A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

Cytogenetic markers using single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes from tandemly repetitive 5S ribosomal RNA (rRNA) and core histone coding sequences to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the 5S rDNA probe was on chromosome 5, whereas that of core histone probe was on chromosome 8. The sequence of the 5S rDNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rDNA. This is the first report of a tandemly repetitive linkage of snRNA and 5S rDNA sequences in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

Single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa: a potential tool for karyotyping

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes for tandemly repetitive 5S ribosomal RNA (rRNA) and core histone genes to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the core histone gene was on chromosome 8, whereas that of 5S rRNA gene was on chromosome 5. The sequence of the 5S rRNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rRNA gene. This is the first report of a tandemly repetitive linkage of snRNA and 5S rRNA genes in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

Phosphorylation of the Canonical Histone H2A Marks Foci of Damaged DNA in Malaria Parasites

ABSTRACT Plasmodium falciparum parasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage; therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate under such conditions. In higher eukaryotes, double-strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X, which marks the site of damaged DNA. We show that in P. falciparum that lacks the H2A.X variant, the canonical P. falciparum H2A (PfH2A) is phosphorylated on serine 121 upon exposure to sources of DNA damage. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the nonphosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and that over time, as the parasite activates the repair machinery, this phosphorylation is removed. Finally, we demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay in P. falciparum. IMPORTANCE Plasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the bloodstream and begins proliferating inside red blood cells, where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such conditions are also the driving force for acquiring genome plasticity that enables them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied for these parasites. The paper addresses our recent discovery that P. falciparum that lacks the histone variant H2A.X phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms.

Nucleosome structural variations in interphase and metaphase chromosomes

SummaryStructural heterogeneity of nucleosomes in functional chromosomes is unknown. Here we report cryo-EM structures of nucleosomes isolated from interphase and metaphase chromosomes at up to 3.4 Å resolution. Averaged chromosomal nucleosome structures are highly similar to canonical left-handed recombinant nucleosome crystal structures, with DNA being selectively stabilized at two defined locations. Compared to free mono-nucleosomes, which exhibit diverse linker DNA angles and large structural variations in H3 and H4, chromosomal nucleosome structures are much more uniform, characterized by a closed linker DNA angle with interactions between the H2A C-terminal tail and DNA. Exclusively for metaphase nucleosomes, structures of the linker histone H1.8 at the on-dyad position of nucleosomes can be reconstituted at 4.4 Å resolution. We also report diverse minor nucleosome structural variants with rearranged core histone configurations, which are more prevalent in metaphase than in interphase chromosomes. This study presents structural characteristics of nucleosomes in interphase and mitotic chromosomes.Highlights3.4~ Å resolution nucleosome structures from interphase and metaphase chromosomesNucleosome structures in chromosomes are more uniform than in free mono-nucleosomesHistone H1.8 binds to the nucleosome dyad axis in metaphase chromosomesNucleosome structural variants are more prevalent in metaphase than in interphaseNOTES TO READERSWe would like to emphasize the importance of supplemental movies S1-S3, which should greatly help readers to understand characteristics of the nucleosome structural variants that we report in this study.

Phosphorylation of the canonical histone H2A marks foci of damaged DNA in malaria parasites

Plasmodium falciparum parasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage, therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate in such conditions. In higher eukaryotes, double strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X which marks the site of damaged DNA. We show that in P. falciparum that lacks the H2A.X variant, the canonical PfH2A is phosphorylated on serine 121 upon exposure to sources of DNA damage in a dose dependent manner. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the non-phosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and as the parasite repairs its DNA over time, this phosphorylation is removed. We also demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay in P. falciparum.ImportancePlasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the blood stream and begins proliferating inside red blood cells where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such condition are also the driving force for acquiring genome plasticity that enable them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied in these parasites. The paper addresses our recent discovery, that P. falciparum that lacks the histone variant H2A.X, phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms.

Plant Histone HTB (H2B) Variants in Regulating Chromatin Structure and Function

Besides chemical modification of histone proteins, chromatin dynamics can be modulated by histone variants. Most organisms possess multiple genes encoding for core histone proteins, which are highly similar in amino acid sequence. The Arabidopsis thaliana genome contains 11 genes encoding for histone H2B (HTBs), 13 for H2A (HTAs), 15 for H3 (HTRs), and 8 genes encoding for histone H4 (HFOs). The finding that histone variants may be expressed in specific tissues and/or during specific developmental stages, often displaying specific nuclear localization and involvement in specific nuclear processes suggests that histone variants have evolved to carry out specific functions in regulating chromatin structure and function and might be important for better understanding of growth and development and particularly the response to stress. In this review, we will elaborate on a group of core histone proteins in Arabidopsis, namely histone H2B, summarize existing data, and illuminate the potential function of H2B variants in regulating chromatin structure and function in Arabidopsis thaliana.

Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.