Functional epigenomic network of Kdm3b and Smug1 during mouse retinal aging

국문 초록 (Abstract)

후성유전학적 변화는 가역적인 크로마틴의 구조 변화를 통해 전사과정을 시간적 그리고 공간적으로 적절하게 유도하는 데 중요한 역할을 한다. 유전체의 후성유전학적 변화들은 매우 역동적이며 짧은 시간동안 빠르게 유전자 발현을 조절한다. 따라서 후성유전학적 변화의 역동성을 특성화하고 그로 인해 영향 받는 서로 다른 유전자들의 발현 조절을 밝히는 것은 어렵다. 대표적인 후성유전학적 메커니즘으로 히스톤 변형과 DNA 메틸화가 알려져 있으며 이는 DNA를 heterochromatin 상태로 응축시키거나 eu-chromatin 상태로 풀어 줌으로써 유전자 발현을 조절한다. 발생과정동안 세포는 세포 유형 특이적인 유전자 발현의 패턴을 확립하기 위하여 광범위한 후성유전체 변화를 통해 세포 분화를 유도한다. 하지만 전사체 그리고 크로마틴 네트워크를 포함한 후성유전학적 정보는 노화과정동안 비정상적으로 조절된다. 노화와 후성유전학의 연관성을 제안하는 모델 중 하나는 ‘heterochroma-tin의 감소’ 이다. 이 모델은 배아 단계동안 확립된 heterochroma-tin 도메인이 노화과정동안 분해되어 전체 핵 구조의 변경을 통해 비 정상적인 유전자 발현 패턴을 유도함을 시사한다.
본 논문에서, 나는 핵심적인 후성유전학 조절인자가 세포 유형 특이적인 기능 유전자들의 조절을 통해 분화된 세포들의 항상성을 유지하는데 중요한 역할을 할 것이라는 가설을 세웠다. 본 가설을 검증하기 위하여 나이든 생쥐 망막에서 heterochromatin의 형태학적 변화를 확인하였다. 흥미롭게도, 나이든 생쥐 망막의 원추세포에서 다른 세포 유형과 달리 명백한 ‘heterochromatin의 감소’가 관찰되었다. Heterochromatin의 감소와 연관된 후성유전학적 조절인자들을 발굴하기 위하여 분화가 완료된 망막 뉴런 세포 들에서 조직 수준 mRNA-seq, 단일세포 수준 mRNA-seq, DNA 메틸화를 위한 WGBS 그리고 ChIP-seq을 이용하여 통합적인 후성유전체 분석을 시행하였다. 그 결과 망막 노화과정동안 H3K9 탈 메틸화 효소로 알려진 Kdm3b와 DNA 탈 메틸화 효소로 알려진 Smug1의 mRNA 발현이 점차 감소하고 있음을 확인하였다. 또한 KDM3B와 SMUG1의 단백질 발현량은 분화가 완료된 원추세포들에서 매우 높게 발현하고 있었다. 노화과정동안 망막에서 Kdm3b와 Smug1의 후성유전학적 조절 메커니즘을 연구하기 위하여 Kdm3b와 Smug1 녹아웃 생쥐를 제작하였으며 분자적 수준에서 다양한 기능적 그리고 생리학적 연구들을 시행하였다.
먼저, 12개월령 Kdm3b 반 감수성 생쥐 망막에서 원추세포와 원추세포의 리본 시냅스의 형태가 변화하는 것이 관찰되었다. 이러한 비 정상적인 표현형은 최종적으로 세포자사를 통해 원추세포의 감소를 유도하며 최종적으로 시냅스 후 신호전달능력을 저하시킴을 확인할 수 있었다. 전사체 그리고 후성유전학적 프로파일링을 종합하여 분석한 결과, 나이든 Kdm3b 반 감수성 생쥐 망막에서 시냅스 조립 그리고 소낭 전달과 관련된 유전자들이 H3K9me1 그리고 H3K9me2의 축적에 의해 발현이 저하된다는 것을 확인할 수 있었다. 또한, 나이든 Kdm3b 반 감수성 생쥐 망막에서 히스톤 탈 메틸화의 비정상적인 조절 외에도 세포자사 관련 유전자들의 발현이 증가하는 것을 확인하였다. Kdm3b 에 의한 생물학적 현상들의 이해를 향상시키기 위하여 KDM3B 중심적인 네트워크를 구축한 결과, Kdm3b 가 결핍된 나이든 생쥐 망막에서 KDM3B 중심적인 네트워크의 특성이 변화하는 것을 확인하였다. 이러한 결과들은 KDM3B 중심적인 네트워크 특성의 미세한 차이에 의한 비 정상적인 전사체 변화는 heterochromatin 특징을 변화 시키며 이는 세포 자사와 망막에서 역할을 하는 유전자들의 기능에 영향을 줄 수 있음을 제시한다.
다음으로, Smug1의 부재가 2개월령 생쥐 망막에서 원추세포의 segment와 시냅스의 형태학적 결핍을 유도하며 이는 12개월령 생쥐 망막에서 원추세포의 퇴화를 유도함을 밝혀내었다. 이러한 생리학적 결핍은2개월령과 12개월령에서 모두 원추세포의 신호 흡수 방해와 신호 전달의 지연을 유도하였다. DNA 메틸화 패턴과 유전자 발현을 통합하여 후성유전학적 네트워크를 분석한 결과 광 전달 그리고 시냅스 구축과 관련된 유전자들의 발현이 감소하였으며 이는 Smug1 결핍에 의한 3’UTR 영역의 CpH 메틸화 축적에 의한 것을 확인하였다. 또한, 18개월령 Smug1 결핍 생쥐 망막에서 단백질 번역과 관련된 유전자들의 발현이 감소하였으며 이는 Smug1 결핍에 의한 프로모터 영역의 CpG 메틸화 축적에 의한 것임을 밝혀내었다. 이러한 발견들을 통해 Smug1에 의한 망막에서 기능을 하는 유전자들의 DNA 탈 메틸화는 망막의 항상성을 유지하며 이러한 기능들은 시간적인 조건 즉 발생단계별로 서로 다른 유전체 영역에서 일어남을 제시하였다.
본 연구는 생쥐 망막 발생과정동안 Kdm3b와 Smug1이 후성유전학적 변화에서 발생 단계에 따라 서로 구별된 역할을 하고 있음을 밝혀내었다. 파트 I 에서는, Kdm3b의 결핍이 나이든 생쥐에서 원추세포 리본시냅스의 비정상적인 형태학적 변화를 일으켜 원추세포의 활성을 손상시킴을 밝혀내었다. 또한 조직 그리고 단일 세포 수준의 mRNA 시퀀싱과 H3K9me1, H3K9me2, H3K9me3 그리고 KDM3B 항체를 이용한 ChIP 시퀀싱 기법을 이용한 전사체 그리고 후성유전학적 프로파일링의 통합적인 분석을 통하여, Kdm3b 반감수성이 H3K9me1 그리고 H3K9me2의 축적을 통한 시냅스 구축 관련 유전자들의 발현 감소를 유도함을 밝혀내었다. 파트 II 에서는, Smug1의 결핍이 원추세포와 원추양극세포사이의 연접성, 광 전달 경로, 그리고 원추세포 생리활성의 결핍을 유도함을 밝혀내었다. 또한 조직 그리고 단일세포 수준의 mRNA 시퀀싱과 SMUG1 항체를 이용한 ChIP 시퀀싱 그리고 전장유전체 메틸화분석 (WGBS) 을 종합 분석하여, Smug1의 결핍이 3’UTR 영역의 CpH 메틸화를 축적시켜 광수용체 세포의 기능과 연관된 유전자들의 발현을 감소시킨다는 것을 밝혀내었다. 더불어, 노화단계에서 Smug1의 결핍은 프로모터 영역의 CpG 메틸화를 축적시켜 단백질 번역과 연관된 유전자들의 발현을 감소시킨다는 것을 밝혀내었다. 이와 같은 결과들을 통해 Kdm3b와 Smug1에 의한 영역 특이적인 후성유전학적 조절은 발생단계 그리고 노화단계에서 망막의 항상성 유지에 기여한다는 것을 제시하였다.

다국어 초록 (Multilingual Abstract)

Epigenetic modifications play a central role in the spatial and temporal induction of appropriate transcriptional pathways via reversible alteration of chromatin. The epigenetic modifications of the genome are highly dynamic and control rapid and short-term regulation of gene expression. For these reasons, it is difficult to characterize the dynamics of epigenetic changes and reveal their impact on differential gene expression regulation. The typical epigenetic mechanism is known as histone modifications and DNA methylation, which regulate gene expression by allowing DNA to condense into heterochromatin or open into euchromatin. During development, the epigenome undergoes extensive modification driving cellular differentiation to establish cell type-specific patterns of gene expression. During the aging process, however, the epigenetic information of transcriptional and chromatin networks was dysregulated. One of the models proposing the involvement of epigenetics in aging was the ‘loss of heterochromatin’. This model proposes that the heterochromatin domain, which is established during embryogenesis degraded during aging, resulting in aberrant gene expression patterns via changes in global nuclear architecture.
In this dissertation, I hypothesized that key epigenetic regulators maintain the homeostasis in differentiated cells through the regulation of cell-type specific functional genes. To test this hypothesis, morphological changes of heterochromatin was first confirmed in aged mice retina. Interestingly, the clear ‘loss of heterochromatin’ in cone photoreceptors of aged retina was found, but not in other cell types. To further identify the epigenetic regulators involved in heterochromatin loss, transcriptomic analysis of fully differentiated retinal neuron cells was performed by the genome-wide analysis such as mRNA-seq, scRNA-seq, whole genome bisulfite sequencing for DNA methylation and ChIP-seqs. The mRNA expression of histone H3 K9 demethylase Kdm3b and DNA demethylase Smug1 was gradually decreased during retinal aging. The protein expression patterns of both KDM3B and SMUG1 was highly enriched in matured cone photoreceptors. To study the epigenetic regulatory mechanisms of Kdm3b and Smug1 during retinal aging, Kdm3b and Smug1 knock-out mice were generated, and various functional and physiological studies at the molecular level were executed in this dissertation.
First, the disruption of Kdm3b of 12-month-old mouse retina changes morphology of cone photoreceptor and cone ribbon synapse. These abnormal phenotypes lead to decreasing number of cone photoreceptors via apoptosis and it impaired synaptic transmission. The integration of transcriptomic and epigenomic profiling demonstrated that synapse assembly and vesicle transport genes were downregulated via the accumulation of H3K9 mono- and di-methylation in aged Kdm3b deficient mouse retina. Also, instead of dysregulation of histone demethylation effects, the apoptotic genes were activated in aged Kdm3b deficient mouse retina. KDM3B-centered network was constructed to improve the understanding of biological phenomena, and its properties were changed in aged Kdm3b deficient mouse retina. These results revealed that potential cause of abnormal transcriptome changes by subtle changes of KDM3B-centered network characteristics modulate the heterochromatin features depend on the gene functions, such as apoptotic and retinal-functional genes.
Second, the ablation of Smug1 leads to the phenotypical defects in cone photoreceptor segment and synapse at 2 months and cone photoreceptors were degenerated through the apoptotic cell death at 12 months. These physiological defects induced signal absorption disruption and synaptic transmission delay in cone photoreceptors at 2 and 12 months. Through the integrated epigenomic network analysis with DNA methylation and gene expression, phototransduction and synapse organization genes were downregulated via the accumulation ‘CpH’ methylation on 3’UTR in 2-month-old Smug1 deficient mouse retina. Moreover, the protein translation genes were downregulated via the accumulation of ‘CpG’ methylation on promoter region in 18-month-old Smug1 deficient mouse retina. These findings suggest that Smug1 mediated DNA demethylation in retina functional genes to maintain the homeostasis, and its functions in differential genomic regions depending on temporal conditions.
Here, the distinct roles of Kdm3b and Smug1 in temporal epigenetic changes were discovered during mouse retina development and aging. In part , I discovered that the disruption of Kdm3b leads to abnormal morphological changes of cone ribbon synapses and impairs cone cellular activity in the aged mouse retina. Through the integration analysis of transcriptomic and epigenomic profiling using mRNA-seq (both bulked and single-cell) and ChIP-seq with H3K9me1, H3K9me2, H3K9me3, and KDM3B, haploinsufficiency of Kdm3b induced downregulation of synapse organization-related genes via the accumulation of H3K9 mono-, and di-methylation. In part , I suggested that the absence of Smug1 causes defects of cone-cone bipolar connection, phototransduction pathway, and cone cellular activity in the adult mouse retina. In addition, the absence of Smug1 induces cone cell degeneration in the aged mouse retinas. By integrative genome-wide analysis using mRNA-seq (both bulked and single-cell), ChIP-seq with SMUG1, and WGBS, the ablation of Smug1 induced downregulation of photoreceptor functional genes via the accumulation of 3’UTR CpH methylation in the adult mouse retina. In addition, the ablation of Smug1 induced the downregulation of protein translation genes via the accumulation of promoter CpG methylation in the aged mouse retina. Taken all together, these results suggest that site-specific epigenetic regulation by Kdm3b and Smug1 contributes to the maintenance of retinal homeostasis during development and aging.

목차 (Table of Contents)

• 1. General Introduction 1
• 1.1 Epigenetic regulation 1
• 1.1.1 Post-translational modifications (PTMs) of histone proteins 1
• 1.1.1.1 Histone H3 methylation at lysine 9 (H3K9 methylation) 4
• 1.1.1.2 The role of histone H3 lysine 9 specific demethylase 3B (KDM3B) 7
• 1.1.2 DNA methylation: CpG and non-CpG (CH) methylation 10
• 1.1.2.1 DNA demethylation: Passive and Active demethylation 13
• 1.1.2.2 Crosstalk between Base-excision repair (BER) pathway and DNA demethylation 17
• 1.1.2.3 The patterns of Histone methylation and DNA methylation during development 23
• 1.2 Structure and function of the retina 26
• 2. General Materials and Methods 32
• 2.1 Immunohistochemistry (IHC) and Image analysis 32
• 2.2 Immunoprecipitation and immunoblot 33
• 2.3 Electroretinogram (ERG) 33
• 2.4 mRNA sequencing and bioinformatical analysis 34
• 2.5 Retina dissection and dissociation for single cell RNA sequencing (scRNA-seq) 36
• 2.6 Single cell library preparation and sequencing 36
• 2.7 Bioinformatics processing (scRNA-seq) 37
• 2.8 Statistical analysis 39
• 3. PART I. Site-specific gains and losses of heterochromatin accelerate the neuronal aging through the cascading destruction of KDM3B-centered epigenomic network 40
• 3.1 Introduction 41
• 3.2 Materials and Methods 44
• 3.2.1 Animals 44
• 3.2.2 Antibodies 44
• 3.2.3 AAV viral vector production and subretinal infection 45
• 3.2.4 Total RNA isolation and RT-qPCR 45
• 3.2.5 Mouse apoptosis antibody array 46
• 3.2.6 Transmission electron microscopic (TEM) analyses 47
• 3.2.7 Chromatin immunoprecipitation (ChIP) sequencin g 47
• 3.2.8 ChIP-seq library preparation and bioinformatical analysis 50
• 3.2.9 Motif discovery 50
• 3.2.10 Epigenomic network construction 51
• 3.2.11 Measurement of network structural dissimilarities 51
• 3.3 Results 53
• 3.3.1 Heterochromatin features are altered in cone cells during aging 53
• 3.3.2 Identification of Kdm3b as heterochromatin modulator in aging mouse retina 59
• 3.3.3 mRNA stability of Kdm3b is gradually decreased in cone cells during aging 63
• 3.3.4 Haploinsufficiency of Kdm3b induces abnormal eye phenotypes 70
• 3.3.5 Kdm3b regulates the expression of cone cell-specific apoptotic and synaptic functional genes 81
• 3.3.6 Haploinsufficiency of Kdm3b induces cone cell degeneration in aging 91
• 3.3.7 Haploinsufficiency of Kdm3b occasion impairment of synapse structure and synaptic transmission between cone and cone bipolar cells 103
• 3.3.8 Kdm3b regulates the expression of synaptic functional genes through H3K9me1 and H3K9me2 demethylation 111
• 3.3.9 KDM3B modulates retinal homeostatic and cell deathrelated genes by int eracting with AP-1 transcription factors 125
• 3.3.10 Haploinsufficiency of Kdm3b accelerates the destruction of epigenomic network robustness 129
• 3.4 Discussion 133
• 4. PART II. SMUG1-mediated temporal and spatial DNA demethylation contribute to photoreceptor homeostasis during mouse retina development 137
• 4.1 Introduction 138
• 4.2 Materials and Methods 140
• 4.2.1 Animals 140
• 4.2.2 Antibodies 140
• 4.2.3 Whole-genome bisulfite sequencing (WGBS) and bioinformatical analysis 141
• 4.3 Results 143
• 4.3.1 Identification of Smug1 as DNA demethylation regulator in mouse retina during development 143
• 4.3.2 Deficiency of SMUG1 induces abnormal morphologies and degenerative phenotypes of cone cells 155
• 4.3.3 Deficiency of SMUG1 impairs visual perception by downregulates phototransduction and synaptic adhesion genes 160
• 4.3.4 SMUG1 regulates 3'UTR DNA demethylation in mouse retina 169
• 4.3.5 SMUG1 regulates retina functional genes -specific constant expression via DNA demethylation 180
• 4.3.6 Depletion of SMUG1 induces photoreceptor degeneration during aging 184
• 4.3.7 SMUG1-mediated CG demethylation maintains photoreceptor homeostasis via regulating protein stabilization-related genes in the aging 190
• 4.4 Discussion 198
• 5. Conclusion 202
• 6. References 204
• 국문초록 214

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