Maternal high-zinc diet attenuates intestinal inflammation by reducing DNA methylation and elevating H3K9 acetylation in the A20 promoter of offspring chicks  Changwu Li, Shuangshuang Guo, Jing Gao, Yuming Guo, Encun Du, Zengpeng Lv, Beibei Zhang  Journal of Nutritional Biochemistry  Volume 26, Issue 2, Pages 173-183 (February 2015) DOI: 10.1016/j.jnutbio.2014.10.005 Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 1 Zn supplementation in broiler breeder and progeny bird diets enhanced MUC2 transcription and sIgA production. (A and B) sIgA levels of jejunal mucosa at days 14 and 35 (n=7).OZS birds consistently exhibited markedly higher MUC2 mRNA expression at day 14 (C; n=6) and day 35 (D; n=6). ZD, OZA and OZS represent breeders supplied by extra 0, 50 or 300 mg/kg organic Zn in basal diet, respectively. IZA and IZS represent the breeders supplied by extra 50 or 300 mg/kg inorganic Zn in basal diet, respectively. Low Zn and normal Zn represent the offspring chicks fed with extra 20 or 70 mg/kg Zn in basal diet, respectively. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 2 Supplemental high dose of organic Zn in hens improved the expression of PCNA and caspase 3 in progeny jejunums. Representative Western blots of PCNA and caspase 3 protein in progeny jejunal tissues at days 14 (A; n=4) and 35 (D; n=4) are shown. β-Actin was utilized as the protein loading control. Maternal Zn deficiency resulted in significant up-regulation of caspase 3 at day 14 (C) and down-regulation of PCNA on days 14 (B) and 35 (E). (F) No changes were noted in caspase 3 at day 35 after maternal exposure to Zn. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 3 Maternal excessive organic Zn intake reduced the proinflammatory response of offspring jejunums at day 14. The expression of proinflammatory cytokines IL-1β (A), TNFα (B), IL6 (C) and IL8 (D) was significantly down-regulated by maternal and offspring Zn addition. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars from six chicks. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 4 Zn addition alleviated proinflammatory cytokine expression of offspring jejunums at day 35. mRNA expression of proinflammatory cytokines IL-1β (A), TNFα (B), IL6 (C) and IL8 (D) was determined by quantitative PCR and significantly up-regulated in the ZD group compared with OZS and IZS groups. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars from six chicks. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 5 Zn supplementation reversed the abnormal expression of NF-κB p65 and A20. (A) The mRNA expression at day 14 (n=6). (B) The mRNA expression at day 35 (n=6). Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 6 Zn supplementation in hen diets decreased genomic methylation levels in progeny jejunum. (A and B) Western blots of DNA methyltransferases (DNMT3a and DNMT3b) normalized by corresponding β-actin expression in jejunal tissues at day 14 and day 35 (n=4). (C and D) Global genomic methylation levels detected using the MethylFlash Methylated DNA Quantification Kit in the jejunum at days 14 and 35 (n=6). Global DNA methylation level was calculated as a percentage of 5-mC to total DNA. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 7 Maternal Zn exposure repressed methylation status of the A20 gene promoter in offspring jejunum. (A) Schematic representation of A20 and its CpG-pattern rich regions is presented. Red horizontal line indicates the input sequence. Red vertical lines show the positions of CpG sites within the 1201-bp fragment. (B and C) Methylation frequency at the A20 promoter at day 14 and 35 (n=4). Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 8 Zn supplementation altered the protein abundances of HAT1, HDAC2 and H3K9Ac. (A and B) Protein blot analysis for HDAC2, HAT1 and H3K9Ac at days 14 and 35 (n=4). (C and D) Quantification of days 14 and 35 jejunal H3K9Ac levels. β-Actin and H3 were used as internal controls for HDAC2, HAT1 and H3K9Ac, respectively. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). Values are means, with standard deviations represented by vertical bars. a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 9 Maternal and offspring Zn treatments suppressed H3K9 acetylation at the A20 promoter. (A and C) Maternal and offspring Zn addition enhanced H3K9Ac at the promoter regions (340–425 and 506–603 bp) of the A20 gene at day 14. (E) Dietary organic Zn supplementation in hens elevated the H3K9Ac levels of offspring jejunums (1691–1745 bp). (B, D and F) ChIP analysis of the A20 gene in jejunal tissues at day 35 from the maternal and offspring Zn-treated groups using the antiacetylated H3K9 (A) antibodies. Levels of A20 promoters in the IPs were measured by quantitative PCR. Statistical analysis was performed by SPSS GLM, and a one-way ANOVA was performed using the Duncan correction for multiple comparisons when the interaction was significant (P<.05). a,bTreatments with unlike letters were significantly different (P<.05). Journal of Nutritional Biochemistry 2015 26, 173-183DOI: (10.1016/j.jnutbio.2014.10.005) Copyright © 2015 Elsevier Inc. Terms and Conditions