Comparative analysis of the secondary structure of non-histone chromatin proteins HMGB1 and HMGB2

The non-histone chromosomal proteins HMGB1 and HMGB2 were found in the cells of all studied eukaryotes. They are involved in cell decision and many biological processes such as replication, transcription, repair, etc. In this work, the secondary structure of the HMGB1 and HMGB2 proteins was studied by the circular dichroism method. It was shown that, despite the high homology between them, the secondary structure of these proteins is different. The revealed structural features, most likely, should influence their functions in the cell nucleus, in particular, the interaction with DNA and other proteins.

Proteome Analysis of Condensed Barley Mitotic Chromosomes

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

SetDB1 and Su(var)3-9 play non-overlapping roles in somatic cell chromosomes of Drosophila melanogaster

We explored functional roles of two H3K9-specific histone methyltransferases SetDB1 and Su(var)3-9. Using DamID approach, we generated the binding profile for SetDB1 in Drosophila salivary gland chromosomes, and matched it to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartment, and is largely restricted to TSSes and 5'UTRs of ubiquitously expressed genes. Significant SetDB1 association is also observed at insulator protein CP190 binding sites. SetDB1 and H3K9me2/3-enriched sites tend to display poor overlap. At the same time, SetDB1 has clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, we speculate that in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains.

Different Proteins Mediate Step-wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

PICH function is required for organization of SUMOylated proteins on mitotic chromosomes

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by abnormal distribution of genetic information. Polo-like kinase 1 interacting checkpoint helicase (PICH) is a DNA translocase essential in chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by Small Ubiquitin-like modifier (SUMO). However, it is unclear how these activities are cooperating to resolve chromosome bridges. Here, we show that PICH specifically promotes the organization of SUMOylated proteins like SUMOylated TopoisomeraseIIα (TopoIIα) on mitotic chromosomes. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in the retention of SUMOylated chromosomal proteins, including TopoIIα, indicating that PICH functions to control proper association of these proteins with chromosomes. Replacement of PICH with its mutants showed that PICH is required for the proper organization of SUMOylated proteins on chromosomes. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taken together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.Summary StatementPolo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH controls association of SUMOylated chromosomal proteins, including Topoisomerase IIα, and that function requires PICH translocase activity and SUMO binding ability.

Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging. In the following report, we describe the complete and easy protocol for the localization of proteins to the male meiotic spindle and male meiotic chromosomes. The protocol is fast, improved, and robust without the use of any harsh enzymes.