globin gene transcription in these cells. Indeed, using this normalization one can see that total amounts of β-globin gene transcripts are comparable in 3-day and 9-day RBCs while the spectrums of transcribed β-globin genes are different.
Interaction of the locus control region and β/Ε enhancer with promoters of β-globin genes in red blood cells of 3-day and 9-day chicken embryos
The quantitative chromosome conformation capture (3C-qPCR) assay was used to investigate the spatial organization of the chicken β-globin gene domain in RBCs of 3- and 9-day-old embryos. It is well known that the accurate analysis and interpretation of raw 3C data is not a trivial task, and it can be difficult to distinguish the relevant chromatin interactions from background and cross-linking artifacts. One problem in the interpretation of the 3C data is that neighboring restriction fragments are always ligated to a certain extent due to cross-links between nucleosomal proteins, even if these fragments are not involved in the assembly of a chromatin hub. To discriminate the functionally relevant interactions from the background cross-linking, we performed the 3C analysis of the β-globin gene domain in chicken embryonic fibroblasts (CEF). These cells do not express globin genes. Thus, there are good reasons to expect that the domain in these cells does not adopt any functionally relevant spatial configuration.
In the main set of 3C experiments, we used MboI restriction enzyme digestion to cleave the DNA before the proximity ligation. This enzyme cuts the β-globin gene domain into a number of relatively small fragments, such that the promoters of β-globin genes and regulatory elements were located in different restriction fragments separated by several cleavage sites. It was thus possible to independently analyze all of these regulatory elements. In some cases, however, it was not possible to place the primers and TaqMan™ probes exactly on the regulatory element under study due to the small size of the corresponding restriction fragment or the presence of repetitive sequences. In these cases, the primers and TaqMan™ probes were placed at the adjacent restriction fragments. For example, for βA and Ε genes, the probes were placed at the fragments located just downstream of the promoters.
We have first fixed the anchor at the restriction fragment containing HS2 (a part of β-globin domain LCR). In the 3-day RBCs, this anchor was shown to interact preferentially with the fragments containing the promoter of the embryonic gene ρ, the adult gene βA and the β/Ε-enhancer (Figure2B, blue curve). In addition, an elevated frequency of interaction of the HS2 with the upstream and the downstream insulators of the β-globin gene domain was observed. Interestingly, no interaction of the HS2 with the embryonic gene Ε was detected. As expected, no interaction between any of the functional elements studied was detected in embryonic fibroblasts (Figure2B, green curve). In 9-day RBCs the frequency of interactions of the HS2 with the βA gene increased. In addition, a strong interaction between the HS2 and the promoter of βH gene was established (Figure2B, red curve). The validity of the observations made with the anchor placed at HS2 was confirmed in experiments with the anchor placed on the promoter of the ρ gene (Figure2C), the upstream portion of the βA gene (Figure2D), the upstream portion of the Ε gene (Figure2F) and the β/Ε-enhancer (Figure2E). The anchor placed on the promoter of the ρ gene demonstrated strong interaction frequencies with the HSs 1‐3 of the LCR in 3-day RBCs (Figure2C, blue curve). The frequency of interaction of this anchor with the HS1 remained about the same in 9-day RBCs while the frequencies of interaction with the HSs 2 and 3 decreased (Figure2C, red curve). Unexpectedly, in 3-day RBCs the promoter of the ρ gene demonstrated a strong interaction with the upstream region of the βA gene. The frequency of this interaction decreased in 9-day RBCs but still remained quite prominent (Figure2C, blue and red curves). The interaction between ρ and βA genes was also observed in embryonic fibroblasts (Figure2C, green curve). It may thus reflect some internal feature of the β-globin gene domain spatial organization that is not related to the transcriptional status of the domain. The interaction between the βA and ρ gene promoters observed in the experiment with the anchor placed on the ρ gene promoter was confirmed in the reciprocal experiment with the anchor placed on the upstream part of the βA gene (Figure2D, all curves). No interaction of the ρ gene promoter with the promoter of the minor adult βH gene was observed in 3-day RBC (Figure2C, blue curve). In contrast, in the same cells (3-day RBC) the upstream region of the βA gene demonstrated strong interactions with both the ρ and βH gene promoters (Figure2D, blue curve). As expected, the upstream region of the βA gene demonstrated a strong interaction with the HSs 1 to 3 of the LCR in 9-day RBCs, but not in 3-day RBCs (Figure2D, red and blue curves). A strong interaction of the βA gene with the β/Ε enhancer was also observed in 9-day but not in 3-day RBCs (Figure2D, red and blue curves). Although in this particular case the anchor (the upstream part of the βA gene) and the test-fragment (β/Ε enhancer) are located close to each other, the much higher interaction frequency observed in 9-day RBCs comparing to 3-day RBCs and CEF argues for the existence of functionally relevant interaction between the βA gene and the β/Ε enhancer in 9-day RBCs. This conclusion is supported by the results of a reciprocal experiment with the anchor placed on the β/Ε enhancer. Again, in 9-day RBCs the frequency of interaction of the β/Ε enhancer with the upstream part of the βA gene was much higher in 9-day RBCs than in 3-day RBCs and CEF (Figure2E). The interaction of the β/Ε enhancer with the three HSs of the LCR and the promoter of ρ gene was observed in both 3-day and 9-day RBCs, but not in CEF. On the other hand, interaction of the β/Ε enhancer with the promoter of gene βH was observed only in 9-day RBCs (Figure2E, red curve). No interaction of the β/Ε enhancer with the gene Ε was observed in either of the cell lines studied (Figure2E). In full accord with this observation, the anchor placed on the upstream area of the gene Ε showed no interaction with any of the test fragments studied in either erythroid or non-erythroid cells (Figure2F). Thus it appears that the Ε gene does not interact with any of the remote regulatory elements of the domain. To verify this interesting observation, we performed 3C experiments using digestion with another restriction enzyme, NlaIII. Distribution of NlaIII recognition sites along the β-globin gene domain allows fixation of the anchor exactly on the promoter of the gene Ε. Using this anchor we performed 3C analysis of the β-globin domain configuration in 3-day RBCs and in CEF. Again, no interaction of the Ε gene promoter with any of the remote regulatory elements was observed (Figure2G). As a matter of fact, the frequencies of interaction of the Ε gene promoter with all tested fragments of the β-globin gene domain were very similar in 3-day RBCs that express the Ε gene and in embryonic fibroblasts that do not express globin genes at all.
Interaction between the upstream and the downstream insulators of the β-globin gene domain
The current model of the enhancer action suggests that enhancers directly interact with target promoters[23, 28, 29, 30]. As a result, an assembly of regulatory elements, called the active chromatin hub (ACH), forms. One or several enhancers present in the ACH may control the activity of one or several promoters recruited to the ACH[23, 29]. The transcriptional switching of murine β-globin gene expression correlates with a reconfiguration of the ACH. The aim of the present study was to determine whether the developmental switching of chicken β-globin gene transcription depends on the spatial reconfiguration of the β-globin gene domain. The study of the chicken β-globin gene domain is particularly interesting for two reasons. First, this domain is the best example of the so-called closed chromatin domains, as the upstream and the downstream insulators perfectly delimit the area of preferential sensitivity to DNase I in erythroid cells, which is not the case in mouse and human β-globin gene domains[32, 33]. Second, the chicken β-globin gene domain contains an internal enhancer element in addition to the upstream locus control region. Once again, this is not the case in the mouse or human. Hence, the regulation of chicken β-globin gene expression may differ from regulation of β-globin gene expression in the mouse and human.
The results of the present study showed that in erythroid cells there is a clear interaction of the upstream and the downstream insulators of the chicken β-globin gene domain. As a result, the domain should be organized in a closed loop. Although we have not studied the mechanism of this loop formation, it is very probable that CTCF is involved, as both insulators possess CTCF-binding sites[9, 10] and it was shown that CTCF might mediate an interaction between distant genomic elements[34, 35]. It is also likely that the β-globin loop ends are fixed at the nuclear matrix, as the 5′ insulator of the chicken β-globin gene domain has been reported to interact with the nuclear matrix. This type of spatial organization perfectly explains the fact that in the chicken β-globin gene domain, the area demonstrating the cell-lineage-specific sensitivity to DNase I perfectly co-localizes with the genomic segment flanked by insulators. In agreement with this model, the interaction between the upstream and the downstream insulators of the chicken β-globin gene domain was not observed in non-erythroid cells where the domain is not preferentially sensitive to DNase I. In should be mentioned, however, that in mouse and human the insulators flanking the β-globin gene domain from the upstream and the downstream also interact with each other[22, 24, 34], although the chromatin loop established by this interaction does not correspond to the region of preferential sensitivity to DNase I.
The most interesting feature of the spatial organization of the chicken β-globin gene domain is that the embryonic β-globin gene Ε interacts with neither the LCR nor the β/Ε enhancer. This does not exclude the possibility that the β/Ε enhancer located at a short distance upstream of the Ε gene can activate this gene promoter by some tracking mechanism. However, an activation effect of the LCR on Ε gene transcription seems very improbable. In this respect, it is of interest that in the chicken α-globin gene domain, the embryonic gene π also does not interact with any of the known distant regulatory elements of the domain. It is thus likely that in the chicken, both α-type and β-type early embryonic globin genes possess an autonomous regulatory system.
In 9-day RBCs, the level of mutual interaction of most of the regulatory elements of the β-globin gene domain increases compared to the levels in 3-day RBCs. These observations may reflect more compact packaging of the domain as a whole. Thus, it should be noted that 9-day RBCs differ drastically from the 3-day RBCs in the total amount of RNA, including rRNA, present in the cell (see the first section of the results). Evidently, the 9-day RBCs are less metabolically active compared to 3-day RBCs. In other words, they possess certain features of mature chicken erythrocytes, which have non-active nuclei. Nevertheless, the globin genes are still transcribed in the majority of 9-day RBCs. Thus, the concept that the apparently more compact spatial organization of the β-globin gene domain in 9-day RBCs may simply reflect the presence of a substantial portion of mature erythrocytes in the analyzed cell population would not be correct. Rather, the β-globin gene domain suffers a certain amount of pressure because it is a part of the chromosomal territory that gradually acquires a more compact configuration. Regardless, it remains active and possesses certain features of an active chromatin domain, including a preferential sensitivity to DNase I, even in the compact nuclei of mature chicken erythrocytes. The spatial isolation of this domain from the surrounding chromatin by the formation of a distinct chromatin loop via interaction of the flanking insulators may be of major importance.
In this work, we have analyzed the spatial configuration of the chicken β-globin gene domain in red blood cells of embryonic and adult lineages. The obtained results strongly support the active chromatin hub model, as it has been found that distant regulatory elements located upstream of the β-globin gene cluster form a complex with the β/Ε enhancer located in the downstream portion of the cluster. Promoters of all β-globin genes, with the exception of embryonic gene Ε, are recruited to this complex, demonstrating a specificity of interaction with the LCR in respect to the developmental stage. The embryonic β-globin gene Ε does not interact with any of the distal regulatory elements; thus, it is likely to possess an autonomous regulatory system. In both 3-day and 9-day RBCs the upstream and the downstream insulators of the chicken β-gene domain interact with each other, thus placing the domain in a separate chromatin loop. This may explain the fact that in chickens, the borders of erythroid cell-specific domain with preferential sensitivity to DNase I perfectly co-localize with the borders of β-globin gene domain marked by insulators.
Real-time reverse transcriptase polymerase chain reaction (RT-PCR)
Primers and TaqMan™ probes used for analysis of β-globin primary transcripts and β-actin mRNA levels
The 3C analysis was performed as described with minor modifications. Briefly, 7.5×107 cells were fixed in DMEM/serum with 2% formaldehyde for 10 min at room temperature. The reaction was stopped by adding glycine to a concentration of 0.125 M and placing the samples on ice. Cross-linked cells were washed with cold PBS and lysed by incubation for 10 min in ice-cold lysis buffer (10 mM Tris (pH 8.0), 10 mM NaCl, 0.2% NP-40, protease inhibitor cocktail R1321 (Fermentas, Vilnius, Lithuania)). The nuclei were harvested, and aliquots containing 3.75×107 nuclei were frozen in liquid nitrogen and stored at ‐70°C.
Approximately 1.25×107 nuclei were suspended in 0.25 ml of an appropriate 1.2x restriction buffer, SDS was added to a final concentration of 0.3%, and the solution was incubated with shaking at 37°C for 1 h. The solution was then diluted 2-fold with the same restriction buffer, and Triton X-100 was added to a final concentration of 1.8% (to sequester the SDS) followed by incubation at the same conditions for another hour. After incubation, 400 U of MboI (NEB, Hitchin, UK) or NlaIII (Fermentas, Vilnius, Lithuania) was added, and the digestion was performed for 20 h at 37°C with continuous shaking. The reaction was terminated by the addition of SDS to a final concentration of 1.3% and incubation at 65°C for 20 min. The solution was diluted by adding 7 ml of 1x ligation buffer (50 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM ATP). Triton X-100 was added to a final concentration of 1%, and the solution was incubated at 37°C for 1 h while shaking. T4 DNA Ligase (100 U, Fermentas, Vilnius, Lithuania) was then added, and DNA was ligated at 14-16°C for 5 h followed by further incubation at room temperature for 30 min with slow agitation. Cross-links were reversed by incubation at 65°C for 16 h in the presence of 500 μg of Proteinase K (Sigma). After cross-link reversion, 300 μg of RNase A was added, and the RNA was digested at 37°C for 45 min. DNA was purified by extraction with phenol (pH 8.0), followed by precipitation with ethanol in the presence of 120 μg of glycogen (Fermentas, Vilnius, Lithuania).
The efficacy of restriction enzyme digestion in 3C reactions was checked using PCR stop analysis and was found to be more than 80% for different sites throughout the locus under study (data not shown).
To generate a random ligation control, 8 μg of a bacterial artificial chromosome containing the chicken β-globin gene domain along with the flanking areas [CHORI BACPAC Resources Center:TAM32-28 J3] was digested with Sau3A or NlaIII and then ligated at a high DNA concentration.
Primers and TaqMan™ probes used for 3C analysis
Primers for MboI 3C analysis
HS4 -|- HS3
HS2 -|- HS1
HS1 -|- Rho
Rho -|- betaH
BetaH -|- BetaA
BetaA -|- Beta/eps enh
Beta/eps enh (anchor)
Beta/eps enh -|- Epsilon
Downstream to 3′HS
Primers for NlaIII 3C analysis
HS4 -|- HS3
HS3 -|- HS2
HS2 -|- HS1
HS1 -|- Rho
Beta/eps enh -|- Epsilon
Epsilon -|- 3′HS
TaqMan™ probes for MboI 3C analysis
TaqMan™ probes for NlaIII 3C analysis
To account for the qualitative and quantitative differences of independently prepared 3C samples, the qPCR data for each sample were normalized to the results obtained by using primers for the housekeeping gene, ERCC3 (encodes the ATP-dependent DNA-helicase, a component of the complex TFIIH), located in an area that presumably possesses the same structure of chromatin in all types of cells. The research was approved by the institute ethics governing board.
This work was supported by the Ministry of Science and Education of the Russian Federation (contracts 16.740.11.0353, 14.740.12.1344 and 16.740.11.0483), by the Russian Foundation for Support of Basic Research (grants 11-04-00361-Ð°, 11-04-91334-NNIO_Ð° and 12-04-00036-Ð°), by the Presidium of the Russian Academy of Sciences (grants from the Program on Molecular and Cellular Biology), by a grant from the President of the Russian Federation for young scientists (MK-3813.2012.4) and by Dmitri Zimin’s foundation ”Dynasty”. Experiments were performed using the equipment of IGB RAS facilities supported by the Ministry of Science and Education of the Russian Federation (contract 16.552.11.7067). The work of Sergey Ulianov was also supported in part by the Russian Federation (Order 220) research grant.
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