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

Селекция Cucumis sativus L. на устойчивость к фузариозу с применением фильтрата культуральной жидкости гриба Fusarium oxysporum Schlectend

Relevance Traditional breeding methods are based on crossing and selection of genotypes among hybrid offspring. In recent decades, along with traditional methods, more and more attention is paid to alternative methods of selection, based on biotechnological manipulations with plants. One of the most important methods of biotechnology is the method of cell selection, which is based on the replacement of the whole plant, as a unit of selection, on its cell. Applying biotechnology techniques from a single plant can produce millions of cells, which increases the chances of finding, eliminating the need for areas for the cultivation of tested plants. As well as accelerating the selection process due to the possibility to carry out the study in the offseason. Methods The studies used the linear material of C. sativus hybrids of All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center and Agroholding "Poisk". Plants were cultivated in laboratory room conditions. As explants used hypocotyl 0.5-1 cm segments isolated from young plants. Results To obtain Cucumis sativus plants with increased resistance to Fusarium by cell selection method, it is recommended to alternate culturing of callus on a non – selective medium containing sucrose in a concentration of 30 g/l, agar – 7 g/l, 0.1 mg/l, NUC – 0.5 mg/l and the filter of the cultural fluid of the fungus in a concentration of 10% within 3 passages.Актуальность В последние десятилетия наряду с традиционными методами все больше внимания уделяется альтернативным методам селекции, в основе которых лежат биотехнологические манипуляции с растениями. Применяя методы биотехнологии из одного растения можно получить миллионы клеток, что увеличивает шансы поиска, исключая потребность в площадях для выращивания испытуемых растений, а также ускоряется селекционный процесс за счет возможности проводить исследования в межсезонье. Методика В исследованиях использовали линейный материал гибридов C. sativus селекции ВНИИО – филиала ФГБНУ ФНЦО и совместной селекции ВНИИО – филиала ФГБНУ ФНЦО с Агрохолдингом «Поиск». Материалом для исследования служили растения C. sativus, которые культивировали в вегетационных сосудах в условиях лабораторного помещения. В качестве эксплантов для получения пролиферирующей каллусной ткани, способной к морфогенезу, использовали гипокотильные сегменты размером 0,5-1 см, изолированные от молодых растений. Результаты Для получения растений Cucumis sativus L. с повышенной устойчивостью к фузариозу методом клеточной селекции рекомендуется чередование культивирования каллуса на неселективной и селективной средах, содержащих сахарозу в концентрации 30 г/л, агар – 7 г/л, БАП – 0,1мг/л, НУК – 0,5 мг/л и фильтрат культуральной жидкости гриба F. oxysporum в концентрации 10% в течение 3-х пассажей

Actual ligation frequencies in the chromosome conformation capture procedure.

Chromosome conformation capture (3C) and derivative experimental procedures are used to estimate the spatial proximity between different genomic elements, thus providing information about the 3D organization of genomic domains and whole genomes within the nucleus. All C-methods are based on the proximity ligation-the preferential ligation of joined DNA fragments obtained upon restriction enzyme digestion of in vivo cross-linked chromatin. Here, using the mouse beta-globin genes in erythroid cells as a model, we estimated the actual frequencies of ligation between the fragments bearing the promoter of the major beta-globin gene and its distant enhancers and showed that the number of ligation products produced does not exceed 1% of all fragments subjected to the ligation. Although this low yield of 3C ligation products may be explained entirely by technical issues, it may as well reflect a low frequency of interaction between DNA regulatory elements in vivo

The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells

<div><p>The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.</p></div

Expansion of nuclei under hypotonic conditions and recovery in the presence of a crowding agent.

<p>(A) Phase contrast images of the nucleus inside the cell (cell), the nucleus isolated in isotonic conditions (nucleus), the nucleus transferred into a law salt buffer (hypotonic nucleus) and the nucleus placed in a law salt buffer with a subsequent addition of 12.5% PEG 8 kDa (recovery). NL—nucleolus, HC—heterochromatin. (B) Mean volume of nuclei under the above-described conditions calculated based on their linear size measurement. The error bars represent SEM for 25 measurements.</p