基于DNA甲基化组学技术分析TET 1基因对小鼠uNK细胞DNA甲基化的影响

doi:10.11843/j.issn.0366-6964.2025.02.039

Abstract

Abstract:

The purpose of this study was to investigate the effect of ten eleven translocation 1 (TET 1) on the methylation of the DNA of mouse uterine natural killer (uNK) cells, and to gain insight into its molecular regulatory mechanism. Mouse uterine metaphase was aseptically collected on the 10th day of gestation, and the uNK cells were isolated and purified for culture. The expression of TET 1 gene was knocked down by RNA interference technology. Then, total DNA from cells of the interference group and the normal control group were extracted, respectively. Sequencing was performed using reduced representation bisulfite sequencing (RRBS) method, and the results were analyzed by bioinformatics software for the statistics and annotation of differentially methylated region (DMR) in the two group samples, and further analyzed by the GO database for DMR-related genes to understand their functions. Also, the sequencing results were analyzed for the differentially methylated region (DMR) annotation, and the enriched signaling pathways in which regulated by the KEGG database. The results showed that there were 14 120 DMRs in the TET 1 interference group when compared with the control group, of which 4 897 were hypermethylated DMRs and 9 223 were hypomethylated DMRs. The largest number of DMRs were distributed in the genebody, with a total of 9 762 DMRs, accounting for 69.14% of the total number of DMRs. DMRs were widely distributed in the different elements of the genome, and some genes had DMRs with both hypermethylated and hypomethylated in different elements. GO annotation results showed that the DMRs were mainly concentrated in ATP binding, nucleic acid binding, cell formation, cell differentiation, embryonic development, RNA polymerase II transcriptional regulation and negative regulation of cell proliferation, etc. KEGG database analysis results revealed that the DMRs were significantly enriched in the metabolic pathways, with a total of 12 key molecules involved in metabolism in the pyruvate metabolic pathway, and 54 DMRs appeared, which was the most significant enrichment of DMRs in the metabolic pathway. Among the 12 key molecules, acetyl-coA synthetase (Acss), lactate dehydrogenase B (Ldhb), and pyruvate kinase L/R (Pklr) exhibited a single hypermethylated status. In addition, significant enrichment of DMRs were also found in the PI3K/AKT signaling pathway and HIF-1 signaling pathway, indicating that DMRs play important roles in mediating pyruvate metabolism. Thus, TET 1 has a methylation regulatory effect mouse uNK cells, and pyruvate metabolism is the main pathway for its regulatory effect. What's more, Acss, Ldhb and Pklr are potential regulatory target molecules, while PI3K/AKT and HIF-1 are important signaling pathways which involved in regulation.

Key words: TET 1, uNK cell, methylomics, pyruvate, mouse

CLC Number:

S857.2

ZHAO Jingxian, YANG Xiaowei, LIU Yanyan, ZHAO Ziliang, ZHAO Guangwei, ZHAO Yongju. Analysis of the Effect of TET 1 Gene on Methylation of Mouse uNK Cells based on DNA Methylation Histology Technique[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 912-924.

Figures/Tables 8

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名称Name	序列(5′→3′) Sequence
GAPDH-F	GTCTCCTCTGACTTCAACAGCG
GAPDH-R	ACCACCCTGTTGCTGTAGCCAA
WPRE-F	CGCTATGTGGATACGCTGCTTTA
WPRE-R	GCAACCAGGATTTATACAAGGAGGA
β-actin-F	CCACTGTCGAGTCGCGTCC
β-actin-R	ATTCCCACCATCACACCCTGG
TET 1-F	TGGGAGAGCTCCCTTTGATGGTTT
TET 1-R	TTGGGTCAATTGTGCTGCGACATC

样本Sample	序列Reads	碱基Bases	Q20	Q20(%)	Q30	Q30(%)
NC_uNK-1	18 661 773	4.95×10⁹	4.80×10⁹	96.99	4.42×10⁹	89.16
NC_uNK-2	17 315 461	4.9×10⁹	4.75×10⁹	96.83	4.42×10⁹	90.28
NC_uNK-3	17 357 287	4.92×10⁹	4.77×10⁹	96.97	4.42×10⁹	89.88
TET 1KD_uNK-1	17 913 483	4.95×10⁹	4.80×10⁹	96.98	4.43×10⁹	89.51
TET 1KD_uNK-2	18 460 166	4.92×10⁹	4.77×10⁹	97.09	4.39×10⁹	89.30
TET 1KD_uNK-3	18 510 276	5.01×10⁹	4.85×10⁹	96.86	4.43×10⁹	88.54

样本 Sample	C位点类型 C site type	全基因组C位点数量 Number of whole genome C site	平均测序深度 Average sequencing depth	有效覆盖深度 Effective depth of coverage	覆盖度Coverage		Msp I酶切效率/% Msp I digestion efficiency
样本 Sample	C位点类型 C site type	全基因组C位点数量 Number of whole genome C site	平均测序深度 Average sequencing depth	有效覆盖深度 Effective depth of coverage	测序深度5×以上位点 Sequencing depth of 5× or more site	测序深度10×以上位点 Sequencing depth of 10× or more site	Msp I酶切效率/% Msp I digestion efficiency
NC-uNK-1	CG	4 477 985	20.956 05	20.956 05	0.638 982	0.516 772	99.53
	CHG	9 963 429	20.892 63	20.892 63	0.597 96	0.501 204
	CHH	23 957 443	19.630 41	19.630 41	0.560 375	0.464 912
NC-uNK-2	CG	4 827 758	14.268 55	14.268 55	0.518 964	0.368 129	99.60
	CHG	12 425 721	15.684 28	15.684 28	0.544 611	0.407 767
	CHH	32 032 980	15.228 59	15.228 59	0.520731	0.387 003
NC-uNK-3	CG	4 739 099	14.328 09	14.328 09	0.500 71	0.356 494	99.51
	CHG	12 170 807	15.762 94	15.762 94	0.522 228	0.391 935
	CHH	31 218 592	15.280 84	15.280 84	0.497 111	0.370 422
TET 1-KD-uNK-1	CG	4 971 883	15.737 09	15.737 09	0.559 367	0.417 201	98.39
	CHG	12 485 237	16.015 49	16.015 49	0.533 318	0.421 735
	CHH	31 158 989	15.422 20	15.422 20	0.506 721	0.398 892
TET 1-KD-uNK-2	CG	4 921 960	18.209 16	18.209 16	0.615 603	0.492 745	98.20
	CHG	11 919 056	17.074 72	17.074 72	0.542 751	0.449 476
	CHH	29 141 027	16.233 09	16.233 09	0.509 518	0.418 160
TET 1-KD-uNK-3	CG	5 326 157	15.892 160	15.892 16	0.560 050	0.434 085	97.61
	CHG	13 524 746	14.945 568	14.945 57	0.492 790	0.399 544
	CHH	33 889 240	14.220 125	14.220 12	0.458 943	0.370 210

起始 Start	终止 End	差异甲基化水平 Diff	启动子 Promoter	2K上游 Upstream2k	外显子 Exon	内含子 Intron	基因体 Genebody	5′非翻译区 5′ UTR	3′非翻译区 3′ UTR
150 647 817	150 647 953	0.418 333	--	--	--	Acss	Acss	--	--
23 204 147	23 204 310	0.340 533	--	--	--	--	Ldhb	--	--
173 153 059	173 153 123	0.263 533	Pck1	Pck1	Pck1	--	Pck1	Pck1	--
4 585 071	4 585 163	0.209 667	--	--	--	Pcx	Pcx	--	--
89 136 449	89 136 466	0.207 2	Pklr	--	--	Pklr	Pklr	--	--
160 137 770	160 137 858	-0.111 93		--		Pdha1	Pdha1	--	--
114 147 897	114 147 964	-0.139 11	--	--	--	Acacb	Acacb	Acacb	--
111 626 585	111 626 779	-0.139 57	--	--	Ldhd	--	Ldhd	--	Ldhd
30 606 560	30 606 690	-0.142 03	--	--	--	Glo1	Glo1	--	--
173 152 732	173153001	-0.142 8	Pck1	Pck1	--	--	--	--	--
160 137 879	160 137 935	-0.173 4	Pdha1	--	--	Pdha1	Pdha1	--	--
121 592 596	121 592 800	-0.186 19	--	--	--	Aldh2	Aldh2	--	--
4 618 284	4 618 430	-0.188 06	--	--	Pcx	--	Pcx	--	--
114 174 985	114 175 011	-0.194 2	--	--	--	Acacb	Acacb	--	--
44 984 450	44 984 669	-0.194 38	--	--	Grhpr	Grhpr	Grhpr	--	--
114 146 703	114 147 413	-0.198 64	Acacb	--	--	Acacb	Acacb	Acacb	--
30 572 773	30 572 987	-0.252 46	--	--	--	Acyp2	Acyp2	--	--

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Analysis of the Effect of TET 1 Gene on Methylation of Mouse uNK Cells based on DNA Methylation Histology Technique

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