(A) study on the gene expression regulation by chromatin organization factor LAP2α through differential recognition of histone modification patterns

국문 초록 (Abstract)

적절한 시공간에서의 유전자 발현은 세포의 생존에 있어서 매우 필수적이기에 유전자가 전사되어 단백질로 만들어지는 일련의 과정 속에서 각 단계마다 유전자 발현은 엄격히 조절되고 있다. 유전자 발현을 조절하는 여러 기작 중 염색질 구조는 일차적으로 여러 인자의 접근성을 결정하는 역할을 하고 있다. 이러한 염색질 구조는 히스톤 코드라고 알려진 다양한 히스톤 잔기의 변형에 의해 조절된다. 핵 단백질인 lamin과 상호작용하는 여러 단백질 중 LAP2α는 유일하게 핵질 내에서 염색질과 상호작용하고 있으며 그 동안 주로 세포 주기를 조절하는 역할을 한다고 알려졌지만 최근 히스톤 잔기의 변형을 유도하여 염색질 구조 변화에 영향을 준다는 것이 밝혀졌다. 하지만 구체적인 분자생물학적 기전은 잘 알려져 있지 않다. 본 연구에서는 CRISPR-Cas9 시스템을 사용하여 LAP2α 단백질이 적게 생성되는 LAP2α녹다운 (KD) H1299 세포주를 제작하였다. 해당 세포주에서는 세포 주기의 G2/M 기의 정지와 세포 사멸의 증가를 포함한 세포 생리학적 변화가 관찰되었다. 어떤 유전자들의 발현 변화가 일어나는지 알아보기 위해 RNA-sequencing 방법을 이용한 전사체 분석을 수행하였다. 총 1,312개의 차별 발현 유전자 (DEGs)를 확인한 결과 DEGs 중 세포 주기 조절 유전자들과 염색질 재구성 유전자들 및 전사 조절 유전자들은 광범위하게 감소되어 있었으며 히스톤 아세틸화 유전자들 및 이질염색질 형성 유전자들과 세포 사멸 유전자들은 증가되어 있었다. 전반적으로 염색질과 히스톤 변형에 관련된 유전자들이 확인되어 LAP2α가 직접 히스톤과 결합하여 전반적인 히스톤 변형에 영향을 주는 것인지 알아보았다. LAP2α가 히스톤 H3와 H4에 직접 결합하는 것을 확인하였지만 광범위한 히스톤 변형 자체에는 영향을 주지는 않았다. 그렇다면 LAP2α가 변형된 히스톤 잔기를 인지하여 결합하는 것인지를 확인한 결과 특이적으로 아세틸화된 H3 라이신 (lysine) 잔기와 메틸화된 H4 아르기닌 (arginine) 잔기와 상호작용하는 것을 발견하였다. 또한, 히스톤 변형자들인 PCAF와 CBP, HDAC1과 직접적으로 결합하는 것을 면역침강 실험을 통해 확인하였다. RNA-seq 및 공공 ChIP-seq 자료를 통합하여 LAP2α가 히스톤 변형자들과 복합체를 이루어 세포 주기 조절 유전자들 및 전사조절 유전자들을 활성화시키고 히스톤 아세틸화 유전자들과 세포 사멸 유전자들을 억제시킨다는 것을 확인하였다.

다국어 초록 (Multilingual Abstract)

Chromatin architecture alteration plays an essential role in the appropriate gene expression regulation. The chromatin architecture is regulated dynamically by histone modifications called histone code. Histone modifications and the enzymes that perform these events can influence chromatin compaction, nucleosome dynamics, and transcription. Histone modifiers and chromatin-bound proteins regulate transcription activation and repression. At the steady state of the cell, a balance between specific modifications and modifiers is preserved to maintain chromatin structure, execute the proper gene expression program, and control the biological outcome. Lamina-associated polypeptide 2α (LAP2α) is known to associate with nuclear lamin A/C in the nuclear interior. The LAP2α-lamin A/C complex regulates epigenetic gene regulation and higher-order chromatin architecture through the interaction with DNA and chromatin. Recently, it has been reported that LAP2α controls the cell cycle via interaction with nuclear lamin A/C. However, the LAP2α-mediated molecular mechanism in gene regulation remains to be fully elucidated.
In this study, I aimed to address the underlying mechanism of how LAP2α selectively prefers histone modification in its specific target gene expression regulation. The LAP2α KD stable cell line was constructed using shRNA-induced gene knockdown, and transcriptome analysis was performed through RNA-seq. 1,407 differentially expressed transcripts (DETs) in LAP2α KD cells were identified. Genes related to cell cycle regulation and transcription regulation were downregulated, and genes related to histone deacetylation and apoptosis were upregulated. As previously, ICC and immunoblot were performed with active and repressive histone markers in LAP2α KD cells. However, alterations in global histone modification were not changed. Then, to confirm whether LAP2α recognizes the specific genomic regions, direct interactions with histones and LAP2α were investigated through GST-pull-down assay. LAP2α was directly bound to H3 and H4, and as a result of performing a histone peptide array, it was confirmed that LAP2α specifically binds to H3 lysine-acetylated residue and H4 arginine methylated residue. An immunoprecipitation assay was performed to determine whether these histone modification-inducing modifiers bind to LAP2α. It was confirmed that LAP2α directly interacts with the histone acetyltransferases PCAF and CBP and the histone deacetylase HDAC1. The target gene sets were selected by integrating public ChIP-seq data of PCAF, CBP, and HDAC1 and LAP2α KD RNA-seq data. LAP2α-PCAF and LAP2α-CBP complexes activate the expression of genes related to cell cycle regulation, chromatin remodeling, and transcription, while LAP2α-HDAC1 complex suppressed histone acetylation and apoptosis-related gene sets. Taken together, LAP2α forms a complex with PCAF, CBP, and HDAC1 and specifically recognizes and binds to H3 acetylated residues and H4 arginine methylated residues near genes to be regulated. PCAF and CBP make the surroundings of sets of genes involved in cell cycle regulation, chromatin remodeling, and transcription euchromatic, turning on those genes. In addition, HDAC1 makes the surroundings of a set of genes related to histone acetylation and apoptosis heterochromatic, turning off the corresponding genes.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1. Regulation of gene expression 1
• 1.2 Chromatin structure 3
• 1.3 Histone modifications 6
• 1.4 Nuclear lamins and LAP2 family 11
• 1.5 Functions of LAP2α 16
• 1.6. View of this study 19
• 2. Materials and Methods 20
• 2.1 Cell cultures 20
• 2.2 Generation of LAP2α knockout (KO) and knockdown (KD) stable cell lines 20
• 2.2.1 Construction of knockout (KO) cell line 20
• 2.2.2 Construction of knockdown (KD) cell line 21
• 2.3 Immunoprecipitation 21
• 2.4 Immunoblotting 22
• 2.5 Immunocytochemistry (ICC) and image analysis 22
• 2.6 Histone purification 23
• 2.7 Total RNA extraction and RT-qPCR 23
• 2.8 Annexin VFITC/propidium iodide (PI) apoptosis assay 25
• 2.9 Cell cycle assay 25
• 2.10 RNA sequencing and bioinformatical analysis 26
• 2.11 Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) 27
• 2.12 Statistical analysis 27
• 2.13 GST-pull down assay 28
• 2.14 Modified histone peptide array 28
• 2.15 Public ChIP-seq data analysis 29
• 3. Results 33
• 3.1 Generation of LAP2α knockdown (KD) and knockout (KO) cell lines 33
• 3.2. Knockdown of LAP2α induce cell cycle arrest at G2/M phase and apoptosis 39
• 3.3 Transcriptome analysis of LAP2α KD cells using RNA-seq 41
• 3.4 Global histone modification changes in LAP2α KD cells 53
• 3.5 Direct interaction between Histone proteins and LAP2α 57
• 3.6 LAP2α tends to bind mainly to H3 acetylated lysine and H 4 methylated arginine residues 59
• 3.7 Addition of H3 lysine acetylation modification or H4 arginine methylation causes LAP2α binds 65
• 3.8 LAP2α directly interacts with histone modifiers 69
• 3.9 LAP2α regulates the expression of chromatin remodeling and cell cycle regulation-related genes by interacting with PCAF or CBP 71
• 3.10 LAP2α regulates the expression of apoptosis and chromatin organization-related genes by interacting with HDAC1 75
• 4. Discussion 78
• 5. References 83
• 국문초록 91