Nucleosome positioning sequence patterns as packing or regulatory

Nucleosome positioning DNA sequence patterns (NPS)—usually distributions of particular dinucleotides or other sequence elements in nucleosomal DNA—at least partially determine chromatin structure and arrangements of nucleosomes that in turn affect gene expression. Statistically, NPS are defined as oscillations of the dinucleotide periodicity of about 10 base pairs (bp) which reflects the double helix period. We compared the nucleosomal DNA patterns in mouse, human and yeast organisms and observed few distinctive patterns that can be termed as packing and regulatory referring to distinctive modes of chromatin function. For the first time the NPS patterns in nucleus accumbens cells (NAC) in mouse brain were characterized and compared to the patterns in human CD4+ and apoptotic lymphocyte cells and well studied patterns in yeast. The NPS patterns in human CD4+ cells and mouse brain cells had very high positive correlation. However, there was no correlation between them and patterns in human apoptotic lymphocyte cells and yeast, but the latter two were highly correlated with each other. By their dinucleotide arrangements the analyzed NPS patterns classified into stable canonical WW/SS (W = A or T and S = C or G dinucleotide) and less stable RR/YY (R = A or G and Y = C or T dinucleotide) patterns and anti-patterns. In the anti-patterns positioning of the dinucleotides is flipped compared to those in the regular patterns. Stable canonical WW/SS patterns and anti-patterns are ubiquitously observed in many organisms and they had high resemblance between yeast and human apoptotic cells. Less stable RR/YY patterns had higher positive correlation between mouse and normal human cells. Our analysis and evidence from scientific literature lead to idea that various distinct patterns in nucleosomal DNA can be related to the two roles of the chromatin: packing (WW/SS) and regulatory (RR/YY and “anti”).

1 Dinucleotide frequency profiles across various conditions 1

.1 Dinucleotide patterns in human and mouse
Similarities between dinucleotide profiles across conditions in human and mouse are prominent in WW ( Figure 1a) and SS (Figure 1b) patterns in corresponding peaks and valleys. Consistently, the peaks in the WW profiles are in phase for human CD4+ and mouse cells. Most of the WW versus SS peaks and most of the RR versus YY peaks are off phase in both human and mouse cells.      2 Synchronicity of peaks and positional preferences

Peak alignment in nucleosomal DNA patterns across various conditions
Dinucleotide peaks in nucleosomal DNA appear in specific locations that are synchronous in human and mouse organism and in different conditions as illustrated in Figure 6. The peaks in general tend to co-occur at certain distances. Frequency distribution of distances between the co-occurring peaks showed that peaks mostly co-occur at intervals separated by a step of 10 +/-1 base pairs. A dendrogram on the left represents patterns grouped by Euclidean distance and clearly represents coinciding maxima and minima across conditions. Dinucleotide frequency patterns in nucleosomal DNA are centered on a dyad. The labels on the right cd4 and apo corresponds to human CD4+ and apoptotic lymphocyte cells; the con, sus and res correspond to mouse NAC in control, susceptible and resilient to stress conditions.

Positional preferences of dinucleotides
A frequency of peaks simultaneously occurring in each position in all conditions is shown in Figure 7. For some pairs of the dinucleotides their maximums occurr together only in human or only in mouse. Figure 8 shows an order of positions that were used more than eight times by different dinucleotide classes. The hub positions occurr at ±10, 11 base pair steps around the dyad. Positional preferences elucidated in this study are summarized in Table 1

Importance of Superhelical Locations
Statistically defined relations between nucleosome positioning and covalent chromatin modification were noted -but it is not clear yet how specific composition of DNA sequence might interact with these processes and affect nucleosome positioning and remodelling. In the following we briefly summarized current knowledge about importance of specific SHL zones trying to provide more context to the regularities observed in this work.
In this study we observed multiple peaks from all organisms in SHL zones ±2 , ±3 and ±4. The SHL ±2, ±3 are zones in which chromatin remodelers interact with DNA and in SHL ±4 zone histone H2A tail interacts with DNA which makes it a less stable zone. Peak distributions across SHLs were different in mouse compared to human in SHL zones ±1.5, 4 and ±4.5 which also play roles in nucleosome dynamics. Most of DNA deformations take place in the SHL±1.5 zone. It has an increased binding of DNA proteins and it is most susceptible to DNA damage.

Nucleosome stability
The H3-H4 tetramer is the most stable part of the formed nucleosome. The H2A and H2B are more mobile and H1 is very dynamic. Main histone tails occur in minor grooves [3]. The nucleosomes can adopt multiple conformations providing a structural basis for nucleosome plasticity which in turn may contribute to chromatin regulation in many processes [4]. The SHL ±1.5, 2.5 and SHL ±6, 7 are the most active zones in which DNA shows the largest movements between the structures and H3 alpha helices show the largest rearrangements. Nucleosomes wrap and unwrap spontaneously interchanging between a 250ms in a compacted state 10-50 ms in an unwrapped state where the unwrapping occurs from the ends of the nucleosome [5]. The out-of-planar bending (by SHLs ±3.5 and ±4.5) and in-planar rolling (by SHLs ±6 and ±6.5) motions of the super helical DNA in general are responsible for the breathing motionwrapping and unwrapping of nucleosome [6].

Histone-DNA interactions
The greatest numbers of interactions between histone core and DNA are located at the central bound DNA region, SHLs ±0.5 at the dyad axis. The least number of interactions are found at the entry-exit regions in SHLs ±5.5 and ±6.5. Other SHLs have an intermediate number of direct hydrogen bonds [7]. Pressure points, are very important for nucleosome stability [8]. The SHL ±2.5 zone has increased unwrapping because of the acetylation of H4 residues and because histone H2B N-terminal extension makes numerous contacts with DNA at that site. Both, H2A/H2B and H3/H4 contact the DNA via their α1 − α1 helices and two L1-L2 loops, with histone H2A/H2B binding to superhelical locations ±3 to ±6. The histone H2A tail exits adjacent to the minor groove at SHL ±4-4.5 and residues in this helix contact the DNA. Histone H2B N-terminal extension makes numerous contacts with both DNA coils at SHL ±4.5 − 5 and SHL ±2.5 − 3 [9]. The DNA deformations predominantly occur around the sites SHL ±1.5 and ±2 (in the central part of nucleosomal DNA interacting with the H3/H4 tetramer), and at the sites SHL ±4.5 (interacting with the H2B histones) [1]. The sites at SHL ±1.5 and ±4.5 are sites where the helix is stretched and it is more reactive to DNA damaging agents [10]. The DNA superhelical location (SHL) ±1.5 is a known hot spot for DNA binding/damaging molecules. Energetically significant interaction stable sites are ±3 and less stable sites are ±4 and ±5 where H2A/H2B bind to the DNA.

Remodeling and post-translational modifications
In addition to DNA sequence other mechanisms modulate nucleosome stability and dynamics. One involves chemically altering the histones themselves and greatly increases the DNA accessibility. These changes can be post-translational modifications (PTMs) the best-studied of which are acetylation, methylation, phosphorylation, ubiquitynation, and ADP-ribosylation of which acetylation of histone tails destabilize nucleosomes. Increased DNA unwrapping from the nucleosome happens with acetylation of two residues, H4(K77) and K4(K79) near superhelical location (SHL) ±2.5. A second mechanism influencing nucleosome stability and dynamics is through remodelling factors that must transiently disrupt DNA protein contacts [2]. Remodelers are grouped into INO80, SWI/SNF, CHD and ISWI families that collectively shape the nucleosome landscape [11]. INO80 binds into SHL -6 and disrupts H2A/H2B DNA contacts. Arp5-Ies6 binds about 7-8 bases at SHL-2/-3. The ISWI and SWI/SNF remodelling enzymes engage nucleosomal DNA at SHL +/-2 [12] at which a detachment of DNA could be achieved inside the nucleosomal structure near the dyad axis. Regions with active H3K4 H3K9 marks are characterized by lower nucleosome density, while inactive chromatin marks such as methylation H3K9 H3K27 are characterized by higher nucleosome density [13].
All these observations taken together show that all SHLs play important role in processes regulating nucleosome stability. A better understanding of how specific dinucleotides at certain SHL may affect these processes is a subject of current and future research.