On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is ‘translated’ to the particular ‘activating’ or ‘repressing’ chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content – such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a ‘polymer’ of individual ‘canonical’ nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions

Comparison of the Isw1a, Isw1b, and Isw2 Nucleosome Disrupting Activities

The three Saccharomyces cerevisiae ISWI chromatin remodeling complexes, Isw1a, Isw1b, and Isw2, are implicated in the regularization of arrayed nucleosomes and regulation of gene activity. Although Isw1a and Isw1b are based on the same catalytic unit, in general, their functions in vivo do not overlap. To better understand the structural consequences of these complexes, we compared the putative nucleosome disrupting activities of the purified Isw1a, Isw1b, and Isw2. To account for the putative effects of nucleosomal environment, we employed reconstituted dinucleosomes in which the histone octamers were specifically positioned by the 146 base pair high-affinity nucleosome sequence “601”. We have compared the MNase and deoxyribonuclease I protection patterns of remodeled nucleosome templates and evaluated the nucleosome destabilizing abilities of the Isw1a/b and Isw2 using restriction endonucleases. Although the Isw2 showed little evidence of nucleosome disassembly, the Isw1b remodeled dinucleosomes exhibited some common features with the ySwi–Snf remodeling products. The nuclease digestion data suggest that Isw1a can also promote ATP-dependent distortion of nucleosome structure, although less efficiently than the Isw1b complex

Comparison of the Isw1a, Isw1b, and Isw2 Nucleosome Disrupting Activities

The three Saccharomyces cerevisiae ISWI chromatin remodeling complexes, Isw1a, Isw1b, and Isw2, are implicated in the regularization of arrayed nucleosomes and regulation of gene activity. Although Isw1a and Isw1b are based on the same catalytic unit, in general, their functions in vivo do not overlap. To better understand the structural consequences of these complexes, we compared the putative nucleosome disrupting activities of the purified Isw1a, Isw1b, and Isw2. To account for the putative effects of nucleosomal environment, we employed reconstituted dinucleosomes in which the histone octamers were specifically positioned by the 146 base pair high-affinity nucleosome sequence “601”. We have compared the MNase and deoxyribonuclease I protection patterns of remodeled nucleosome templates and evaluated the nucleosome destabilizing abilities of the Isw1a/b and Isw2 using restriction endonucleases. Although the Isw2 showed little evidence of nucleosome disassembly, the Isw1b remodeled dinucleosomes exhibited some common features with the ySwi–Snf remodeling products. The nuclease digestion data suggest that Isw1a can also promote ATP-dependent distortion of nucleosome structure, although less efficiently than the Isw1b complex

SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

The trithorax (trxG) and Polycomb (PcG) group proteins recognize and propagate inheritable patterns of gene expression through a poorly understood epigenetic mechanism. A distinguishing feature of these proteins is the presence of a 130-amino-acid methyltransferase domain (SET), which catalyzes the methylation of histones. It is still not clear how SET proteins distinguish gene expression states, how they are targeted, or what regulates their substrate specificity. Many SET domain-containing proteins show robust activity on core histones but relatively weak activity on intact nucleosomes, their physiological substrate. Here, we examined the binding of two SET domain-containing proteins, ALL1 and SET7, to chromatin substrates. The SET domains from these proteins bind and methylate intact nucleosomes poorly but can recognize disrupted nucleosomal structures associated with transcribed chromatin. Interestingly, the remodeling of dinucleosomes by the ISWI class of ATP-dependent chromatin remodeling enzymes stimulated the binding of SET domains to chromatin and the methylation of H3 within the nucleosome. Unexpectedly, dinucleosomes remodeled by SWI/SNF were poor substrates. Thus, SET domains can distinguish nucleosomes altered by these two classes of remodeling enzymes. Our study reveals novel insights into the mechanism of how SET domains recognize different chromatin states and specify histone methylation at active loci

Remodeling of Nucleosome-Dimer Particles with yIsw2 Promotes Their Association with ALL-1 SET Domain in Vitro

Functioning of histone lysine methyltransferases (HKMTs) involves interactions of their catalytic domain “SET” with the N-termini of histone H3. However, these interactions are restricted in canonical nucleosomes due to the limited accessibility of H3 termini. Here we investigated whether nucleosome remodeling with the yeast Isw2 affects nucleosome affinity to the SET domain of ALL-1 HKMT. Reconstitution of mononucleosomes by salt dilutions also produces some nucleosome-dimer particles (self-associated mononucleosomes, described by: Tatchell and van Holde (1977) <i>Biochemistry</i>, <i>16</i>, 5295–5303). The GST-tagged SET-domain polypeptide of ALL-1 was assayed for binding to assembled mononucleosomes and nucleosome-dimer particles, either intact or remodeled with purified yeast Isw2. Remodeling of mononucleosomes does not noticeably affect their affinity to SET domain; however, yIsw2 remodeling of nucleosome-dimer particles facilitated their association with GST-SET polypeptide. Therefore, it is conceivable that nucleosome interactions in trans could be implicated in the maintenance of chromatin methylation patterns in vivo

A Motif within SET-Domain Proteins Binds Single-Stranded Nucleic Acids and Transcribed and Supercoiled DNAs and Can Interfere with Assembly of Nucleosomes

The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trx(Z11) homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription

Remodeling of Nucleosome-Dimer Particles with yIsw2 Promotes Their Association with ALL-1 SET Domain in Vitro

Functioning of histone lysine methyltransferases (HKMTs) involves interactions of their catalytic domain “SET” with the N-termini of histone H3. However, these interactions are restricted in canonical nucleosomes due to the limited accessibility of H3 termini. Here we investigated whether nucleosome remodeling with the yeast Isw2 affects nucleosome affinity to the SET domain of ALL-1 HKMT. Reconstitution of mononucleosomes by salt dilutions also produces some nucleosome-dimer particles (self-associated mononucleosomes, described by: Tatchell and van Holde (1977) <i>Biochemistry</i>, <i>16</i>, 5295–5303). The GST-tagged SET-domain polypeptide of ALL-1 was assayed for binding to assembled mononucleosomes and nucleosome-dimer particles, either intact or remodeled with purified yeast Isw2. Remodeling of mononucleosomes does not noticeably affect their affinity to SET domain; however, yIsw2 remodeling of nucleosome-dimer particles facilitated their association with GST-SET polypeptide. Therefore, it is conceivable that nucleosome interactions in trans could be implicated in the maintenance of chromatin methylation patterns in vivo