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2020 Sodium Butyrate: A study on a commonly prescribed HDACi on chromatin structure Alana Helen Chang

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS & SCIENCES

SODIUM BUTYRATE: A STUDY ON A COMMONLY

PRESCRIBED HDACI ON CHROMATIN STRUCTURE

ALANA HELEN CHANG

A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded: Spring, 2020

The members of the Defense Committee approve the thesis of Alana Helen Chang’s defended on November 24, 2020

Dr. Jonathan Dennis Thesis Director

Dr. Debra Ann Fadool Outside Committee Member

Dr. Justin G. Kennemur Committee Member

Table of Contents

Abstract 2

Abbreviation List 3-4

Introduction 5-9

Materials and Methods 10-14

Results 15-16

Future Directions and Discussion 17-18

Figures 19-27

References 28-31

Abac Butyric acid sodium salt (sodium-butyrate; NaBu), is a powerful anti-psychotic noteworthy for its anti-inflammatory and cancer properties. NaBu is produced naturally in the human gut as a by-product of insoluble fiber digestion by bacteria in the phyla Firmicutes and Fusobacteria in concentrations upwards of 10-20 mM (Nepelska et al., 2012; Jiminez et al., 2017). High concentrations of any substance are likely to cause widespread changes in chromatin structure, exhibited through a genome-wide chromatin remodeling event. As a strong histone deacetylase inhibitor (HDACi), NaBu alters the underlying histone code in the cell causing an inappropriate chromatin remodeling response (Davie, 2003). As an anti-inflammatory NaBu inhibits the Nuclear factor-B (NF-B) pathway preventing stimuli such as lipopolysaccharide (LPS), found in gram-negative bacteria, from triggering an immune response (Lee et al., 2017). This inhibitory mechanism is explored in my thesis by exposing HCT116 Colorectal Epithelial Cancer cells to NaBu, followed by LPS to determine what structural and biochemical changes in chromatin structure are produced by NaBu inflammatory response inhibition. NaBu’s ability to alter gene expression, coupled with its high concentration in the human gut, may cause the downregulation of the effects of LPS signaling through Toll-like receptor 4 (TLR4) receptors, thus decreasing the inflammatory response in HCT116 cells. It is expected that nucleosomal sensitivity to micrococcal nuclease (MNase) will decrease in TLR4 receptor-pathway related genes caused by NaBu inflammatory response inhibition of LPS. The role of NaBu as an inhibitor of inflammation through the diversification of gut bacterial flora and introduction of a high-fiber diet could potentially benefit inflammatory bowel disease (IBD) and Crohn’s disease patients that are afflicted with high instances of colorectal inflammation. This natural option is a promising alternative to other drug therapies with unknown predictability on gene expression.

Abbreviations List

Term Abbreviation

Histone deacetylase inhibitor HDACi

Histone deacetylase HDAC

Lipopolysaccharide LPS

Sodium butyrate NaBu

Nuclear factor-B NF-B

Toll-like receptor 4 TLR4

Micrococcal Nuclease MNase

Inflammatory bowel disease IBD

Genomic transient intermediate state GTIS

RNA polymerase II RNA Pol II

GATA binding protein 1 GATA-1

Tumor protein p53 P53

Tumor protein p21 P21

Short Chain Fatty Acids SCFA

Cardiac-derived mesenchymal stromal cells CMC

Fetal bovine calf serum FBS

Penicillin-streptomycin-glutamine PSG

Dulbecco’s modified eagle medium DMEM

Toll/interleukin-1 receptor domain-containing TIRAP adapter protein

Tumor necrosis factor receptor associated TRAF6 factor 6

Myeloid differentiation primary response MYD88 protein

Solid phase reversible immobilization SPRI

Polyethylene glycol PEG

Guanidinium hydrochloride GuHCl

Nucleus Isolation Buffer NIB

Ethylenediaminetetraacetic acid EDTA

Sodium dodecyl sulfate SDS

Tris-EDTA TE

Ethanol EtOH

5-hydroxytryptamine 5-HT

Introduction

Chromatin Structure and Nucleosome Positioning

DNA is packaged in the nucleus and regulated based on chromatin structure in eukaryotic cells, with nucleosomes as its most fundamental components. Nucleosomes are composed of ~150 bp of negatively charged, double-stranded DNA wrapped ~1.5 times around a positively charged histone octamer protein (Sexton et al., 2016). Nucleosomes allow the cell to both organize compacted DNA measuring roughly 2 meters in length and averaging ~6.8 billion base pairs per cell (Davie, 2003). Genetic regions composed of tightly wound DNA around a histone can be inaccessible to most transcription and regulatory factors resulting in a decreased potential of gene accessibility. Conversely, unwound regions of linker DNA readily accessible to most transcription and regulatory factors allow an increased potential for gene regulation. Nucleosomes accordingly act as transcriptional regulatory factors of genetic material within the cell by exposing or shielding DNA, affecting the overall genetic potential of genes within the genome. Due to high variation in gene expression for different phenotypes, it was previously hypothesized nucleosome architecture was unique to each cell type. However, findings from the Dennis Laboratory demonstrate that nucleosome positioning is largely universal across all human cell types studied thus far (Sexton et al., 2016).

Currently the disconnect between observed uniform genomic structure and extensive phenotypic variation amongst various eukaryotic cell types is poorly understood. The Dennis Lab addresses this disconnect by exposing various cell types to stimuli and mapping their nucleosome distribution during a genomic response at high temporal resolution. It’s been found that nucleosome remodeling, in response to a stimulus, is both transient and widespread; denoted by the Genomic Transient Intermediate State (GTIS) or Chromatin Remodeling Event. The Dennis Lab is the first to describe this GTIS defined as: a widespread and transient remodeling event coupled with altered regulatory factor binding, favoring conditions for the nucleus to suitably respond to developmental or environmental changes (Sexton et al., 2016).

Nucleosomal positioning is determined by cis-acting elements and trans-acting factors, with the former referring to the underlying DNA sequence determining both basal and transient positions and the latter involving regulatory proteins such as ATP-dependent chromatin remodelers and transcription factors that alter chromatin structure (Reuveni et al., 2018). Cis-acting elements affect nucleosome positioning through intermittent AA/AT/TT/TA dinucleotide sequences that create sharp bends in the DNA double helix. These sharp bends produce favorable regions of the genome for wrapping around histone octamers. Trans-acting factors affect nucleosomal contribution to gene regulation by influencing the translational positioning of nucleosomes and include regulatory proteins such as chromatin remodelers and transcription factors like RNA Pol II (Wright & Cui, 2017).

Nucleosomal Sensitivity and the Genomic Transient Intermediate State

Once a GTIS is triggered by a stimulus, nucleosomes move transiently and return to their basal position position by trans-acting factors with biochemically altered sensitivity to nucleases (Sexton et al., 2016). Nucleosome sensitivity is measured by MNase titration in which chromatin is exposed to varying concentrations of MNase, with previously remodeled nucleosomes requiring lighter concentrations to release from adjacent nucleosomes compared to those which remained at basal position requiring heavier concentrations. Nucleosome changes in nuclease sensitivity can be tracked by observing nucleosome occupancy profiles after MNase digestion (Mieczkowski et al., 2016). In addition to recently remodeled nucleosomes participating in a GTIS showing a significantly higher signal under light MNase digestion; areas of the genome associated with highly transcribed genes that have undergone altered chromatin conformation show high sensitivity to MNase cleavage, specifically hypersensitive nucleosomes located at promoter regions. The presence of nucleosomes at these promoter regions indicates their crucial role in regulating transcription factor binding at these sites (Struhl & Segal, 2013). This further complicates the reliance on genome-wide nucleosomes occupancy studies to determine which nucleosomes have undergone remodeling due to a GTIS vs. altered chromatin conformation. The mechanisms by which nuclease sensitivity are altered are currently unknown, but it is known that

altered nuclease sensitivity suggests a GTIS has occurred and establishes different biochemical potentials for cellular response. Therefore nuclease sensitivity can be used as a tool to explore cellular predispositions to nucleosome remodeling and predict possible changes in gene expression. Micrococcal nuclease (MNase) is a favored enzyme to unearth these cellular predispositions as it preferentially digests linker DNA and releases nucleosomally protected fragments from adjacent chromatin segments and MNase concentrations needed to release these fragments can be easily identified using MNase titrations.

Sodium butyrate as an anti-inflammatory and histone deacetylase inhibitor (HDACi)

Histone deacetylase inhibitors (HDACi) are a class of chemical compounds that act to inhibit histone deacetylases (HDACs) by preventing the removal of acetyl groups from histone octamers, increasing acetylation of histones and resulting in an overexpression of genes. NaBu inhibits Class I HDACs specifically HDAC1, HDAC2, and HDAC3 with HDAC1 involved in tumor suppression by enhancing transcription factors such as p53 and GATA-1 to induce cell apoptosis and inhibit cell proliferation in cancer cells (Condorelli et al., 2008). These mechanisms are utilized in a number of cancer therapy treatments.

NaBu is a short-chain fatty acid metabolite naturally occurring in the human gut via bacterial breakdown of long-chain fatty acids and dietary fibers in the Firmicutes and Fusobacteria phyla (Steliou et al., 2012). Firmicutes are gram-negative bacteria that are abundantly found in the human gut microbiome, with F. prausnitzii making up nearly 5% of the total gut microbiome (Chakraborti, 2015). These phyla digest dietary fiber via fermentation and produce short-chain fatty acids (SCFAs) such as NaBu along with acetate, and propionate. Synthesis of these products can be both detrimental and beneficial to human health. Reduced gut inflammation, energy sources for colonic mucosa cells, and inhibition of cell proliferation in cancer cells are attributed to propionate and NaBu. Simultaneous support of colonic mucosa cells and inhibition of cancer cell proliferation are due to the Warburg Effect in which cancer cells favor specialized fermentation over anaerobic respiration (Vander Heiden et al., 2009).

Propionate, along with Acetate, act as substrates for gluconeogenesis and lipogenesis in the liver and nearby organs. Consequently SCFAs including amines, thiols, phenols, and ammonia can build up and cause an increased risk of colorectal cancer in the absence of saccharolytic bacteria (Williams et al., 2017). NaBu provides the connection between chromatin structure and dysbiosis in IBD and Crohn’s disease patients affected by increased gut inflammation due to a lack of diverse anaerobic bacteria.Note that the same Firmicutes and Fusobacteria phyla that produce LPS responsible for inflammation, are also involved in the production of a biochemical that reduces inflammation. This further highlights the intricate nature of the human microbiome and the importance of bacterial diversity. NaBu inhibits the activity of HDAC1 bound to the Fas gene promoter in T cells resulting in the hyperacetylation of the Fas promoter and the upregulation of the Fas receptor on T cells. This in turn reduces colonic inflammation by inhibiting IFN-/STAT1 signaling pathways (Kitamura et al., 1999). SCFAs such as NaBu also affect mental health through means of vagus nerve stimulation, with increased vagal stimulation lowering instances of depression and anxiety (Pierce & Alviña, 2019).

Sodium butyrate (C3H7COO) is a sodium salt of butyric acid with a molecular weight of 110.1 g/mol and is a crystalline-solid state at ambient conditions (Cayman Chemical, n.d.). Structurally it is composed of a propyl group attached to a carboxylate (the conjugate base of a carboxylic acid) with a N a+ counter-cation. A lack of stereocenters makes this molecule achiral (National Center for Biotechnology Information, n.d.). Pharmacologically as a HDACi it works as a histamine antagonist, blocking mechanisms of histamine agonists (Figure 1).

Other studies have investigated the transcriptional regulatory effects of NaBu. It has been found NaBu inhibits HDAC1 in cardiac-derived mesenchymal stromal cells (CMCs) and induces cardiac-endothelial specific gene transcription via the tumor suppressor p53, implicating HDAC1 as a potential modulatory of CMC cell fate decisions making HDAC inhibitors helpful for cardiac cell therapies (Moore et al., 2016) . In HSC-3 and HSC-4 oral cancer cells, NaBu inhibits cancer cell proliferation in a concentration dependent manner by regulating expression of cyclin/cdk inhibitor protein p21 C i1/ p WAF1 cell cycle inhibitory genes thus indirectly resulting in

G2/M cell cycle arrest (Miki et al., 2007). In HCT116 colon epithelial cancer cells, NaBu inhibits cancerous cell proliferation by increasing H3 histone deacetylation and cyclin-dependent kinase inhibitor p21 tumor suppression. HDAC activity downregulates transcriptional activity by condensing chromatin, thus silencing tumor suppressor genes like p53 and p21 (Zeng et al., 2017). As a HDACi NaBu strongly upregulates transcriptional activity of these genes. Ultimately these findings have confirmed NaBu’s effectiveness in having a substantial effect on transcriptional activity involved in cell apoptosis of cancer cells in numerous cell types.

Lipopolysaccharide (LPS) and the inflammatory response

HCT116 Colorectal Epithelial Cells, the cell line used in this experiment, were derived from colorectal metastatic tissues obtained from a male patient (ATCC, 2020). The stimulus used, Lipopolysaccharide (LPS), is composed of an oligosaccharide core, Lipid A, and an O antigen unique to each bacterial strain (Raetz & Whitfield, 2002). LPS is found on the outer membrane of the gram-negative bacteria firmicutes and activates Toll-like receptor 4 (TLR4), a transmembrane protein responsible for the recruitment of NF-B transcription factors responsible for the widespread cytokine cascade involved in the inflammatory response via the MyD88 Dependent Pathway (Brubaker et al., 2015) (Figure 2).

The structural and biochemical changes in chromatin structure of HCT116 cells caused by NaBu inflammatory response inhibition to the LPS stimulus are observed by measuring nucleosomal repositioning and sensitivity respectively. MNase titration experiments reveal widespread changes in nucleosomal sensitivity through genome-wide nucleosome occupancy studies, and heat maps reveal altered nucleosomal distribution within the genome. Due to an expectation of nucleosomal sensitivity to decrease in nucleosomes located at promoter regions of genes involved in inflammation as a result of NaBu inflammatory response inhibition, TLR4 receptor pathway involved genes are heavily focused on in this study (Figure 2).

Materials and Methods

Cell Culture and Treatment

HCT116 human colorectal cancer cells, commonly used in tumorigenicity studies, were cultured in twenty eight 100mm plates and grown to a density of 5.0 x01 6 cells at approximately 90% confluence in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin-glutamine (PSG) antibiotics to a final concentration of 1% (Green BioResearch LLC, 2016). This cell line expresses TLR4 receptors and receptor pathways related genes including TIRAP, TRAF6, MYD88, and NFKB2. TLR4 transmembrane proteins are responsible for the recruitment of NFkB transcription factors, which in turn recruit cytokines during the inflammatory response to LPS stimulation (Doan et al., 2009). fourteen plates of cells

were grown in a 5% CO 2 incubator at 37°C and separated into 4 groups: [A] Untreated, [B] Treated with 2.5 mM Sodium Butyrate (NaBu), [C] Treated with 1.0 ug/mL Lipopolysaccharide (LPS), [D] Treated with 2.5 mM NaBu + 1.0 ug/mL LPS. Group A was left untreated. Group B was treated 2.5 mM NaBu for 12 hours. Group C was treated with 1.0 ug/mL LPS for 30 min, 1 hour, 4 hours, 12 hours, 24 hours, and 48 hours respectively. Group D was initially treated with 2.5 mM NaBu for 12 hours followed by 1.0 ug/mL LPS for 30 min, 1 hours, 4 hours, 12 hours, 24 hours, and 48 hours respectively. This procedure was repeated once more for the other 14 plates and denoted by a * next to each group A = control, B = NaBu, C = LPS, D = NaBu + LPS (Figure 2) (Figure 3) (Figure 4) (Table 1).

Cell Cross-Linking, Cell Harvesting, and NIB Suspension

Post-treatment HCT116 cells in twenty eight plates, groups A/A*-D/D*, were crosslinked with 37% formaldehyde (Sigma Aldrich) to a final concentration of 1% and incubated at room temperature for 10 minutes while rocking gently. Crosslinking refers to the formation of covalent bonds between the functional groups of two macromolecules, DNA and protein, by reacting with formaldehyde (Hoffman et al., 2015). Cells were washed with 2.5M glycine to a final volume of

2.5 mM glycine at room temperature to stop the crosslinking reaction. All media was subsequently removed to begin the harvesting process. 2mL of Nuclei Isolation Buffer with ++ added Calcium (NIB + C a ) consisting of 2 mM MgOAc2, 0.3 M sucrose, 1 mM C a C2 l , 10 mM HEPES at pH 7.8, were added to each plate. The cells were scraped off using a razor and placed in a 15 mL conical then centrifuged for 10 minutes forming a pellet. The supernatant was removed followed by resuspending the pellet in 2 mL NIB, centrifuging for 10 minutes, and removing the supernatant once more. The pellet was finally resuspended in 500 L of final volume NIB and transferred to a labeled tube. This process was repeated for Groups A/A*-D/D* for a total of 28 tubes. Each labeled tube contained 500L of 5.0 x6 01 cross-linked HCT116 cells corresponding to their experimentally treated plates. The tubes were preserved at -40°C prior to micrococcal nuclease (MNase) digestion.

MNase Digestion and Decrosslinking

Each cross-linked HCT116 treated sample (A/A*-D/D*) containing 500L of 5.0 x 6 01 cells was thawed at room temperature and aliquoted into (2) 1.5mL centrifuge tubes each containing ~250L of 2.5 x6 01 cells respectively. The cells were placed in a 37°C incubator for 5 minutes to reach optimal temperature for MNase enzymatic digestion. Heavy and Light MNase digest solutions for each sample were prepared by adding 10 units MNase (0.5L of 20 units/mL stock MNase ) to 250 L NIB for Heavy digests, and 5 units MNase (0.25 L of 20 units/mL stock MNase) to 250 L NIB for Light digests. The added calcium in the NIB buffer activates the MNase reaction. 250 L of 10 unit MNase/NIB solution were added to 250 L of incubated cells for Heavy digests, and 250 L of 5 unit MNase/NIB solution were added to 250 L incubated cells for Light digests. Linker DNA was digested by the MNase leaving mononucleosomally protected DNA fragments in solution. The Heavy and Light MNase solutions were allowed to digest the cells for 28 minutes and 5 minutes respectively and the reactions were stopped with 100 L ethylenediaminetetraacetic acid (EDTA) in excess which chelates divalent cations such as calcium binding them indefinitely. Each sample was vortexed for 10 seconds and 25 L of 20% Sodium Dodecyl Sulfate (SDS) was added to a final

concentration of 1% which acts as a detergent to lyse cell membranes and denatures proteins making them easier to remove later on. The samples were incubated in a 65° C overnight to decrosslink DNA-protein complexes and denature histone proteins.

Spin-Column Isolation and Purification of mononucleosomal and small fragment DNA

The samples containing digested and lysed cells were removed from the 65° C incubator the following day and vortexed for 30 seconds. 500 L Guanidinium HCl/70% isopropanol were added to each sample and incubated at room temperature for 16 minutes. Guanidinium HCl is a chaotropic salt that denatures biomolecules by destabilizing H-bonds, hydrophobic interactions, and van der waal forces thus destabilizing proteins such as nucleases (BiteSize Bio, 2010). By destabilizing these forces it increases the solubility of hydrophobic molecules such as amino acids, stopping them from aggregating in aqueous solution. It also disrupts the association of nucleic acids with water, allowing the nucleic acids in solution to adhere more easily to silica beads and membranes (Tian et al., 2000). 10 L of silica beads were added to each sample and evenly mixed. The solutions were transferred 500 L at a time to individual silica membrane spin-columns for each sample and centrifuged on max (16,873 RPM) for 2 minutes, saving the supernatant each time.

DNA binds to the silica via GuHCl acting as a chaotropic agent by disrupting the shell of hydration around biomolecules allowing a positively charged ion salt bridge to form between the negatively charged silica and negatively charged DNA backbone. The DNA remains bound to the silica and all other biomolecules are washed away in the supernatant (Karp et al., 1998). 750 L of 70% ethanol was added to each spin column and centrifuged on max for 2 minutes, dumping the supernatant and repeating once more. Ethanol, present in a high salt GuHCl environment, is used to wash excess particles off nucleic acids by forcing the nucleic acids out of aqueous solution leaving all other molecules in the supernatant. Nucleic acids are hydrophilic due to the negatively charged phosphate groups along the sugar-phosphate backbone. GuHCl salt is needed in ethanol precipitation to neutralize negatively charged phosphate groups, making the

molecules less soluble in aqueous solution . Water, having a high dielectric constant , makes it + − difficult for H and PO 3 to bind. Ethanol is needed as it has a lower dielectric constant than + − water, allowing H and PO 3 to come together more easily making nucleic acids less hydrophilic thus dropping out of aqueous solution (BiteSize Bio & New England Biolabs, 2007).

The spin columns were dry-spun for 10 minutes at 15,000 RCF’s to remove excess ethanol from the silicone membranes. 50 L ofT 10E 1 (10 mM Tris-CL, 0.1 mM EDTA, pH 8.0), the elution buffer, was added to each spin-column to elude the DNA off the silica beads and silica membrane. The spin-column samples were centrifuged for 2 minutes on max into new 2 mL labeled tubes. Each sample was nano dropped in an optical density test using a Nanodrop UV-Vis Spectrophotometer by Thermo Science to determine the concentration of DNA present and check for protein impurities. This test relies on the Beer-Lambert Law which correlates light absorbance and concentration of a sample. Nucleic acids have peak UV light absorbance at 260 nm, therefore the amount of light absorbed within this region coupled with the Beer-Lambert law can be used to determine the concentration of DNA or RNA within a sample (University of Arizona, 2014) (Figure 5) (Figure 6).

Mononucleosome SPRI-Peg Bead Selection and Glycogen Pull Down

Solid phase reversible immobilization (SPRI) bead selection in the presence of polyethylene glycol (PEG) was used to separate DNA based on fragment size for library preparation for Next Generation sequencing. SPRI beads are paramagnetic metal beads made up of polystyrene and coated in a layer of magnetite with carboxyl molecules (succinic acid) that reversibly bind to DNA in the presence of a PEG crowding agent and salt (Hadfield, 2012; BiteSize Bio, 2013). The PEG crowding agent causes negatively charged DNA to bind with carboxyl groups on the SPRI bead surface, annealing larger sized DNA fragments and leaving smaller sized fragments in solution. The target size for small fragments in SPRI selection is ~150 bp, roughly the size of a mononucleosome (Kornberg & Lorch, 1999). The specific size of DNA fragments that anneal to the SPRI bead surface depends on the SPRI-PEG:DNA ratio. To bind

DNA fragments greater than ~150 bp a 1.2x ratio is needed. By increasing the ratio of SPRI-PEG:DNA, the efficiency of binding smaller fragments increases (Fred Hutchinson Cancer Research Center, 2012). Each sample contained 1 part DNA at a concentration of ~20-50 ng/ L diluted with TE along with 1.2 parts SPRI beads to a final volume of 500L . The samples were vortexed, pop-spun, mixed by pipetting up/down 10 times and allowed to incubate for 5 minutes at room temperature. The samples were then placed on a magnetic stand until clear, indicating nucleic acid fragments larger than ~150 bp along with the SPRI beads they’re attached to have successfully adhered to the side of the tube. 90L of 85% EtOH for every 100L of reaction were added to each tube while still on the magnet and incubated at room temperature for 30 seconds. The supernatant containing smaller fragments was transferred to a 2 mL tube. 75L of TE was added to elute the larger fragments off beads by pipetting up/down 10 times. The samples were centrifuged and placed back on the magnet until the beads settled once more for 5 minutes. The supernatant containing larger fragments was transferred to a fresh 2 mL tube.

Glycogen pull-down is used to bind small DNA fragments in supernatant solutions with high EtOH concentration. Glycogen, insoluble in ethanol, is used to recover the small DNA fragments from alcohol precipitation by forming a precipitate that traps nucleic acids (Gold Biotechnology, 2019). 1 L of 10M Glycogen, 1/10th of solution volume of 5 M NaOAc, and 1 mL of 100% EtOH were added to each sample then vortexed and centrifuged for 5 minutes on max. The solution was decanted by dumping the supernatant and washing with 1 mL of 70% EtOH followed by vortexing and centrifugation once more for 5 minutes on max. The samples were placed in the DryVac for 15 minutes and resuspended with 50 L of TE.

Results

Purified DNA samples from each experimental group were checked for DNA concentration and protein contamination using a UV-Vis spectrophotometer (NanoDrop). An absorbance of 260 nm indicates the presence of nucleic acids as both pyrimidines and purines have peak absorbance at this wavelength (University of Arizona, 2014). The absorbance values of samples A1-C3 show a strong peak of 2.9 Abs at 260 nm indicating a strong nucleic acid presence. The A260/280 ratio identifies nucleic acid contamination of proteins, with an acceptable value falling between 1.8 and 2.1 with a value of 1.8 indicated pure DNA. The A260/280 ratio for samples A1-C3 was an acceptable value of 1.96 indicating the likelihood of protein contamination was low. The A260/230 ratio identifies contamination of nucleic acids with organic compounds with an acceptable range of 1.8-2.0. Values closer to 2.0 indicate a pure sample free of contamination. The A260/230 ratio for samples A1-C3 was 1.18, outside the acceptable range. This is possibly due to significant organic compound contamination likely caused by excess GuHCl from spin-column DNA purification. Samples C4-D2 show a slightly weaker peak of 1.3 Abs at 260 nm when compared to samples A1-C3, indicating a weaker nucleic acid presence in this sample range. The A260/280 of these samples was an acceptable value of 1.97 indicating unlikely protein contamination. The A260/230 ratio was 1.02 which was significantly lower than the acceptable range of 1.8-2.0, even lower than that of sample groups A1-C3. This may be due to significant organic compound contamination also likely caused by excess GuHCl from spin-column DNA purification (Figure 5) (Figure 6).

The cross-linked cells from each experimental group were harvested and digested with micrococcal nuclease in concentrations of 5 and 10 units for light and heavy digests respectively. MNase preferentially digests linker DNA between nucleosomes, creating a continuous pattern of DNA fragments incrementally increasing by roughly ~150 bp as each nucleosome contains roughly 150 bp of double stranded DNA. Gel electrophoresis can be used to authenticate MNase digestion. We expect DNA samples digested with light concentrations of MNase to have faint banding around ~150 bp around mononucleosomally sized fragments and darker banding >150

bp as MNase only digest DNA around the most sensitive nucleosomes. Samples digested with heavy concentrations will conversely have heavier banding around ~150 bp and faint banding >150 bp demonstrating a greater success in digesting resistant nucleosomes (Figure 7).

Mononucleosomal and subnucleosomal fragments were separated using SPRI bead selection in the presence of a PEG crowding agent and salt. A SPRI:DNA ratio of 1.2x was used to precipitate sub nucleosomal sized DNA fragments larger than ~150 bp to the SPRI bead surface, leaving mononucleosome sized DNA fragments ~150 bp in PEG solution. A magnet was used to hold the sub nucleosomal sized DNA fragments to the side of the tube while the supernatant containing smaller fragments was removed and transferred to a new tube. The small DNA fragments were bound with 1 L 10M glycogen and precipitated in EtOH to form a precipitate trapping nucleic acids. The samples were placed in a DryVac and resuspended with TE.

Future Directions and Discussion

We will produce indexed libraries of the isolated mononucleosomally sized DNA fragments in preparation for next-generation DNA sequencing by ligating distinctive adaptors to the 3’ end of each DNA fragment and ligating a universal adaptor to the 5’ end in all samples. The purpose of the unique ligated adaptors is to serve as barcodes for each DNA fragment so we can identify them during next-generation sequencing. We will index these libraries to aid in identifying each sample by highlighting the relationship between related samples along with distinguishing these samples from other sample pools during sequencing. After ligation indexed DNA libraries are amplified using PCR and quantified by Qubit assay.

DNA fragments located at transcription start sites for roughly 20,000 human protein coding genes will be targeted and enriched during TSS sequence capture using the NovaSeq6000 to identify their sequences. This will map the distribution of nucleosomes in the genome at each time point under each experimental condition. We can compare the nucleosomal occupancy at each time point to determine if/when a GTIS occurs in response to NaBu, LPS, and NaBu + LPS stimuli. A Bioanalyzer 2100 dsDNA High Sensitivity Assay will be conducted to verify that library preparation and TSS sequence capture were successful.

The nucleosome occupancy profiles at each time point will be compared within each treatment group and between treatment groups. Nucleosome occupancy will be analyzed using R (+/- one kilobase) of each transcription start site for ~20,000 protein coding genes in the human genome. The corresponding function for genes affected by a chromatin remodeling event can be identified using gene ontology software. Identifying genes affected by the GTIS is extremely valuable as it provides insight to the probability of potential changes in gene expression in response to LPS, NaBu, and LPS + NaBu stimuli. This helps us to determine what genes were affected by LPS and NaBu stimuli alone along with identifying genes resistant to chromatin remodeling events due to NaBu inflammatory response inhibition, highlighting NaBu’s ability to inhibit changes in chromatin structure caused by LPS. Nucleosome occupancy profiles only tell

us the potentiality of gene expression, not if they will definitely occur. RNA sequencing will allow us to see these changes in gene expression both before and after each experimental stimulus is applied along with their immediate response to stimulus exposure.

The purpose of this study is to understand how NaBu inhibits potential changes in gene expression from the LPS stimulus in HCT116 colorectal cells. The data obtained from this study will give us insight into both short-term and long-term effects of NaBu inflammatory inhibition of LPS along with NaBu and LPS alone by observing the biochemical properties of HCT116 cells. The former refers to gene expression and chromatin remodeling events and the latter refers to changes in chromatin sensitivity we can use to predict gene expression changes in the future.

Future studies involving inflammation on the human colon may be pursued to determine what genes related to dopamine and serotonin production are affected by NaBu inflammatory response inhibition of LPS. About 50% of the body’s dopamine is produced in both the gastrointestinal tract and intestinal epithelial cells and more than 90% of the body’s serotonin is produced in the gastrointestinal tract (Eisenhofer & Åneman, 1997) (Yano et al., 2015). Nucleosomes located at TSS involved in serotonin and dopamine production such as the synthesis of 5-hydroxytryptamine (5-HT) and ghrelin will be mapped in HCT116 cells after exposure to NaBu, LPS, and NaBu followed by LPS. 5-HT peripheral dysbiosis is strongly tied to the pathogenesis of several diseases including irritable bowel syndrome (Stasi & Bellini, 2014). This study will be done to see if A.) LPS exposure inhibits 5-HT and ghrelin production and B.) if NaBu inflammatory response inhibition prevents LPS from causing a chromatin remodeling event in nucleosomes occupying 5-HT and ghrelin producing genes. (Yang & Zhao, 2019).

Figure 1:

Figure 1: Structure of Sodium Butyrate (above). HDACis inhibit the inflammatory response involved in signaling of the NF-KB pathway.

Figure 2:

Figure 2: TLR4/5/7/8 Signaling Cascade. Reprinted from “TLR4/5/7/8 Signaling Cascade”, by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates Visually demonstrates the TLR4 signaling cascade in addition to TLR 5, 7, 8 signaling cascades. Includes genes mentioned in Table 1, pro-inflammatory cytokines and Type I IFNs.

Table 1:

Gene Protein Function Citation

TLR4 Toll-like Cooperates with LY96 and CD14 to (Weizmann Institute of receptor 4 mediate the innate immune response to Science, 2020a) bacterial lipopolysaccharide (LPS) Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response Involved in LPS-independent inflammatory responses triggered In complex with TLR6, promotes sterile inflammation in monocytes/macrophages

TIRAP TIR Domain It activates NF-kappa-B, MAPK1, MAPK3 (Weizmann Institute of Containing and JNK, which then results in cytokine Science, 2020b) Adaptor secretion and the inflammatory response. Protein

TRAF6 TNF Functions as a signal transducer in the (Weizmann Institute of Receptor NF-kappaB pathway that activates IkappaB Science, 2020c) Associated kinase (IKK) in response to Factor 6 proinflammatory cytokines.

MYD88 MYD88 Adapter protein involved in the Toll-like (Weizmann Institute of Innate receptor and IL-1 receptor signaling Science, 2020d) Immune pathway in the innate immune response. Signal Transduction Adaptor

NFKB2 Nuclear encodes a subunit of the transcription factor (Weizmann Institute of Factor complex nuclear factor-kappa-B (NFkB). Science, 2020e) Kappa B Subunit 2

Table 1: Genes involved in innate immune response to LPS and proteins encoded by each gene along with their relevant function.

Figure 3:

Figure 3: HCT116 cell culture stimulus groups Each treatment group contains HCT116 cells grown to a confluency of 5.0x01 6 cells in 100mm plates. Groups A*-D* denote replicate plates following the same treatment plans as Groups A-D. I anticipate that nucleosomal sensitivity in TLR4 pathway-related genes will remain at basal state in group A, decrease in group B, increase in group C, and increase slightly more than group A but not as drastically as group C.

Figure 4:

Figure 4: Visual representation of experimental design for NaBu, LPS, and NaBu + LPS exposure Timepoints for stimulus exposure, cell cross-linking and harvesting are mapped above.

Figure 5:

Figure 5: Optical density test of purified nucleic acid sample C3L. A Absorbance at 260 nm confirms presence of DNA or RNA due to both pyrimidine (adenine and guanine) and purine (cytosine, thymine and uracil) having peak absorbance at this wavelength (University of Arizona, 2014). A strong peak of 1.9 Abs at 260 nm present within this sample indicates a strong nucleic acid presence. Absorbance between 210 nm and 230 nm indicates organic compound contamination including chaotropic salts (GuHCl) and peptide bonds. Absorbance at 280 nm indicates protein and phenolic compound contamination including aromatic amino acid side chains (tyrosine, histidine, tryptophan, phenylalanine) found within proteins. B A260/280 ratio is used to identify nucleic acid contamination of proteins, with a value of 1.8 indicating pure DNA and 2.1 indicating pure RNA. In this sample there is likely no protein contamination due to an acceptable value of 1.96. A260/230 ratio identifies contamination of nucleic acids with organic compound contaminants as mentioned above, with values close to 2.0 indicating a pure sample and

those below 1.8 indicating contamination. In this specific sample there is significant organic compound contamination with a value of 1.18 likely present from excess GuHCl.

Figure 6:

Figure 6: Optical density test of purified nucleic acid sample D2L. A. A peak of 1.3Abs at 260 nm present within this sample indicates a strong nucleic acid presence. B. In this sample there is likely no protein contamination due to an acceptable value of 1.97. C. In this specific sample there is significant organic compound contamination with a value of 1.02 Abs likely present from excess GuHCl.

Figure 7:

Figure 7: MNase Titration of Untreated HCT116 Cells. 1 Kb molecular weight marker in 150 bp intervals was used in the first well. HCT116 cells digested with micrococcal nuclease in concentrations of 10 Units and 5 Units for 28 minutes and 5 minutes, are referred to as Heavy digestion and Light digestion respectively. Light digests show lower concentrations of mononucleosomally protected DNA, with heavy digests showing higher concentrations of these fragments.

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