The Role Of Histone Acetylase MOF In Intestinal Functions

The intestine is where nutrients are digested and absorbed.The intestine development would have an effect on intestinal functions that might be genetically driven.The intestinal homeostasis might be triggered by environmental factors such as intestinal microbiota and feed enzymes.Epigenetic modification plays an important role in regulating the intestinal metabolism and immune system.Histone acetylase Mof,which sepecifically acetylates histone 4 at lysine 16 site（H4K16ac）,is involved in regulating multiple physiological process in both human and animals.However,the effect of Mof on the intestinal development and intestinal microbiota in animals including livestock and poultry is still unknown.The first objective of this study was to explore the role of Mof in intestinal morphology,function and microbiota using the intestinal epithelial cells（IECs）-specific mof knockout mice.The addition of exogenous feed enzymes in feed is another way to maintain the intestinal functions and resultantly homeostatic balance of intestine.Inceasing feed digestibility is another effective way to improve the animal intestinal functions.This is due to the destruction of anti-nutrient factors such as cellulose and thus,promoting the utilization of nutrients such as protein,starch and fat in feed.Lignin is the limiting factor for use of roughage.Laccases have been characterized as efficient lignin degraders.However,laccases have not been used in animal feed,due to lack of suitable commercial enzymes.Thus,the second objective of this study was to isolate and characterize new laccases.Several bacteria and fungi isolates containing laccase activities were isolated.A laccase-like gene（atm）was further identified from a ligninolytic strain,Agrobacterium sp.S5-1;the enzymatic properties of Atm were investigated and its ablity to improve ruminal degradability of maize straw in vitro was measured.The results are shown as follows:1.The effect of Mof in intestinal development.By using the Cre-loxP technology,the IEC-specific mof knockout mice were generated.The mof deletion in intestinal epithelial cells was confirmed by both qPCR and Western blot analyses.The deletion of mof led to significant reduction of H4K16 acetylation in IECs.To explore the role of Mof in intestinal development,we performed immunoblotting,histology assay and immunofluorescence assay using wildtype（WT）and knockout（KO）mice.Compared to WT mice,the length of the small intestine was significantly decreased in KO mice（WT female:36.17±2.75;KO female:28.83±2.10;WT male:37.77±3.35;KO male:31.67±1.76）.In addition,both the amount of Paneth cells and the levels of lysozyme in Paneth cells were decreased due to the mof knockout in IECs.To detetmine the relationship between mof deletion and the proliferation and/or apoptosis in IECs,we performed the immunofluorescence analysis and found that Ki67 and Caspase-3 were increased in intestinal cells of KO mice,suggesting the increased turnover of the intestinal cells.Furthermore,the mRNA expression of cldn5,cldn8 were markedly reduced in IECs of KO mice indicating the destroyed intestinal barrier function.2.The variation of intestinal microbiota in IEC-specific mof knockout mice.By removing low-quality,chimeric sequences and pair-end joined,a total of 1,540,380 reads were obtained from 8 ileal samples from KO mice,8 ileal samples from WT mice,11 colonal samples from KO mice,6 colonal samples from WT mice,9 cecal samples from KO mice,and 4 cecal samples from WT mice.After removing the rare OTUs（<0.005%of total OTUs）,a total of 2,506 OTUs remained for downstream analyses.Theα-diversity analysis among the samples was performed,both the Faith-pd methods and Shannon-Wiener Index showed the highest abundance in cecal samples and the lowest abundance in ileal samples.To determine the similarities between pairs of microbial communities（β-diversity）,a principal component analysis（PCoA）was performed using unweighted UniFrac distance matrices.Unweighted Unifrac PCoA of OTUs from the ileum,colon and cecum indicated that the bacterial community structure differed significantly in different sampling sites.Differential abundance testing with ANCOM was used to determine which,if any,sequence variants and genera were differentially abundant across the gut samples in the KO and WT mice.In this case,the proportion of Prevotella and Helicobacter genera was dramatically increased in the knockout mice across different sample sites.3.Characterization of a novel recombinant laccase Atm and its ability to enhance in vitro digestibility of maize straw.First,seven fungal isolates and seven bacterial isolates with high ligninolyitic activities were isolated from wood decay samples.A novel laccase-like gene（atm）in Agrobacterium sp.S5-1 with the highest laccase antivity was amplified using TAIL-PCR method and was heterologously expressed and purifed.The laccase activity of purified Atm was 185.4 U mg^-1 when using ABTS as the substrate.According to the Linerweaver-burk plots,Km and kcat values were 230.8μM and 32.8 s^-1,respectively.Atm belongs to type[B]laccases.Atm exhibited its maximal activity at pH 4.5 and at 50°C.The activity of Atm was markedly improved by the addition of Cu²⁺.This enzyme was tolerant to high temperature,a broad range of pH,heavy metal ions and all tested organic solvents.Next,effects of Atm on maize straw was investigated.Atm significantly（P<0.05）increased dry matter digestibility of maize straw from 23.44%to 27.96%and from 29.53%to 37.10%after8 or 24 h of digestion,respectively.The combination of Atm and fibrolytic enzymes significantly increased in vitro dry matter digestibility（P<0.05）and neutral detergent fiber digestibility from 19.02%to 24.55%after 24 h digestion.Addition of Atm and the fibrolytic enzymes enhanced acid detergent fiber digestibility from 5.81%to 10.33%and from 12.80%to 19.07%after 8 or 24 h of digestion,respectively.In conclusion,the IEC-specific mof knockout led to decrease of Paneth cells and less expression of the lysozyme,the increased levels of Ki67 and Caspase-3 and the abnormal expression of Claudins and thereby affect the intestinal development and function.Meanewhile,the proportion of Prevotella and Helicobacter genera in intestinal microbiota was also increased in mof-knockout mice.In addition,a novel recombinant laccase Atm from Agrobacterium sp.S5-1was purified and it was tolerant to high temperature,a broad range of pH and organic solvents.The combination of Atm and fibrolytic enzymes significantly increased in vitro dry matter digestibility,neutral detergent fiber digestibility and acid detergent fiber digestibility of maize straw,suggesting that Atm has a great potential for biotransformation of lignin rich agricultural-by products into animal feed.Overall,our study lay the foundation for further studies to improve feed utilization efficiency through maintaining intestinal homeostasis and adding of exogenous enzymes.