Α-globin Gene Domain Research Articles

Главный регуляторный элемент (MRE) домена α/β-глобиновых генов Danio rerio проявляет энхансерную активность по отношению к промоторам глобиновых генов эмбрионально-личиночного и взрослого субдоменов

In warm-blooded vertebrates, the α- and β-globin genes are organized in domains of different types and are regulated in different fashion. In cold-blooded vertebrates and, in particular, the tropical fish Danio rerio, the α- and β-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and β-globin genes of embryonic-larval and adult types. The region upstream of the cluster contains c16orf35, harbors the main regulatory element (MRE) of the α-globin gene domain in warm-blooded vertebrates. In this study, transient transfection of erythroid cells with genetic constructs containing a reporter gene under the control of potential regulatory elements of the domain was performed to characterize the promoters of the embryonic-larval and adult α- and β-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio reriowas detected a functional analog of the warm-blooded vertebrate MRE. This enhancer stimulated activity of the promoters of both adult and embryonic-larval α- and β-globin genes.

Detection of complementary transcripts for the intergenic region of the chicken α-globin gene domain

Detection of complementary transcripts for the intergenic region of the chicken α-globin gene domain

Обнаружение двунаправленных транскриптов в межгенной области домена α-глобиновых генов кур

Using strand-specific reverse transcription followed by Real Time PCR analysis we have characterized the transcription profile of the segment of chicken α-globin gene domain harboring embryonic gene π, adult gene αD and spacer region separating these genes. It has been demonstrated that in erythroid cells of adult lineage the spacer region is transcribed in both directions. These results suggest a possibility that switching of α-globin genes expression is controlled by RNA-interference mechanism.

Three-Dimensional Chromatin Model of α-Globin Gene Locus in Differentially Activated States

The global architecture of cell nucleus and the spatial organization of chromatin play important roles in gene expression and nuclear function. By measuring the frequency of physical proximity between pairs of chromosomal regions on a locus scale, Chromosome Conformation Capture Carbon Copy (5C) technique revealed a wealth of information about the spatial organization of chromatin. However, the three-dimensional structure of the chromatin and how it changes at different activation states are largely unknown. We have developed an algorithm to reconstruct the three-dimensional structure of α-globin gene locus, which is expressed in K562 cell line and inactive in GM12878 cell line based on publicly available 5C interaction data [1]. Our reconstructed three-dimensional chromatin models show an open conformation in the active cells, whereas they fold into more compact conformations in inactive cells. Furthermore, our models not only can capture known long-range interactions, but can also predict new interactions that were not in the 5C data, but were experimentally observed in an independent study. Analysis of ensemble of model chromatin chains reveals an activation mechanism of α-globin gene that is highly influenced by the folding landscape and the spatial organization of the locus. Clusters of interacting regions suggest that the accessibility of specific regions of regulatory elements in three-dimensional space is essential for cells to maintain the necessary long-range interactions for gene activation.[1] Bau, D. et al. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat Struct Mol Biol. 18, 107-114 (2011)

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.

The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules

We developed a general approach that combines chromosome conformation capture carbon copy (5C) with the Integrated Modeling Platform (IMP) to generate high-resolution three-dimensional models of chromatin at the megabase scale. We applied this approach to the ENm008 domain on human chromosome 16, containing the α-globin locus, which is expressed in K562 cells and silenced in lymphoblastoid cells (GM12878). The models accurately reproduce the known looping interactions between the α-globin genes and their distal regulatory elements. Further, we find using our approach that the domain folds into a single globular conformation in GM12878 cells, whereas two globules are formed in K562 cells. The central cores of these globules are enriched for transcribed genes, whereas nontranscribed chromatin is more peripheral. We propose that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as previously observed by microscopy.

An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression

Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5′ non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF.

Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a ‘guest’ compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited.

Early replication timing of the chicken α-globin gene domain correlates with its open chromatin state in cells of different lineages

The vertebrate α-globin gene domain is an open chromatin domain overlapping a neighboring house-keeping gene. The tissue-specific cluster of α-globin genes and the overlapping housekeeping gene share the same replication origin. We have studied the replication timing of chicken α-globin genes in cells of different lineages using the FISH-based approach and found that α-globin genes replicate early both in erythroid and in non-erythroid cells, i.e. regardless of their transcriptional activity. Early replication timing of chicken α-globin genes in cells of different lineages was in good correlation with the open chromatin configuration of the α-globin gene domain in both erythroid and non-erythroid cells. We propose that active transcription of the housekeeping gene overlapping the α-globin gene domain enables an access of Origin Recognition Complex (ORC) proteins to the replication origin resulting in early replication of α-globin genes even in non-erythroid cells.

Study of spatial organization of chicken α-globin gene domain by 3c technique

This work deals with 3C (Chromosome Conformation Capture) analysis of the chicken alpha-globin gene domain in embryonic erythrocytes and lymphoid cells. Ligation products were quantitatively analyzed by real-time PCR with TaqMan probes. It was found that in lymphoid cells, where alpha-globin gene is not active, the domain has a relatively extended configuration. In embryonic erythrocytes that transcribe alpha(D) and alpha(A) genes, simultaneous interaction of several domain elements was revealed including the major regulatory element, the erythroid-specific DNase I hypersensitive site at a distance of 9 kb upstream from the alpha-globin gene cluster (-9 DHS), promoter of the housekeeping gene CGTHBA, the alpha(D)-globin gene promoter, and the erythroid-specific enhancer located after the alpha-globin gene cluster. We suppose that such interaction is necessary to provide for the active transcription status of the chicken alpha-globin gene domains in erythroid cells.

Spatial configuration of the chicken α-globin gene domain: immature and active chromatin hubs

The spatial configuration of the chicken α-globin gene domain in erythroid and lymphoid cells was studied by using the Chromosome Conformation Capture (3C) approach. Real-time PCR with TaqMan probes was employed to estimate the frequencies of cross-linking of different restriction fragments within the domain. In differentiated cultured erythroblasts and in 10-day chick embryo erythrocytes expressing ‘adult’ αA and αD globin genes the following elements of the domain were found to form an ‘active’ chromatin hub: upstream Major Regulatory Element (MRE), −9 kb upstream DNase I hypersensitive site (DHS), −4 kb upstream CpG island, αD gene promoter and the downstream enhancer. The αA gene promoter was not present in the ‘active’ chromatin hub although the level of αA gene transcription exceeded that of the αD gene. Formation of the ‘active’ chromatin hub was preceded by the assembly of multiple incomplete hubs containing MRE in combination with either −9 kb DHS or other regulatory elements of the domain. These incomplete chromatin hubs were present in proliferating cultured erythroblasts which did not express globin genes. In lymphoid cells only the interaction between the αD promoter and the CpG island was detected.

Mechanisms controlling activation of the α-globin gene domain in chicken erythroid cells

In this review we consider the organization of the chicken alpha-globin gene domain and mechanisms regulating the activity of this tissue-specific gene domain located in a potentially active (characterized by an increased sensitivity to nucleases) chromatin configuration in cells of all lineages. Both regulatory mechanisms ensuring repression of alpha-globin genes in non-erythroid cells and mechanisms responsible for activation of transcription of these genes during erythroid cell differentiation are discussed. The analysis of the structure-function organization of the chicken alpha-globin gene domain presented in this review is based mainly on the authors' own results obtained over the last 20 years. On discussing the hypotheses explaining the mechanisms controlling the functional activity of chicken alpha-globin gene domain, data obtained in studies of alpha-globin gene domains of other vertebrates are also analyzed.

A CTCF-Dependent Silencer Located in the Differentially Methylated Area May Regulate Expression of a Housekeeping Gene Overlapping a Tissue-Specific Gene Domain

The tissue-specific chicken alpha-globin gene domain represents one of the paradigms, in terms of its constitutively open chromatin conformation and the location of several regulatory elements within the neighboring housekeeping gene. Here, we show that an 0.2-kb DNA fragment located approximately 4 kb upstream to the chicken alpha-globin gene cluster contains a binding site for the multifunctional protein factor CTCF and possesses silencer activity which depends on CTCF binding, as demonstrated by site-directed mutagenesis of the CTCF recognition sequence. CTCF was found to be associated with this recognition site in erythroid cells but not in lymphoid cells where the site is methylated. A functional promoter directing the transcription of the apparently housekeeping ggPRX gene was found 120 bp from the CTCF-dependent silencer. The data are discussed in terms of the hypothesis that the CTCF-dependent silencer stabilizes the level of ggPRX gene transcription in erythroid cells where the promoter of this gene may be influenced by positive cis-regulatory signals activating alpha-globin gene transcription.

Spatial Organization of the Chicken α-Globin Gene Domain in Cells of Different Origins

Hybridization with an oligonucleotide array was used to map the regions of DNA anchorage to the nuclear matrix. Matrix-associated DNA served as a hybridization probe. To obtain the oligonucleotide array, 60-mer oligonucleotides regularly distributed throughout the genome region of interest at 2-kb intervals were immobilized on a nylon filter. The organization of DNA into loop domains was studied in a 100-kb region of chicken chromosome 16, including the α -globin gene cluster. A 40-kb DNA loop, which was fixed to the nuclear matrix and harbored all α-globin genes, was observed in erythroid cells. One of its anchorage regions colocalized with matrix associated region (MAR) and an insulator found previously in the 5′ region of the chicken α-globin gene domain. The spatial (domain-loop) organization of the α-globin gene cluster in lymphoid cells proved to be strikingly different from that in erythroid cells.

The upstream area of the chicken α-globin gene domain is transcribed in both directions in the same cells

It was demonstrated previously that in erythroid chicken cells an extended upstream area of the α-globin gene domain is transcribed in both directions as a part of ggPRX gene and a part of a full domain transcript of the α-globin gene domain. Here, we show that both DNA chains of the above-mentioned region are transcribed in the same cells and that the corresponding transcripts coexist in nuclei. The data obtained suggest that cells possess a molecular mechanism which in some cases prevents the formation of dsRNA and subsequent destruction of both transcripts in spite of the presence of complementary RNA chains in the cell nucleus.

Study of the regulatory sequences associated with DNase I-hypersensitive sites in the 5′ region of the chicken α-globin gene domain

The functional activity of DNA sequences from the 5′ region of the chicken α-globin gene domain was studied. Silencers and transcription terminators were found in a fragment located 11–14.5 kb upstream of the π gene. A fragment located 3–4.5 kb upstream of π was shown to contain a CpG cluster, a part of which proved to be selectively methylated in nonerythroid cells. Cloned upstream of the αD gene promoter, this fragment suppressed the promoter activity when all its CpG were methylated.

Extensive methylation of a part of the CpG island located 3.0–4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of α-globin genes is preceded by a CpG island of which the downstream part (∼0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called “uCpG”) placed upstream to a reporter CAT gene expressed from the promoter of the αD chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the αD promoter were methylated. Methylation of CpG dinucleotides within the αD gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken α-globin gene domain may play an essential role in silencing the α-globin genes in non-erythroid cells.

A novel gene is transcribed in the chicken alpha-globin gene domain in the direction opposite to the globin genes.

A novel gene transcribed in the direction opposite to that of the globin genes was found in the chicken alpha-globin gene domain. Northern hybridisation with single-stranded riboprobes revealed that a 4.5-kb poly(A)+ RNA is transcribed in antisense polarity with respect to the globin genes. The transcription unit encoding this RNA seems to overlap the entire cluster of alpha-globin genes and extends at least 15 kb upstream from pi, the first of the alpha-globin genes. This new transcript shows partial sequence homology with that encoded by the human "-14" gene. An oligonucleotide based on part of a restriction fragment of chicken DNA that is 80% homologous to exon 4 of the human "-14" gene hybridises with a 4.5-kb RNA molecule. In situ hybridisation of globin-antisense probes, that detect polyribosomal mRNAs of 1.7 and 2.5 kb on Northern blots, shows these "antisense" transcripts to be present in the cytoplasm. The 4.5-kb RNA is absent in polyribosomal poly(A)+ RNA and may, hence, represent a nuclear pre-mRNA transcribed from the chicken gene that is homologous to the human "-14" gene. The expression of this gene is not specific to erythroid cells; analogous transcripts were also detected in poly(A)+ RNA extracted from a chicken lymphoblastoma cell line (HP50). Taken together, these data allow us to postulate the existence in the chicken genome of a novel gene, for which we suggest the name "ggPRX" in analogy to the murine mProx1, a gene identified in the upstream region of the alpha-globin gene domain in mice.

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain.

The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the alpha-globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the alpha-globin gene domain and the function of the replication origin is discussed.

Preliminary characterization of a nuclear factor interacting with the silencer element at the 3′-side of the chicken α-globin gene domain

A silencer element has been identified previously on the 3′-side of the chicken α-globin genes placed next to the major enhancer in this domain (Recillas Targa et al., unpublished). Deletion fragments of this negative element show the requirement of the entire DNA segment for maximum silencing activity. Additionally, the sub-fragments including the previously defined SF1 (Silencer Factor 1) sequence seem to play an important role on the negative action of this silencer. Preliminary characterization of the nuclear factor interacting with the SF1 sequence was carried out. Methylation interference experiments show the contact points and, by electrophoretic retardation analysis with SF1 and SF1-mutated oligonucleotides, the critical role of these contacts points on the formation of the DNA-protein complex could be demonstrated. These results provide a basis for further experiments on the characterization and understanding of the mechanisms of action of the coupled positive and negative regulatory elements.