Evolution of histone 2A for chromatin compaction in eukaryotes.

During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer

Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells

Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets

The Evolutionary Roles of H2A and H2B in Genome Compaction in Eukaryotes

As eukaryotes have evolved from simple, unicellular organisms to more complex multicellular species, both the genome size and the nuclear volume have increased. However, the rates of increase of these two parameters have not been equal with genome size increasing much faster than the volume of the nucleus. This disproportionality has necessitated an increase in chromatin compaction for larger genome organisms. Although histones, through formation of the nucleosome, are the basic building blocks of chromatin, it is unknown whether histones have evolved to facilitate these higher compaction ratios. Analysis of histone sequences from 160 organisms representing all eukaryotic kingdoms revealed that there are significant changes in both the H2A N-terminus and H2B C-terminus sequences that have systematically evolved as genomes expanded. In the H2A N-terminus, larger genome species have acquired more arginines, and in the H2B C-terminus, lysines. In parallel with acquisition of positively charged residues, both H2A and H2B have lost polar residues. To determine whether these evolutionary sequence changes contribute to increased genome compaction, a series of in vivo and in vitro molecular biological and biochemical experiments were carried out using both budding yeast and human cell lines as model systems. Insertion of precisely positioned arginines into the H2A N-terminus of a small-genome organism substantially increased chromatin compaction while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated using in vitro assembled nucleosomal arrays with unmodified histones, indicating that H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter compaction. Insertions of lysines into the H2B C-terminus of a small genome organism also increased compaction. Combinations of H2A-H2B sequence changes revealed that chromatin compaction is enhanced as compared to single histone mediated compaction and that the H2A N-terminus and H2B C-terminus work additively. An area physically near the H2B C-terminus in the nucleosomal structure, which I have termed the ABC Domain (H2A/H2B Compaction Domain), accounts for the majority of the chromatin compaction mediated by H2A and H2B. These findings reveal a simple evolutionary mechanism for regulation of chromatin compaction that has enabled organisms with larger genome achieve higher compaction ratios

Evolution of histone 2A for chromatin compaction in eukaryotes.

During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer