In Silico Insight into Nucleosome Repositioning Via Oxygen Delivery and Extracellular Movement


 OBJECTIVEHistones and resulting nucleosomes occur within DNA regulating gene expression by slowing, pausing, or halting transcriptional machinery. Positions within the genome have been found with higher affinity for the histone octamer than others. Histone/nucleosome repositioning is adjusted via energy dependent remodeling complexes, and a harmonizing array of constellation proteins and molecules. The energy required to create transcriptional environments is created through oxygen intake, nutrient presence, and extracellular movement. In this paper we aim to help facilitate an in silico framework for further experimentation into how partial pressures of oxygen and other gases impact genetic transcription along with extracellular movement and nutrient delivery.RESULTSCell and tissue culture experimentation with biomechanical strain and variable partial pressures of oxygen and other gases can be made into the expression levels of genes such as PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), and Neuroligin 1 (NLGN1). These genes show in silico to have a higher affinity for a histone octamer binding motif, needing adequate cellular energy to be expressed. Extracellular movement and adequate cellular oxygenation are required to properly reposition nucleosome sequences for transcription.


Introduction
Nucleosomes are known to in uence transcriptional regulation via presence and a nity of the underlying DNA-histone interactions. [1][2][3] Nucleosome repositioning is required for gene expression, either by inherent characteristics of RNA Polymerase or ATP-dependent remodeling complexes. 4 In vivo, repositioning will occur to occupy high a nity regions, in uencing neighboring nucleosomes via linker DNA. 5 Oxygen intake and extracellular muscle movement is required for su cient levels of intracellular ATP. 6,7 Chromatin structure is achieved by a harmonizing array of DNA-protein interactions and DNA shape. [8][9][10][11] The nucleosome core particle has sequence a nities proven in vitro. 12,13 When reintroducing PHLPP protein into tumorigenic cells, tumor growth was reduced by approximately 50% 20 . The identi cation of NLGN1 expression as part of regulating the dynamic processes of memory consolidation and strengthening 23 , along with the antitumor properties of PHLPP1 spurred this study. Possible mechanistic insights found from further experimentation may help discover novel therapies and research opportunities.

Methods And Procedures
Simple Python scripts utilizing regular expressions were used to compare the 1998 Lowary and Widom 12 consensus sequence with 82bp segments of hg38 from the National Center for Biotechnology Information. A pattern of 8 "ATC or G" nucleotides between a group of "TA" nucleotide sequences with anking "ATC or G" nucleotide sequence were discovered and then processed to be scored against the Lowary and Widom consensus sequence. The pattern is noted in Table 1, where bolded and underlined nucleotides are the "TA" discrete pattern used, and all other nucleotides are "\w" (any letter) characters in regular expression notation. Scoring criteria is as follows according to Lowary and Widom 12 : +1 for a match with a higher identity character, + .5 for a match to a lower identity character, and + 0 for all others. Scored sequences were joined into one le and sorted in descending order by score to determine a liturgical highest a nity to the histone octamer ranking. Resulting genes of interest were input into the Interactive Genomics Viewer from the Broad Institute to determine the viewing of genetic locations; gene information was obtained from GeneCards and UniProt. A cysteine-aspartic acid protease that plays a central role in the execution-phase of cell apoptosis. The encoded protein cleaves and inactivates poly(ADP-ribose) polymerase while it cleaves and activates sterol regulatory element binding proteins as well as caspases 6, 7, and 9. This protein itself is processed by caspases 8, 9, and 10. It is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease.

TACAAGCAAAATACCATGGATAGCTGATTTTAGTTGATTCTATAAATTATTATGTAGTCATAAAGGCAAAAATAAGCATAAA STATISTICAL SIGNIFICANCE
Finding one of these sequences, a 5x TA dinucleotide repeat motif with a 20bp anking sequence for consensus analysis, is 1 in 6,533,600 through standard statistical probability in random chance. This probability results in approximately 460 total probable sequences to be found in the human genome with a purported histone binding motif per the Lowary and Widom experimentations 12 .
There are 41,158 sequences like this found, resulting in an NP-hard problem and also a statistically enriched sequence by sheer observation. These results show the top 9, or top 2% most likely to be bound within a histone octamer from the Lowary and Widom 12 experiments, scored as noted.

Discussion
Histones, or nucleosomal formations must be changed in different cellular environments for different environmental outputs.
Optimally, genes involved with tumor suppression would necessitate tumor suppressing genes to be expressed in a cancerous state.
These data show that tumor suppressing genes may have an a nity for histone binding; needing adequate levels of ATP or other metabolic aids to functionally remodel or encode through the nucleosome complexes potentially present, either through added oxygenation of the cell, or other functional metabolic constituents added through biomechanical strain and adequate blood ow.
These data can predict that each cellular environment requires different chromatin structures, most likely dependent on local ATP availability. This differential in ATP availability may be a strong in uencing factor into the nature and general expression of different cell or tissue types. The aim of these data and research is to hopefully inspire new ndings and possible experimentation into the properties of ATP and other metabolic molecular availability for various cell and tissue types relating to histones and transcription.
Different types of tissue and cell types have varying levels of extracellular movement and resulting blood ow. Bone marrow and ngertip cell types would undoubtedly have different levels of biomechanical stress, resulting blood ow, and extracellular movement. A hypothesized difference in chromatin structure and a resulting different genetic expression pro le would likely follow, as most would understand is unanimous in the literature and in vivo.
Standard air is comprised of Oxygen, Nitrogen, and Argon among other trace gases. Once standard air is deposited within the body, various biological processes would alter the concentrations of these compounds to be available to different cell types. Notably, oxygen through the properties of red blood cells, will oxygenate cells and tissues through various levels of extracellular movement. In standard cell and tissue cultures, pH is a dominant factor in the proliferation and differentiation of cell types, which is a main perspective of this research with other gas types. Different partial pressures and concentrations of standard air gases may change individual cell type environments. How does intracellular ATP affect transcription and translation? Results PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), a gene which is established to suppress tumors [14][15][16][17][18][19][20] contains the highest computationally scored sequence match to the Lowary and Widom histone octamer binding consensus sequence 12 , shown in Table 1 and Fig. 1. This is a sequence proposed to have the highest a nity for the histone octamer within the genome. ATP has been shown to impact mRNA transcription in vitro. 21,22 Su cient levels of ATP may allow greater expression of PHLPP1 and other genes in Table 1.

Critical Discussion
Researchers may look at the genes in Table 1 to analyze in the eld of cellular stretching, biomechanical strain, oxygenation, aspiration, and supplemented blood ow to analyze the hypothesized increase in expression from the highlighted association with a nity for histone binding.
Many experiments can be made from these observations, providing cell cultures of different types to different levels of cell stress, along with partial pressures of oxygen and other gases. One can infer from these in silico experiments that both higher levels of extracellular movement, and partial pressures of oxygen or other atmospheric gases would induce higher levels of expression for the genes in Table 1, and others with similar binding motifs. These higher levels of expression can potentially elucidate novel treatments for varying states of the cell. Extracellular movement, experimentally provided by cellular stretching, is a proposed mechanism for enhancing the blood ow, oxygenation, and nutritional delivery to different types of cell. This research proposes that increased overall absorption of available extracellularly available molecules would impact genetic expression, and potentially be a target for novel methods of therapy for varying cellular phenomena.

Conclusion
In silico Genome analysis, along with RNA-Seq is undoubtedly a useful tool in proposing further in vitro and in vivo experimentation into these results. Computational histone binding motif searches of the human genome as shown in this research provides an adequate base to begin projects examining expression levels from discovered genes of interest, notably PHLPP1, a tumor suppressing gene. The enumeration of these expression levels may lay a foundation for future treatments for various cellular environments that require extracellular nutrient delivery in vivo.

Consent for publication
Not applicable.

Data Availability
The datasets generated and/or analyzed during the current study are available in the NCBI RefSeq repository, https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/.