Project summary/Abstract: Histone post translational modifications (PTMs) are fundamental epigenetic mechanisms to modulate chromatin structure and functions. Because chemical structure and position of PTMs dictate diverse functional outputs, which is known as “histone code theory”. To expand our knowledge of histone code, our lab have identified and characterized nine new histone PTMs. Histone lysine palmitoylation (Kpal), which is recently discovered by our lab, appears to be closely associated with lipid metabolism. Our preliminary data showed that histone Kpal is derived from palmitoyl-coA, which is an important intermediate in lipid metabolism. While excessive palmitic acid have been shown altered gene expression profile in many cell types, palmitic acid treatment also lead to a dose-dependent increase of histone Kpal level. In addition, our preliminary data showed that SIRT6 removes palmitoyl groups from H3 peptide with a catalytic efficiency more than 600–fold better than that for deacetylation in vitro, and SIRT6 is a critical enzyme required to maintain lipid homeostasis. Despite the intriguing relationship between histone Kpal and lipid metabolism, it is not known exactly what are histone Kpal’s functions.
Histone PTMs alters nucleosome structure, thereby modulating the access of transcription machinery to DNA. Multiple histone PTMs, including lysine acetylation (Kac) and methylation, has long been recognized to play roles in transcription regulation. Histone Kpal disrupt nucleosome to a greater extent than Kac due to the increased size and hydrophobicity. Therefore, our central hypothesis is that transcription regulation represent one major function of histone Kpal. We will test our hypothesis with two specific aims: (1) to identify histone lysine palmitoylation sites and characterize the distribution of lysine palmitoylation mark on a genome-wide scale (2) to investigate the roles of histone lysine palmitoylation in regulating transcription in vitro and in vivo. The long term goal of this project is to establish histone Kpal as a fundamental epigenetic mechanism that is particular important for lipid metabolism.
Specific Aims: Lipid serves as one of the major energy sources of the cells, building block for cell membranes and signaling molecules. Maintaining homeostasis of lipid metabolism is thus critical for cell integrity and proliferation. One of the central node of lipid metabolism is palmitic acid (PA). PA can be synthesized from other fatty acids, carbohydrates and amino acids, and provide the substrate for glycerolipid synthesis, glycerophospholipid synthesis and fatty acid elongation. PA is also the most common saturated fatty acid found in the food and human body. To support its physiological functions, PA concentration is tightly controlled. Disruption of PA homeostasis is implicated in multiple pathological conditions such as atherosclerosis, neurodegenerative diseases and cancer1. At molecular level, it was shown that PA activates cell surface receptors such as CD36 and TLR4, or provide substrate for S-palmitoylation of cytosolic proteins. However, these mechanisms cannot fully capture the complicated and controversial functions of PA.
Histone post translational modifications (PTMs) are fundamental epigenetic mechanisms to control chromatin structure and functions. Emerging evidence suggests that histone PTMs is closely connected with metabolism. As the best characterized example, Kac is derived from acetyl-coA, an central metabolite in citric acid cycle, and Sirtuins, the enzymes that remove Kac, requires NAD+ as a co-substrate. In addition, our lab has reported eight new histone PTMs2–8, which as known as histone lysine acylations, and all of them are derived from intermediates in crucial metabolic pathways. Together, these evidence support the model that histone integrate metabolic signals by incorporating various PTMs, which in turn help to coordinate homeostatic transcriptional response9–13.
Recently, we identified and verified histone lysine palmitoylation (Kpal) as an unreported histone PTM derived from PA. After PA treatment, we observed a dose-dependent increase in global histone Kpal level in mice embryonic fibroblast (MEF) cells. Given that histone PTMs are known to regulate transcription, in this project, we hypothesize that histone Kpal plays a role in transcription regulation analogous to histone lysine acetylation, methylation and acylations, representing a fundamental mechanism by which PA exert it’s physiological functions. We propose to test this hypothesis by two specific aims:
Aim 1: To identify histone Kpal sites and characterize the distribution of Kpal marks on a genome-wide scale. The same post translational modification, if added on different sites of histone, may have different, even opposite effects on transcription regulation, as exemplified by histone lysine tri-methylation. Therefore, to accurately describe the function of histone Kpal, we will identify and quantify histone Kpal sites. We will treat MEF cells with either PA or PA analogous probe, isolated and proteolytic digest core histones, enrich palmitoylated peptides by either pan-anti-Kpal antibody or click chemistry, and map the histone Kpal sites using HPLC-MS/MS. Once we determine the sites of histone Kpal marks, we will generate site-specific anti-Kpal antibodies. To establish a correlative relationship between histone Kpal marks and transcription regulation, we will perform Chip-seq using pan- and site-specific histone Kpal antibodies and compare the distribution of Kpal marks with the gene expression profile described by RNA-seq.
Aim 2: To investigate the roles of histone Kpal in regulating transcription in vitro and in vivo. Because the fatty acyl chain of palmitoylation is much increased in size and hydrophobicity compared to that of acetylation, it is likely to open up chromatin structure to a greater extent. Therefore, we hypothesize that histone Kpal activate transcription. To test this hypothesis in vitro, we will incorporated either modified or unmodified core histones in the reconstituted chromatin-based cell-free transcription system, and measure the transcription activity. To manipulate histone Kpal level in MEF cells (in vivo), we seek to identify the enzymes that add (“write”) or remove (“erase”) histone Kpal marks, and overexpress and knockdown these enzymes in MEF cells. We will then perform ChIP-seq using both pan- and site-specific anti-Kpal antibodies and RNA-seq to determine whether the transcription is up- or down-regulated as histone Kpal level increase or decrease.
Impact: This project will establish the roles of histone Kpal in regulating transcription and identify the group of genes whose expression is controlled by histone Kpal, which helps to infer the functional consequences of histone Kpal. By comprehensively characterizing the histone Kpal sites and identifying the regulatory enzymes, this project will lay a solid foundation for manipulating histone Kpal to study it’s function in other cell types, under physiological or pathological condition. In addition, we will optimize HPLC-MS/MS to boost the detection sensitivity and quantification accuracy for protein long-chain fatty-acylation. This technological advance can be readily applied to studying Kpal on non-histone proteins, or other types of long-chain fatty-acylation on histones. Overall, this project will uncover an important mechanism that connects lipid metabolism to gene expression regulation.
Research Strategy
Significance: Palmitic acid (PA) is the most common saturated fatty acid found in the food and human body. Because PA can be synthesized from other fatty acids, carbohydrates and amino acids, and provide the substrate for glycerolipid synthesis, glycerophospholipid synthesis and fatty acid elongation, PA act as one of the hubs of lipid metabolism. Under physiological condition, PA concentration is tightly controlled. Disruption of PA homeostasis, most frequently accumulation of excessive PA, is implicated in pathological conditions such as aging, cardiovascular diseases, neurodegenerative diseases and cancer1. Palmitic acid alters expression of a large number (many hundreds) of genes in peripheral blood mononuclear cells14, macrophage15, beta-cell16, cardiomyocyte17, and hepatocyte18. The transcription regulation function of PA has been attributed to PARP activation, however, evidence suggest that palmitic acid can alter gene expression independent of PARP transcription factors19. Yet the molecular mechanisms by which PA regulate gene expression remains to be defined.
(I’ll expand more on palmitic acid biology)
Core histones (H2A, H2B, H3, H4) are known for possessing a large number and variety of post translational modifications (PTMs)20. Histone PTMs affect not only the local nucleosome structure but also the higher-order chromatin architecture, thereby play roles in numerous chromatin based processes, such as gene expression, gene silencing, DNA replication, DNA damage repair and chromosome condensation21. Histone lysine acylation is a group of structurally related histone PTMs, exemplified by acetylation. Given that most acylations derive from metabolic intermediate coA species, it is speculated that histone acylations connect metabolism to gene expression22. Our lab recently identified an unreported histone PTM, histone lysine palmitoylation (Kpal), in commercial calf thymus histones and mice embryonic fibroblast (MEF) cells. Interestingly, our preliminary data showed that SIRT6 removes palmitoyl groups from synthetic H3 peptide with a catalytic efficiency more than 600–fold better than that for deacetylation in vitro23, suggesting that histone Kpal is an important histone PTM that is specifically regulated by SIRT6 and potentially other enzymes in vivo. We showed that histone Kpal level increase in a dose-dependent manner after treating cells with palmitic acid. Given the well-characterized role of histone PTMs in transcription regulation, we hypothesize that histone Kpal represent a previously unknown mechanism by which palmitic acid alters gene expression in cells. Because palmitoylation neutralize the positive charge of lysine residue as acetylation and cause striking increase in the size and hydrophobicity of the modified residue, it is likely to unravel the chromatin to a greater extent. Therefore, we speculate that histone Kpal activate transcription. We will integrate quantitative MS, reconstituted cell-free transcription system, ChIP-seq and RNA-seq analysis to investigate histone Kpal’ functions in transcription in vitro and in vivo. This project will expand our knowledge on how cells sense and respond to palmitic acid concentration variation through epigenetic mechanism.
Innovations: A) A novel epigenetic mechanism that connects lipid metabolism to gene expression: PARP activation account for some but not all effects of palmitic acid on transcription regulation, and evidence suggest that palmitic acid can alter gene expression independent of PARP transcription factors19. Our preliminary data demonstrated a dose-dependent increased of histone Kpal level following palmitic acid treatment. Given the well-established roles of histone PTMs in transcription regulation, this project is likely to identify histone Kpal as an unexamined yet important connection between palmitic acid metabolism and gene expression regulation. This project will identify the genes controlled by histone Kpal, which helps to infer the functional consequences of histone Kpal. By comprehensively characterizing the histone Kpal sites and identifying the regulatory enzymes, this project will lay a solid foundation for manipulating histone Kpal to study it’s function in other cell types, under physiological or pathological conditions.
B) Technological advances that promote protein Kpal researches. Studying protein long-chain fatty-acylation was stalled by lacking of effective detection and quantification methods. Current technology to detect protein lysine is metabolic labeling with chemical probes, yet the use of chemical probes unavoidably changes the metabolic status of the cells, and these probes are challenging to use in whole animals24. Generating anti-Kpal antibodies will solve the challenge because it does not request chemical treatment prior to detection. However, attempts were fail due to poor immunogenicity of the antigen cause by non-specific association with host cell membranes. Our lab have overcome this challenge, generated a pan-anti-Kpal antibody and verified it’s specificity by testing the cross-reactivity against other PTMs, which provides the community with a powerful tool to detect and enrich lysine palmitoylated proteins. To facilitate studying the functions of histone Kpal on individual residues, we further propose to generate site-specific anti-Kpal antibodies. Given that our lab has extensive experience in generating antibodies for protein PTMs2,5,6,25,26, we anticipate no major technical difficulties.
The Zhao lab is at the forefront of developing highly sensitive MS methods for detecting and quantifying histone PTMs. We have developed a unique pipeline for analysis of histone lysine fatty acylated peptides, which have been applied to identification and quantification of seven histone lysine fatty acylations2,5,6,8,25,26. In this pipeline, we will isolate chromatin-bound core histones followed by proteolytic digestion, chemical derivatization of the peptide and HPLC/MS/MS. We will systematically optimize the pipeline by adjusting HPLC column, mobile phase, chemical derivatization methods and MS/MS parameters to further improve the sensitivity and accuracy of palmitoylated peptide quantification.
Research approach:
Aim 1: To identify histone Kpal sites and characterize the distribution of Kpal marks on a genome-wide scale.
Rationale: Extensive researches on histone lysine acetylation and methylation showed that the function of a given histone PTM is context dependent, for example, whether the PTM is added on the tail or the globular region of the core histones, distributed to gene body or promoter/enhancer, etc27. Therefore, to comprehensively dissect the functions of histone Kpal, we first need to determine which lysine residues can be modified, and where are the histone Kpal marks distributed across the genome. In addition, since cysteine palmitoylation was shown to promote the membrane association of soluble signaling proteins, such as Hedgehog28, Wnt28, RAS29 and G-alpha30, we hypothesize that histone Kpal may similarly tether histones to inner nuclear membrane, thus altering large-scale chromatin structure. This hypothesis will be tested by characterizing the spatial distribution of histone Kpal marks using immunofluorescent imaging.
Experimental design: To identify which lysine residues in core histones are modified by palmitoylation, we will couple quantitative mass spectrometry with chemical or antibody enrichment. Once the Kpal sites are identified, we will generate site-specific anti-Kpal antibodies for at least some of the Kpal marks and test if they cross-react with any other known PTMs by dot blot. We will then determine the genome-wide distribution of histone Kpal using ChIP-seq and compare the results with histone lysine acetylation (Kac). We will then use immunofluorescent imaging to determine whether histone Kpal has an unique intranuclear localization.
We will continue to use MEF cell in which we demonstrated the presence of histone Kpal as the model system, because MEF cell allows rapid proteomic and biochemical analysis, has a normal ploidy and is widely used in biomedical studies. The MEF cells will be cultured in DMEM with 10% FBS. To prevent the interference from palmitic acid in FBS, cells will be switched to serum free media (SFM) 24 hours prior palmitic acid treatment unless specified.
Mapping histone lysine palmitoylation sites using quantitative mass spectrometry and generating site-specific anti-Kpal antibodies. Palmitoylated peptides and their unmodified counterpart exhibit dramatic difference in hydrophobicity. Conventional HPLC-MS/MS system either fail to efficiently elute palmitoylated peptide, which reduce detection sensitivity, or elute the palmitoylated peptide by strong organic solvent at the expense of losing the unmodified peptides, which prevent relative quantification. To address this problem, we will adopt a strategy developed by Lin group31, i.e. chemically derivatize the unmodified peptides to enhance their hydrophobicity, and use a less hydrophobic stationary phase for peptide separation. We will tailor the quantitative MS pipeline for histone Kpal analysis by adjusting chemical derivatization methods (propionic anhydride, butyric anhydride), stationary phase with different hydrophobicity(C18, C8, C4), percentage of organic solvent in mobile phase, and MS/MS parameters.
Our lab have developed a unique pipeline for analysis of histone lysine fatty acylated peptides and applied it to analyzing multiple histone PTMs2,5,6,8,25,26. Here, we will use a similar pipeline to analyze histone Kpal: chromatin-bound core histones isolation followed by proteolytic digestion, enrichment of modified peptides, chemical derivatization of peptides and HPLC/MS/MS. Specifically, for histone Kpal, we will use two independent enrichment methods: click chemistry based enrichment and antibody based enrichment. In the first method, MEF cells will be labeled by alkyne-tagged palmitic acid analogue Alk16 at physiologically relevant concentration (100 μmol/L). The labeling time will be optimized to achieve sufficient labeling efficiency and avoid beta-oxidation of the probe. The core histones will be purified and digested by proteasome. the modified peptides will be conjugated to a cleavable biotin-azo-N3 using click chemistry and enriched using streptavidin beads. After washing, conjugated histones will be selectively eluted with Na2S2O4, purified by SDS PAGE, digested and subjected to HPLC/MS/MS analysis. We will use cells cultured with palmitic acid as a negative control. H3 S-palmitoylation at Cys110, which was identified using this method, will be used as a positive control32. In the antibody enrichment methods, MEF cells will be cultured in 100 μM palmitic acid, the plasma palmitic acid concentration in healthy human33 prior to analysis. For both chemistry and antibody based enrichment methods, histone Kpal sites that are consistently detected in three biological replicates will be preserved. We anticipate that antibody enrichment method will identify all histone Kpal sites, whereas chemistry enrichment method will highlight the sites that are more dynamically regulated. The results from the two methods will be merged.
Once we determined histone Kpal sites, we will then synthesize histone peptides which are palmitoylated on a specific site, and generate site-specific anti-Kpal antibodies according to a previously described method34. Our lab have extensive experience in generating pan and site-specific antibodies for histone PTMs2,5,6,8,25,26, so we do not anticipate any major technical problems in generating site specific-histone Kpal antibodies. Antibody specificity will be verified using dot blot assay against untargeted Kpal sites, as well as the acylation marks listed in the “ChIP-seq analysis” section.
ChIP-seq analysis of histone Kpal and Kac. Because the quality of ChIP-seq analysis depends heavily on antibody specificity, we will thoroughly verify the specificity of pan anti-Kpal antibody using dot blot assay against acetylated, propionylated, butyrylated, myristoylated, and cysteine-palmitoylated peptides. To map the genome wide distribution of histone Kpal and Kac (control) in a biologically relevant cellular context, MEF cells will be treated with 100 μM or 300 μM palmitic acid in SFM, to resemble the plasma palmitic acid concentration of healthy and type II diabetes human. Chromatin preparation, immunoprecipitation, and ChIP-seq analysis using pan-anti-Kpal and pan-anti-Kac antibody will be performed as previously described3. Each ChIP-seq should be repeated twice (biological replicates, n=2)35. After peak calling, total Kpal and site-specific Kpal peaks will be annotated for chromosome-scale distribution, such as which chromosomes are over-represented for Kpal, in heterochromatin or euchromatin region; gene structure categories, such as intron, exon, intragenic, intergenic, etc; distance from transcription start sites; DNA sequence motif.
Immunofluorescent imaging of histone Kpal. We will immunostain all histone Kpal marks where site-specific antibody is available as previously describe36. We will co-stain nuclear peripheral marker LaminB1, heterochromatin marker H4K20me3 and histone Kac mark at the corresponding site.
Expected results: We anticipate more than one Kpal site (“Kpal mark”) will be identified in histone tail and/or the globular region. The location of palmitoylation may help us to predict it’s function: it was proposed that modifications located in tail region usually act indirectly and recruit factors to regulate downstream signaling and chromatin remodeling, whereas modifications on the solute accessible surface affect histone-histone interaction and histone-DNA interaction both directly and indirectly.37–39
For the ChIP-seq experiment, we anticipate that at least some genomic regions will have a dose dependent upregulation of histone Kpal. Although we cannot predict exactly how histone Kpal and Kac distribution changes from low (100 μM) to high (300 μM) amount of palmitic acid, the sequences that interest us most should exhibit one of the two features below: (1) Kpal level changes in an opposite direction compared with Kac (2) whereas Kac does not change, Kpal up- or down-regulates for more than 2 fold. We perform gene ontology and ingenuity pathway enrichment analysis of the genes that are nearest to these regions.
We will be most interested in histone Kpal marks which do not colocalize with the corresponding Kac mark. We anticipate that distribution of Kpal marks either be enriched in certain regions, such as nuclear peripheral (indicated by LaminB1), heterochromatin (indicated by H4K20me3), unspecified hotspots, or be diffusive in the entire nucleus. The enrichment of a histone Kpal mark in certain hotspots suggest that this mark may have specific functions. In this case, we will validate the observation in three independent replicates. We will mutate the lysine of interest to arginine and perform the immunostaining again. If the point mutation abolish the phenotype, we would know that the lysine is required for the clustering.
Potential pitfalls and alternative approaches:
We’ve validated that our histone pan-anti-Kpal antibody has more than 10-fold enriched binding signal for Kpal relative to Kac. However, if stoichiometry of histone Kac is more than 10 times higher than that of histone Kpal, which is possible, there will still be considerable amount of Kac be pulled down non-specifically by anti-Kpal antibody. Fortunately, we will be able to tell if Kac is indeed precipitate by anti-Kpal antibody by MS analysis. To address this problem, we will culture MEF cells with 100 μM or 300 μM Alk16 for 2 hours to prevent Alk16 β-oxidation40 and precipitate the crosslinked histone-DNA complex by click chemistry. This approach can pull downed palmitoylated histone with less acetylated histone contamination, at the expense of losing regions where histone Kpal turnover rate is low.
Aim 2: To investigate the roles of histone lysine palmitoylation in regulating transcription in vitro and in vivo
Rationale: Histone PTMs modulate chromatin folding and thereby transcription activity. As an example, histone Kac neutralizes the positive charge on lysine residue, weaken the histone-DNA interaction and expose DNA to transcription machinery.41 In line with this observation, histone Kac is associated with transcription activation, whereas deacetylation is associated with transcription suppression.42 Other type of histone lysine acylation, such as butyrylation and crotonylation, although less well-studied, were also demonstrated to promote transcription in a cell-free transcription system, even more efficiently as compared to histone acetylation.22 We hypothesize that palmitoyl group is likely to perturb chromatin structure to a greater extent, thereby affecting transcription activity, with a hydrophobic fatty-acyl chain that is eight-fold longer than that of acetyl group.
In this project, we aim to investigate the role of histone Kpal in transcription activation or suppression. Unlike genes, RNA or proteins, post translational modifications cannot be knocked down or overexpressed. To manipulate histone Kpal, we will first identify the regulatory enzymes, including the enzymes that add (“writers”) or remove(“erasers”) histone Kpal. We will identify histone Kpal erasers by screening histone deacetylases (HDACs). Although originally annotated as erasers for acetylation, some HDACs (Sirtuins 1-7, HDAC 1-3)3,22,23 are capable to catalyze removal of fatty-acylation. In particular, Sirt6 removes palmitoylation group from histone H3K9 peptide with a catalytic efficiency more than 600–fold better than that for deacetylation23. Based on these findings, we reason that Sirt6 and other HDACs may serve as erasers for histone Kpal in vivo. However, although several histone acetyltransferases (HATs), especially p300/CBP, were found to catalyze the addition of fatty-acylation, the enzyme kinetics is lower for substrates with increased fatty acyl chain length22. Therefore, instead of screening HATs, we seek to purify histone palmitoyl-transferase(s) from the MEF nuclear extract, using an assay that mirrors previously described histone acetyl-transferase assay43.
Experimental design:
Identifying histone Kpal writer(s) from MEF nuclear extract. MEF nuclear extract will be prepared by an ammonium sulfate extraction protocol as previously described44, and separated in sequential by affinity chromatography (heparin column), anion exchange chromatography (Mono Q column), and canion exchange chromatography (Mono S cloumn). Fractions were collected over a linear KCl gradient from 0.15 M to 1 M, and tested by histone lysine palmitoyl-transferase (HPT) assay: each fraction will be incubated with commercialized unmodified histones and palmitoyl-CoA, and the HPT activity of each fraction will be measured by immunoblot using pan-anti-Kpal antibody. We will use MEF nuclear extract as positive control and heat denatured nuclear extract as the negative control. The nuclear extract fraction with the highest HPT activity will be analyzed using MS/MS. The identified proteins will be purified and validated as HPT in three biological replicates by incubating with unmodified histones and palmitoyl-CoA followed by immunoblot using pan-anti-Kpal antibody.
Investigating the role of histone Kpal in transcription regulation in vitro. We expect the experiments above will allow us to either identify HPT(s) or demonstrate that histone Kpal is an non-enzymatic reaction (detailed discussion in “Expected results” section). In either case, we will directly test whether histone Kpal plays a role in transcriptional regulation using a cell-free transcription assay developed by Dr. Roeder45,46, a close collaborator of our lab47–51. This chromatin-based transcription assay will be performed in the following steps: chromatin assembly, activator binding, HPT-mediated or non-enzymatic histone palmitoylation, transcription. The positive control will be transcription in the presence of HAT and acetyl-coA. The negative controls will be transcription in the absence of HPT or palmitoyl-coA. This experiment will be performed in the Roeder lab.
Identifying histone Kpal eraser(s) using HDAC screening. The HDAC screening is modified based on a previous publication3. The reactions will be performed in a 96-well microplate. For each reaction, one HDAC (HDACs 1-11, Sirtuin 1–7) will be incubated with a pool of single palmitoylated synthetic histone peptides at 37˚C for 30 minutes. The reactions is then stopped by adding HCl and acetic acid in methanol. The samples will be dried and analyzed by HPLC-MS/MS to quantify changes in Kpal stoichiometry at each sites. We will further validate the depalmitoylation activity of each eraser candidate in MEF cells by knocking down (KD) the expression level of the eraser using siRNA and analyze the change in histone Kpal level using a combined approach of stable isotope labeling with amino acids in cell culture (SILAC) and HPLC-MS/MS.
Investigating the role of histone Kpal in transcription regulation in vivo. Where the KO mice is available, for example in the case of Sirt6, we will derive KO MEF cells from mice embryo52. If KO mice is not available, we will KD the expression level of the eraser using siRNA. The KO or KD will be validated by western blot. We will perform ChIP-seq using pan-anti-Kpal antibody and RNA-seq using WT, Kpal eraser KO or KD MEF cells. We will plot the fold-change of gene expression versus the fold-change of Kpal level at promoter/enhancer regions, in Kpal eraser KO/KD cell compared with WT cell, to see if increase in Kpal level after eraser KO/KD correlates with increase or decrease in gene expression.
Expected results: For HPT identification, a drastic increase of palmitoylation level in positive control (diluted nuclear extract) compared with negative control (heat denatured nuclear extract) indicates that histone Kpal is catalyzed by enzyme(s). In this case, we anticipate to see enrichment of HPT activity in one or more nuclear extract fractions. However, although histone lysine acylation can be catalyzed by HATs22, protein acylation also occur through non-enzymatic mechanism53–55. If we observe no significant difference of HPT activity in all fractions of nuclear extraction, positive control and negative control, we can conclude that lysine palmitoylation of histones is predominantly a non-enzymatic process.
For histone Kpal eraser identification, we will likely validate result from Lin’s group23 and identify Sirt6 as an eraser for histone Kpal. We may identify other HDACs as erasers in addition to Sirt6. By screening HDACs using a pool of palmitoylated histone peptides instead of a specific peptide (e.g. H3K9), we will be able to determine the substrate selectivity of HDACs for peptide sequences. Based on the crystal structure of Sirt6-H3K9 myristoyl peptide-ADPR complex (PDB: 3ZG6), we predict that Sirt6 to be a sequence independent eraser for histone Kpal, because most inter-molecular interactions between Sirt6 and the modified peptide come from the C=O and N-H of the main chain of the peptide, while the side chain of the peptide only contribute one hydrogen bond.
While we hypothesize that histone Kpal plays a role in transcription regulation, we cannot predict either in vitro or in vivo whether the presence of Kpal confers a positive or negative effect on transcription activity. Whereas the long fatty-acyl chain of palmitoylation may destabilize histone:histone and histone:DNA interaction and loose the nucleosome, the bulk may impede the assembly of transcription factors and RNA polymerase as well. If the stoichiometry of histone Kpal is sufficiently high in the cell-free transcription system, driven by hydrophobic interaction between fatty-acyl chains, palmitoyled nucleosomes may aggregate. If in Aim 1 we observe that histone Kpal marks colocalize with laminB1 in vivo, it is possible that the fatty-acyl chain tether the nucleosome to inner nuclear membrane, where transcription repression factors are known to accumulate36,56. These mechanisms have counteracting effect on transcription, yet can theoretically co-exist. Therefore, whether histone Kpal activates or represses transcription may depends on the relative contribution of these mechanisms.
Potential pitfalls and alternative approaches: We foresee no major challenge in identifying histone Kpal eraser(s), as Sirt6 has already been demonstrated to remove palmitoyl group from H3K9-palmitoyl23 in vitro. However, it is possible that histone palmitoylation cannot occur without enzymatic catalysis but for some reason we fail to purify the HPT from MEF nuclear extract. In this case, we will use synthetic palmitoylated histones to construct the cell-free transcription system.
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