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

AbstractIt 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

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

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

TMEM8 – a non-globin gene entrapped in the globin web

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs

Gene functioning and storage within a folded genome

Abstract In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome. This article was specially invited by the editors and represents work by leading researchers

Genome-Directed Cell Nucleus Assembly

The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA’s genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the “box” in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses

Enhancer Function in the 3D Genome

In this review, we consider various aspects of enhancer functioning in the context of the 3D genome. Particular attention is paid to the mechanisms of enhancer-promoter communication and the significance of the spatial juxtaposition of enhancers and promoters in 3D nuclear space. A model of an activator chromatin compartment is substantiated, which provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements. The mechanisms of selective activation of individual promoters or promoter classes by enhancers are also discussed