Dynamic nature of active chromatin hubs

Aim. In order to get more information about organization of active chromatin hubs and their role in the regulation of gene transcription we have studied the spatial organization of the α-globin gene domain in cultured chicken erythroblasts. Methods. The chromosome conformation capture (3C) protocol was employed to analyze the 3D configuration of the chicken α-globin gene domain. Results. We have demonstrated that in the same cell population the chicken domain of α-globin gene may be organized in two different active chromatin hubs. One of them appears essential for the activation of the α-globin gene expression while the other – for the activation of TMEM8 gene which constitutes a part of the α-globin gene domain in chicken, but not in human and other mammals. Importantly, two regulatory elements participate in the formation of both active chromatin hubs. Conclusions. The assembly of the same genomic area into two alternative chromatin hubs which share some regulatory elements suggests that active chromatin hubs are dynamic rather than static, and that regulatory elements may shuttle between different chromatin hubs.


Introduction.
Although the mechanism of enhancer action is far from being clear, most of the current models postulate that an enhancer physically interacts with the target promoter, while sometimes it is located a considerable distance away, up to several hundred kilobases.Therefore, the segment of the chromatin fibre that separates the promoter and the enhancer must be looped out.In multigene loci a single enhancer or block of enhancers (locus control region) appears to activate simultaneously several promoters.For example, in erythroid cells of adult lineage, the mouse b-globin locus control region (LCR) stimulates expression of both the b-major and b-minor globin genes.The promoters of these genes are located at a distance of 14 kb from each other and could not simultaneously interact with the same LCR if only a single loop was formed.It was therefore proposed that LCR forms short-living alternating complexes with these promoters [1,2].
Another model postulates association of an LCR and several depended promoters in an active chromatin hub, a multicomponent complex, from which several chromatin segments are looped out [3,4].This model, although presently widely accepted [3,4], still remains hypothetical due to the intrinsic limitations of the chromosome conformation capture (3C) approach, which can only establish pairwise interactions between distant chromosome elements [5].Thus, multicomponent chromatin hubs can be only predicted, not proved, based on the results of 3C analysis.Besides, most of the present knowledge about active chromatin hubs is based on the studies of one model system, the murine domain of b-globin genes [4,[6][7][8].In order to get more insights into the nature of active chromanin hubs we studied the spatial organization of the a-globin gene domain in chicken erythroid cells producing globins of an adult type.The results obtained demonstrate that in this domain two alternative active chromatin hubs may be assembled.Most interesting, two regulatory elements (the -9kb DNase I hypersensitive site (-9 kb DHS) and the downstream enhancer) participate in the formation of both active chromatin hubs.They should thus shuttle between these hubs as predicted by the «flip-flop» model [1,2].
3C analysis.3C analysis was performed as described [11,12].A random-ligation control was generated using DNA of a bacterial artificial chromosome containing the chicken a-globin gene domain along with the flanking areas (Gallus gallus BAC clone CH261-75C12, CHORI BACPAC Resources Center).
The ligation products were analysed using the realtime PCR with TaqMan probes.Primers and TaqMan probes for PCR analysis were designed using the DNA sequence of Gallus gallus BAC clone CH261-75C12 (AC172304, GeneBank) and Primer Premier 5 computer software (PRIMER Biosoft International).The sequences of the primers and TaqMan probes are available upon request.Each PCR reaction was carried out in quadruple repetition and the corresponding results were averaged.Once a resulting 3C curve representing the spectrum of interactions between an anchor fragment and other fragments throughout the domain was obtained, the experiments starting with living cells were repeated twice more in order to check the reproducibility of the results.
To take into account the differences in the efficiency of crosslinking/restriction/ligation and in the quantity of DNA in the 3C templates obtained from cells of different types, the internal standard was used [11].A house-keeping gene ERCC3 situated on another chicken chromosome was chosen as such a standard.

Results and discussion.
The domain of chicken aglobin genes (Fig. 1, A) contains three alpha-type globin genes and several regulatory elements, including the major regulatory element (MRE) of the domain located in an intron of the apparently house-keeping gene C16orf35, and the downstream enhancer located right after the a A gene.We have demonstrated that this domain also harbors a non-globin gene TMEM8.It was relocated to the vicinity of the a-globin cluster due to the inversion of a ~170-kb genomic fragment.Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquires an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts [13].In the intron 7 of chicken TMEM8 gene an erythroid-specific enhancer is located [13].
AEV-transformed chicken erythroblasts (line HD3, clone A6 of line LSCC) correspond to chicken hemopoietic cells of the red lineage arrested at early stages of differentiation [9,14].They do not express globin genes, although the a-globin gene domain resides in an active configuration supported by low-level transcription of the whole domain [15].After induction of terminal erythroid differentiation, HD3 cells stop proliferation and start production of globins.To study the spatial organization of the a-globin gene domain in proliferating and differentiated HD3 cells we used the 3C method [5,8,16].The combination of BglII and BamHI restriction enzymes was used for 3C analysis.These enzymes recognize different consensuses but produce compatible DNA ends which can be crossligated.
We first investigated the interaction of the LCRlike MRE with each of the downstream restriction fragments (with the exception of very short fragments).As shown in Fig. 1, B, in both proliferating and differentiated HD3 cells this element interacts with the -9 kb DHS, the upstream CpG island of the a-globin gene cluster and the a D gene promoter.In differentiated (i.e. expressing globins) HD3 cells the frequencies of all above-mentioned interactions increased.In addition, a clear interaction between the MRE element and the downstream enhancer was established.Notably, the MRE did not interact with upstream CpG island of TMEM8 gene and with the enhancer located in the body of TMEM8 gene (Fig. 1, B).In contrast, in experiments with the anchor fixed at the -9 DHS or at the downstream enhancer of the a-globin gene domain the interactions with the CpG island of TMEM8 gene and with the enhancer located in the body of TMEM8 gene were clearly visible (Fig. 1, C, D).Again, the apparent frequency of the interactions was much more prominent in differentiated HD3 cells.The significance of the above-described observations was verified in reciprocal experiments when the anchor was placed on the CpG island of TMEM8 gene and on the enhancer situated in the body of TMEM8 gene (Fig. 1, E, F).
Taking together, the results of our 3C analysis can not be explained by the assembly of a single activation complex harboring all known regulatory elements of the a-globin gene domain.There should exist at least two alternative activation complexes stimulating expression of globin genes and of TMEM8 gene (Fig. 2).The «globin hub» includes the MRE, the -9 kb upstream DHS, the -4 kb upstream CpG island of the aglobin gene domain, the a D gene promoter and the downstream enhancer.The «TMEM8 hub» includes the -9 kb DHS, the downstream enhancer, the upstream CpG island of the TMEM8 gene and the erythroid-specific enhancer located in one of the TMEM8 gene introns.Two regulatory elements (the -9 kb DHS and the downstream enhancer) participate in the formation of both active chromatin hubs.They should thus shuttle between these hubs as predicted by the «flip-flop» model [1,2].This model was proposed to explain the ability of the b-globin gene domain LCR to activate transcription of several genes which appeared to be transcribed simultaneously.Although this model was never disproved, it was almost forgotten after advancement of the active chromatin hub model [4,[6][7][8]17].Indeed, the assembly of several enhancers and promoters into a single active chromatin hub suggests a simpler explanation for the ability of an enhancer to activate simultaneously several promoters.

Fig. 1 .
Fig. 1. 3C analysis of the chicken a-globin gene domain: A -the scheme showing positions of important functional elements in the chicken a-globin gene domain (the scale is in kb and «0» point of the scale is arbitrary placed at the 3'-end of the C16orf35 gene); B-F -relative frequencies of cross-linking between the anchor fragments bearing B -MRE; C --9 kb DHS; D -the downstream enhancer of the a-globin gene domain; E -the upstream CpG island of the TMEM8 gene; F -the erythroid-specific enhancer located in the body of the TMEM8 gene and other fragments of the locus.The x axis shows distances in kb.On the top of each graph a scheme of the domain with the same symbols as in A is shown.The results of 3C analysis for cycling (non-induced) and differentiated (induced) HD3 cells are shown by closed and open circles, respectively.The frequency of cross-linking the fragment bearing MRE with the fragment bearing -9 kb DHS in differentiated HD3 cells was taken to equal 100 relative units.The dark gray rectangle in the background of each graph with the anchor drawn above indicates a fixed (anchor) DNA fragment, and the light gray rectangles -test-fragments.Error bars represent SEM