基于基因及调控区进化保守性评估细胞和组织发育潜能的定量分析

doi:10.3969/j.issn.1674-8115.2023.11.006

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (11): 1384-1395.doi: 10.3969/j.issn.1674-8115.2023.11.006

• 论著 · 基础研究 • 上一篇

基于基因及调控区进化保守性评估细胞和组织发育潜能的定量分析

王志明¹(), 童冉¹, 杨晨¹, 焦慧媛¹, 王一好², 李林颖³, 王烨欣⁴, 张丰¹(), 李令杰¹()

^1.上海交通大学医学院组织胚胎学与遗传发育学系，上海市生殖医学重点实验室，教育部细胞分化与凋亡重点实验室，上海 200025
^2.上海交通大学医学院附属第九人民医院眼科，上海 200011
^3.上海市儿童医院，上海交通大学医学院附属儿童医院中心实验室，上海 200062
^4.上海交通大学医学院附属第九人民医院口腔外科，上海 200011

收稿日期:2023-06-09 接受日期:2023-10-25 出版日期:2023-11-28 发布日期:2023-11-28
通讯作者: 张丰,李令杰 E-mail:james.wong@sjtu.edu.cn;fzhang@shsmu.edu.cn;lingjie@shsmu.edu.cn
作者简介:王志明（1998—），男，白族，硕士生；电子信箱：james.wong@sjtu.edu.cn。
基金资助:
国家重点研发计划(2021YFA1100400)

Quantitative analysis of the developmental potential of cells and tissues based on evolutionary conservation of genes and regulatory regions

WANG Zhiming¹(), TONG Ran¹, YANG Chen¹, JIAO Huiyuan¹, WANG Yihao², LI Linying³, WANG Yexin⁴, ZHANG Feng¹(), LI Lingjie¹()

^1.Department of Histoembryology, Genetics and Developmental Biology; Shanghai Key Laboratory of Reproductive Medicine; Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Department of Ophthalmology, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^3.Department of Central Laboratory, Shanghai Children′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200062, China
^4.Department of Oral Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Received:2023-06-09 Accepted:2023-10-25 Online:2023-11-28 Published:2023-11-28
Contact: ZHANG Feng,LI Lingjie E-mail:james.wong@sjtu.edu.cn;fzhang@shsmu.edu.cn;lingjie@shsmu.edu.cn
Supported by:
National Key Research and Development Program of China(2021YFA1100400)

摘要/Abstract

摘要：

目的·在DNA序列的保守度层面探讨物种进化与发育之间的关系及其内在规律。方法·分析编码基因的氨基酸序列在100个物种中的保守程度，并建立保守率（conservation rate，CR）这一量化基因进化保守程度的指标，进一步使用胚胎干细胞通路特征基因验证保守率与发育潜能的关系。分析早期三胚层（内胚层、中胚层、外胚层）及其对应的成熟器官（肝脏、心脏和大脑等）的转录组测序（RNA sequencing，RNA-seq）数据，寻找差异表达基因，研究其保守性特点。收集人类早期胚层和成熟器官H3组蛋白第27位赖氨酸乙酰化（histone H3 acetylated at lysine 27，H3K27ac）这一增强子表观遗传标志物的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq）数据，寻找增强子位点，使用ROSE程序鉴定各种细胞和组织中的超级增强子（super enhancer，SE）。使用基因通路富集分析研究超级增强子调控的基因与对应的细胞特征的身份相关性，以明确所鉴定的超级增强子是否符合已有研究报道的特点。使用PhastCons程序计算非编码调控区的DNA保守性评分（conservation score，CS），研究其与发育潜能的关系。结果·在基因编码区，成功建立保守率这一对基因保守程度进行量化的指标。早期三胚层和成熟器官的基因表达数据分析显示：保守率越高的基因与干性和早期发育过程相关性越大，基因保守率指标能区分出发育前后的组织差异。在基因非编码区，发现调控区的保守性也与发育具有相关性：发育早期三胚层的超级增强子序列的保守性评分显著高于对应的成熟器官的超级增强子序列；但细胞特异的普通增强子（typical enhancer，TE）没有呈现出这样的趋势。结论·随着发育进行，在基因编码区特异表达的基因在进化中的保守率下降，非编码调控区的超级增强子DNA保守性评分下降。

关键词: 胚胎发育, 物种进化, 超级增强子, 发育遗传学, DNA保守性

Abstract:

Objective ·To study the relationship between evolution and the developmental process from the perspective of DNA sequence conservation, and explore their inherent principles. Methods ·First, conservation rate (CR) was established by analyzing the conservation of amino acid sequences of coding genes in 100 species to quantify the evolutionary conservation of genes. The relationship between CR and developmental potential was verified by using the feature genes involved in embryonic stem cells pathways. Secondly, cell type-specific genes and their characteristics in conservation were studied by analyzing the RNA sequencing (RNA-seq) data of the three early germ layers (ectoderm, mesoderm and endoderm) and their corresponding mature organs (brain, heart, liver, etc). Then, chromatin immunoprecipitation sequencing (ChIP-seq) data of enhancer histone H3 acetylated at lysine 27 (H3K27ac) from early germ layers and mature organs were collected to search for enhancer sites and identify super enhancers in various cells and tissues by using the ROSE procedure. Functional enrichment and signaling pathway analysis of genes was used to examine the identity correlation between SEs-regulated genes and the corresponding cell characteristics, to clarify whether the SEs identified in this study were consistent with the characteristics reported in previous studies. Finally, PhastCons program was used to calculate the DNA conservation score (CS) of non-coding regulatory regions to study their relationship with developmental potential. Results ·In the coding region of DNA, CR was successfully established to quantify the conservation of genes. The gene expression data of early germ layers and mature organs showed that the genes with higher conservation rate were more relevant to the stemness and early developmental process, and the differences between the tissues from early and late development could be distinguished by using CR. In the non-coding regions of DNA, it was found that the conservation of regulatory regions was also correlated with development. The CS of the SE sequences in the early developmental germ layers was significantly higher than that of the SE sequences in the corresponding mature organs. However, cell-specific typical enhancers (TEs) did not show such a trend. Conclusion ·During the developmental process, CR of genes expressed in the coding region decreases, and CS of super-enhancer DNA in the non-coding region decreases.

Key words: embryonic development, evolution, super enhancer, developmental genetics, conservation of DNA

中图分类号:

R394

王志明, 童冉, 杨晨, 焦慧媛, 王一好, 李林颖, 王烨欣, 张丰, 李令杰. 基于基因及调控区进化保守性评估细胞和组织发育潜能的定量分析[J]. 上海交通大学学报（医学版）, 2023, 43(11): 1384-1395.

WANG Zhiming, TONG Ran, YANG Chen, JIAO Huiyuan, WANG Yihao, LI Linying, WANG Yexin, ZHANG Feng, LI Lingjie. Quantitative analysis of the developmental potential of cells and tissues based on evolutionary conservation of genes and regulatory regions[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(11): 1384-1395.

图/表 12

图1 基因保守率的计算及将基因分配到189个集合的流程Note： A. Calculation of CR and bins of genes. B. GO analysis of gene bins on aging and embryonic stem term. Vertical axis—calculation of percentage of Bin′s genes in terms. CR—conservation rate.

Fig 1 Process of calculating genes′ CR and assigning genes to 189 bins

图2 发育过程细胞类型特异性基因和增强子的保守性分析Note： A. Schematic diagram of germ layer specification and organ development. hESC—human embryonic stem cell. B. CR of cell type-specific genes. C. CS of cell type-specific enhancers.

Fig 2 Conservation analysis of cell type-specific genes and enhancers in development

表1 多种器官发育前后特异表达基因的保守率统计

Tab 1 CRs of genes specifically expressed in early and later development of multiplex organs

CR	Ectoderm development			Mesoderm development			Endoderm development
CR	Brain	Skin	Spinal cord	Heart	Skeletal muscle	Spleen	Liver	Lung	Pancreas
Progenitor-specific	0.72	0.72	0.73	0.72	0.74	0.75	0.71	0.74	0.75
Organ-specific	0.70	0.69	0.66	0.71	0.72	0.65	0.66	0.65	0.68
P value	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
Fold change	1.02	1.05	1.10	1.02	1.02	1.16	1.08	1.14	1.11

表2 多种器官发育前后特异增强子的保守性评分统计

Tab 2 CSs of cell type-specific enhancers in early and later development of multiplex organs

CS	Ectoderm development			Mesoderm development			Endoderm development
CS	Brain	Skin	Spinal cord	Heart	Skeletal muscle	Spleen	Liver	Lung	Pancreas
Progenitor-specific	0.15	0.15	0.14	0.15	0.15	0.15	0.18	0.18	0.18
Organ-specific	0.20	0.11	0.16	0.15	0.12	0.11	0.17	0.11	0.12
P value	0.000	0.000	0.000	0.614	0.001	0.000	0.000	0.000	0.000
Fold change	0.72	1.33	0.87	1.00	1.27	1.37	1.10	1.66	1.54

图3 早期三胚层向相应器官发育过程中细胞特异性超级增强子的鉴定Note： A. The procedure of SE identifications. B. ChIP-seq signal of H3K27ac in TE regions. C. ChIP-seq signal of H3K27ac in SE regions. D. Biological process of SE-related gene. SE—super enhancer; TE—typical enhancer.

Fig 3 Identification of SEs during the development of three early germ layers to corresponding organs

表3 三胚层和相应多种器官中超级增强子、超级增强子元件和普通增强子的数量

Tab 3 Numbers of SEs， SE elements and TEs in three germ layers and corresponding organs

Tissue type	SE/n	SE element/n	TE/n
Ectoderm	839	5 078	74 570
Brain	825	5 654	50 322
Spinal cord	942	6 362	67 360
Skin	1 390	12 679	73 065
Mesoderm	508	4 054	79 411
Heart	1151	11 050	72 112
Spleen	1 712	15 581	78 865
Skeletal Muscle	1 496	12 534	80 847
Endoderm	671	3 770	57 547
Liver	777	3 424	43 749
Lung	538	4 304	57 040
Pancreas	1 657	16 475	94 829

图4 超级增强子的保守性与发育潜能的相关性Note： A. CR of SE-related genes. B. CS of SE elements. C. CS of SEs and TEs.

Fig 4 Correlation between conversation of SEs and development potential

表4 多种器官发育前后超级增强子元件的保守性评分

Tab 4 CSs of SE elements in early and later development of multiple organs

CS	Ectoderm development			Mesoderm development			Endoderm development
CS	Brain	Skin	Spinal cord	Heart	Skeletal muscle	Spleen	Liver	Lung	Pancreas
Progenitor-specific	0.17	0.17	0.17	0.16	0.16	0.16	0.20	0.20	0.20
Organ-specific	0.16	0.11	0.15	0.13	0.11	0.10	0.15	0.10	0.11
P value	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
Fold change	1.09	1.60	1.14	1.22	1.42	1.59	1.37	1.93	1.75

表5 三胚层及相关器官超级增强子元件与普通增强子的保守性评分统计

Tab 5 CSs of SE elements and TEs in three germ layers and corresponding organs

CS	Ectoderm development				Mesoderm development				Endoderm development
CS	Ectoderm	Brain	Skin	Spinal cord	Mesoderm	Heart	Skeletal muscle	Spleen	Endoderm	Liver	Lung	Pancreas
TE	0.06	0.16	0.06	0.08	0.08	0.11	0.07	0.06	0.10	0.12	0.05	0.06
SE	0.08	0.05	0.11	0.15	0.10	0.05	0.06	0.04	0.12	0.06	0.04	0.05
P value	0.001	0.000	0.000	0.000	0.206	0.000	0.000	0.000	0.000	0.000	0.000	0.000
Fold change	1.31	0.31	0.84	0.84	1.18	0.47	0.83	0.72	1.25	0.55	0.82	0.83

图5 超级增强子和附近区域的保守性Note：A. CS signal of SEs and nearby regions. B. The SEs of ectoderm and brain showed on the UCSC genome browser. Vertical axis—signal of ChIP-seq or degree of conservation (UCSC).

Fig 5 Conservation of SEs and nearby regions

表6 高保守增强子在超级增强子和附近普通增强子中的百分比

Tab 6 Percentages of high-conservation enhancers in SEs and nearby TEs

Tissue type	SE/%	Nearby SE/%	Fold change
Ectoderm	3.18	2.83	1.12
Mesoderm	1.04	0.93	1.12
Endoderm	2.32	2.04	1.14
Brain	1.67	1.70	0.98
Skin	0.61	0.66	0.93
Spinal cord	1.60	1.77	0.91
Heart	1.10	0.81	1.36
Muscle	0.42	0.56	0.75
Spleen	0.78	0.92	0.85
Liver	1.35	1.07	1.26
Lung	0.78	1.28	0.61
Pancreas	0.96	0.77	1.25

图6 发育潜能与DNA序列的保守性

Fig 6 Developmental potential and conservation of DNA sequence

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基于基因及调控区进化保守性评估细胞和组织发育潜能的定量分析

Quantitative analysis of the developmental potential of cells and tissues based on evolutionary conservation of genes and regulatory regions

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