Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This “elementary” fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30–40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called “immature chromatin,” which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

В работе изучена динамика изменений структуры хроматина на различных стадиях дифференцировки сперматид человека. Показано, что в ядрах ранних сперматид хроматин полностью декомпактизован, его структурной единицей является фибрилла толщиной около 8 нм. На начальных стадиях дифференцировки эта “элементарная” фибрилла является преобладающей структурой в ядрах, а на последующих этапах она постепенно замещается крупными глобулярными комплексами диаметром около 60 нм. В дальнейшем глобулы ассоциируют в фибриллярные структуры, толщина которых уменьшается до 30–40 нм. На всех стадиях дифференцировки, кроме конечных, глобулярно-фибриллярные комплексы представляют собой центры ассоциации элементарных фибрилл. Процесс конденсации хроматина имеет векторный характер – он направлен от апикальной зоны ядра к базальной. В зрелых сперматидах преобладающим компонентом являются фибриллы толщиной около 40 нм, тонкие фибриллы практически отсутствуют. По своей структурной организации ядра зрелых сперматид полностью соответствуют ядрам сперматозоидов с “незрелым” хроматином, которые в небольшом количестве обнаруживаются в эякуляте здоровых доноров и могут преобладать над нормальными клетками при паталогиях спермиогенеза. Причина такой остановки дифференцировки остается непонятной. Возможно, в части сперматид блокируется формирование межмолекулярных дисульфидных связей, которые необходимы для завершающей фазы компактизации генома. Полученные результаты обсуждаются в связи с известными моделями организации нуклеопротаминового хроматина в сперматозоидах человека.

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.

The DNA methylation pattern and chromatin structure of the alpha-amylase gene have been studied in barley aleurone layer native cells with regard to gene expression. Probed by a cloned 3'-end sequence, alpha-amylase genes have been found to be completely unmethylated (or weekly methylated) on the internal C residue in CCGG sites prior to induction of gene expression. Induction of gene expression by gibberellic acid (GA) and/or increase in Ca2+ concentration does not significantly change the DNA methylation level of alpha-amylase genes. These genes are totally devoid of any distinct nucleosome structure both prior to and after induction of gene expression. The alpha-amylase genes seem to be in an expression competent ("activated") state with regard to DNA methylation and chromatin structure; hence, the phytohormone GA is not an alpha-amylase gene expression inducer, but rather a part of some still unknown trigger mechanism.

A biochemical and ultrastructural stereo-morphological analysis, with special reference to spatial organization and length of nucleonema and Ag-positive zones, was performed for various modifications of nucleolonemal type nucleoli in normal and regenerating (6 and 22 hours after partial hepatectomy) rat hepatocytes. To determine possible disorders on nucleosomal and supranucleosomal levels, chromatin DNA degradation was carried out during micrococcal nuclease hydrolysis, followed by analysis of electrophoretically separated particles. Functional characterization of intranucleolar chromatin was performed by testing the rate of DNA degradation after DNAase I treatment as well as by detection of free G-C pairs during titration with actinomycin D. Transcriptional activity of nucleoli was determined according to the intensity of [14C]-UTP uptake with isolated nucleoli. It is shown that the total chromatin from control nucleoli contains nucleosomal fibrils, although deprived of high compactization level. Nucleosomes themselves are strongly destabilized. In activated nucleoli structural differences of chromatin are more perceptible. In 6 hour preparations the bulk of chromatin fibrils (about 70%) undergo a further relaxation and lose the nucleosomal structure. Therefore at this point of experiment, the maximum length of nucleolonema and Ag-positive zones was registered in addition to the highest quantity of free G-C pairs, and sensibility to DNAase I transcriptional activity of isolated nucleoli. 22 hours after hepatectomy, the transcriptional activity and functional parameters of intranucleolar chromatin markedly decreased compared to the 6 hour period. Simultaneously, the share of chromatin restituting the nucleosomal structure increased, while the length of nucleolonema was shorter than in nucleoli 6 hours after hepatectomy. The main results could be resumed in the following way: the general composition of nucleolonemal type nucleolus variations described in our experimental conditions is in close relation with the with the compactization grade of ribosomal DNP-fibrils.