Characterization of Histone Lysine β-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through the histone post-translational modifications. The ketone body β-hydroxybutyrate (BHB) was shown to cause a novel type of epigenetic modification, Histone Lysine β-hydroxybutyrylation (Kbhb), associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation and its effects on cellular epigenome are largely unknown. To unravel these effects, we sought and identified that Kbhb is present in bovine tissues in vivo and further confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. We also demonstrated that the maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation neither to develop until blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but this modification was only faintly detected in oocytes. Profiling the transcriptome in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The down-regulated genes are mainly involved in glycolysis and ribosome assembly pathways, while the up-regulated genes are involved in mitochondrial metabolism and oocyte development. The specific genes and pathways altered by BHB treatment will provide entry points to carry out functional experiments aiming to mitigate problems and improve fertility in cattle suffering metabolic disorders. A key goal for future work will be to understand mechanistically how BHB transmits signals from the environment to affect cellular functions and the bovine epigenome. Summary sentence Beta-hydroxybutyrate induces Histone Lysine β-hydroxybutyrylation in fibroblasts and cumulus-oocyte complexes, it alters the transcriptome in cumulus cells, but does not affect oocyte’s competence to resume meiosis and develop until blastocyst stage.


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Histone Lysine β-hydroxybutyrylation (Kbhb) is a novel epigenetic post-translational 148 modification that was identified in the liver of mice subjected to fasting or streptozotocin-induced 149 diabetic ketoacidosis [5]. Moreover, Kbhb was identified in the brains of depressive mice, 150 indicating a role in modulating mood and suggesting it is physiologically necessary even at 151 basal levels [21]. Dairy cows commonly undergo ketosis episodes during lactation and so far, 152 the post-translational modification (PTM) Kbhb has not been demonstrated in cattle. To 7 investigate whether Kbhb is present in vivo in cattle, we collected samples from dairy cow ovary, 154 cumulus cells, liver, mammary gland, kidney, heart, brain, and from a fibroblast cell culture. We 155 acid-extracted histones and run an SDS-PAGE gel to confirm histone enrichment by Coomassie 156 blue staining ( Figure 1B). Next, we carried out western blot to identify Kbhb utilizing an antibody 157 for the H3K9bhb residue. The choice for the H3K9bhb antibody was due to its robust response 158 to starvation in mouse liver, and specificity by dot blot assays and competition experiments [5].

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As a result, we were able to confirm that Kbhb (hereafter we will use H3K9bhb or Kbhb 160 interchangeably throughout the manuscript) is present in all organs and cells investigated 161 ( Figure 1B). As controls, we used immunoblot for total H3 and H3K9ac, another PTM of the 162 same lysine residue, and, as expected, found that both were present in all samples analyzed.

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Regarding physiological relevance, mice starved for 48 hours showed an increase in 182 liver histone Kbhb that was highly correlated with the transcription of genes involved in a 183 starvation-responsive pathway [5]. Kbhb is reduced in the brain of two mouse models of 184 depressive behavior, i.e. dexamethasone and spatial restraint stress models. BHB increment 185 using a ketogenic diet or through exogenous BHB supplementation led to the reexpression of 186 the neurotrophic factor BDNF, increased H3K9bhb and showed antidepressant effect [21].

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During fasting, levels of histone acetylation increase in the kidney, are maintained in the liver 188 and decrease in the heart, indicating different responses in each organ [19]. These findings 189 suggest that the inhibition of HDACs by BHB may be context-dependent and also that each

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The H3 migrates slower, followed by H2B, H2A and H4 ( Figure 2H). Another observation from 284 our western blots is that the antibody becomes less specific and recognizes other β- 292 among other acylations [5]. Recent work has demonstrated that the radical responsible for 293 Kbhb, β-hydroxybutyryl-CoA is highly reactive and can tag histones by nucleophilic attack [33].

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Our data indicates that exogenous supplementation of BHB dramatically increases Kbhb levels,

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Kbhb is a nutrient-sensitive epigenetic mark that has not been investigated in bovine 630 oocytes so far. To seek for this mark, we exposed COCs to high concentration of BHB during 631 the entire in vitro maturation (~22h). First, we immunostained the whole COC (n>15) using an

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Next, we removed the cumulus cells and focused our search for the effects of BHB on 649 Kbhb levels in oocytes (Figure 8A-H). Curiously, after denuding the oocytes and staining for 650 H3K9bhb we did not observe any Kbhb signal in oocyte metaphase plates (n=44) in the control 651 group (Figure 8A-D). Regarding the treated group (n=43), the signal was only faintly detected in 30 ~53% of the oocytes analyzed (23/43) (Figure 8E-H). We repeated the experiment 4 times and 653 also stained the oocytes in parallel with either H3K4me (n=19, Figure 8I

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Second, BHB influx to the oocyte is controlled to avoid excessive levels. Albeit broadly 687 accepted that BHB is a short fatty acid and can cross freely the membranes, its uptake seems