How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.The bacterial RecA protein is multifunctional (11,17,25). In Escherichia coli, the RecA protein is first a recombinase, promoting homologous recombination and recombinational DNA repair. Second, RecA filaments formed on DNA facilitate the autocatalytic cleavage of the LexA protein to allow induction of the SOS response. This activity is referred to as the RecA coprotease function. Finally, RecA is directly involved in the activation of DNA polymerase V, the polymerase responsible for SOS mutagenesis (11,17,25). The present study focused on RecA function in recombination and recombinational DNA repair.The key steps in homologous recombination promoted by a RecA nucleoprotein filament are (i) formation of the presynaptic filament (a ternary complex including RecA, ATP, and single-stranded DNA [ssDNA]) in the presence of magnesium ion, (ii) pairing of the presynaptic filament with homologous double-stranded DNA (dsDNA), and (iii) DNA strand exchange between ssDNA and dsDNA that results in a new heteroduplex dsDNA and a displaced ssDNA (11,15,17,25,37). To promote these reactions, RecA has two main activities, the DNA-dependent ATPase, activated with both ssDNA and dsDNA, and DNA strand transferase. The latter activity can be broken down into several characteristics commonly used for the comparison of different RecA proteins. These include RecA protein binding to ssDNA to form a presynaptic complex (PC), RecA protein-mediated displacement of SSB protein from ssDNA, the stability of PCs (for ex...

The cell nucleus comprises a number of chromatin-associated domains. Certain chromatin-associated domains are nucleated by nascent RNA and accumulate non-nascent transcripts in the form of ribonucleoprotein (RNP) aggregates. In the transcriptionally active nucleus of the growing avian oocyte, RNP-rich structures, here termed giant terminal RNP aggregates (GITERA), form at the termini of lampbrush chromosomes. Using GITERA as an example, we aimed to explore mechanisms of RNP aggregate formation at certain chromosomal loci to establish whether they accumulate non-nascent RNA and to analyze protein composition in RNP aggregates. We found that GITERA on chicken and pigeon lampbrush chromosomes do not contain nascent transcripts. At the same time, RNA fluorescent in situ hybridization (FISH) and in situ reverse transcription demonstrated that GITERA accumulate poly(A)-rich RNA. Moreover, subtelomere chromosome regions adjacent to GITERA are transcriptionally active as shown by detection of incorporated BrUTP and the elongating form of RNA polymerase II. GITERA on both chicken and pigeon lampbrush chromosomes are enriched in splicing factors but not in heterogeneous nuclear RNP (hnRNP) L and K. A subtype of GITERA concentrates hnRNP I/PTB and p54nrb/NonO. Interestingly, hnRNP I/PTB and p54nrb/NonO in such subtype of GITERA were revealed in long threads. The resemblance of these threads to amyloid-like fibers is discussed. Our data suggest that transcription of subtelomeric sequences serves as a seeding event for accumulation of non-nascent RNA and associated RNP proteins. Such accumulation leads to GITERA formation in terminal chromosomal regions in avian oocyte nucleus. 3'-processed transcripts derived from other chromosomal loci may be attracted to GITERA by binding to the same RNP proteins or to their interaction partners.