Table of Contents
Chap 1. Basic Epigenetic Mechanisms and Phenomena #
1.1 Introduction #
1.2 Basic Epigenetic Mechanisms #
1.3 Epigenetic (Re)Programming #
1.4 Genomic Imprinting as a Model of Epigenetic Silencing #
1.5 Dosage Compensation in Mammals #
1.6 PEF in Drosophila #
1.7 Transgenerational and Intergenerational Epigenetic Inheritance #
1.8 Epigenetics and Disease #
Chap 2. Cancer epigenetics #
2.1 Background #
Epigenetic abberrations in cancers can be typically described as changes in
- DNA methylation
- Histone modification
- Histone variant usage
- Small and noncoding RNA
- Nucleosome positions
While, more recently, it has become clear that an additional layer of epigenetic damage probably occur as a consquence of genetic mutations in chromatin-modifying proteins.
Cancer development에서 epigenetic 기전은 널리 받아들여지고 있으나, 기반하는 molecular etiology는 추가 설명이 더 필요하다.
In particular, the high level of concordant changes emphasized the intricacy of epigenetic control over the genome but highlights the necessity to identify the primary epigenetic events leading to cancer onset and progression.
This task remains a challenge as we must now collate and evaluate ever-increasing amounts of sequencing data, which continue to identify novel epigenetic defects as well as emphasizing roles for epigenetic changes that appear in almost all cancers.
2.2 DNA methylation #
Tumor progression is driven by abnormal transcriptional profiles resulting from both genetic and epigenetic changes.
Ageing and environmental contributors to cancer progression may causes changes to the epigenome, which in turn influence expression of the genome.
Mapping methylation profiles of normal versus tumor cells has provided a great deal of insight into the potential epigenetic mechanisms driving cancer progression.
Epigenetic reprogramming via DNA methylation in cancer cells is typically characterized by global hypomethylation along with promoter-specific hypermethylation.
- Global hypomethylation: Oncogene activation and genomic instability, increasing the rate of mutatioin, resulting in cells accumulating even more genetic damage.
- Locus-specific promoter hypermethylation: involved in silencing Tumor suppressor genes. - Aberrant TSG promoter methylation can contribute to the functional loss of the second TSG, acting as the "second hit".
In addition, while global hypomethylation is a characteristic feature of the cancer epigenome there is also recent evidence for long-range epigenetic silencing in cancer.
Certain regions of the genome are more prone to hypermethylation, for example Polycomb target genes. Differential methylation of certain region may also be attributed to replication timing and attachment to the nuclear lamina.
유전적 손상은 DNA methylation에 기여하고, 또 역으로도 그러하다. - Genetic changes can trigger epigenetic mutations by impacting levels or activity of DNA methyltransferases (DNMT). Meanwhile, anepigenetic lesion, such as hypermethylation and silencing of TSG, can lead to chromosomal instability and genetic mutations.
Epigenetic change에도 driver와 passenger가 있다. 어떤 methylation mark는 oncogene or TSG에 직접적인 영향을 주지만, 또 어떤 것은 단순한 변이의 결과로만 나타나며, tumor cell growth와 evolution에 필수적이지 않다.
대장암에서의 연구가 잘 정립되어 있다 (암 초기에 hyper, hypo 모두). Localized promoter CpG island methylation has been detected in normal tissue, aberrant crypt foci, and adenomas, with evidence demonstrating epigenetic silencing of TSGs.
Genome-wide methylation changes appear to occur early in disease progression and are often highly heterogeneous. Most methylation changes occur before progression to cancer, perhaps consolidating the transcriptome necessary to drive transformation.
Inactivation of DNA mismatch repair (MMR) pathways via DNA methylation can promote genetic instability. Defects in the MMR pathways are reflected by variation in DNA microsatellite length between cells.
This genetic instability is referred to as Microsatellite instability (MSI) and oftern has implications in terms of prognosis and response to therapy.
MSI maligancies tend to responde well to chemotherapy and radiotherapy with increased DNA damage following genotoxic insult pushing cells toward Apoptosis. MSI in colorectal cancer is observed in both familial and sporadic diseases.
The relatively high frequency of gene hypermethylation in colorectal cancer has been used to identify a subgroup termed CpG island methylator phenotype (CIMP). CIMP accounts for around 20% of colorectal cancer cases. It is characterized by numerous features such as BRAF mutation; the concurrent hypermethylation of marker genes RUNX3, SOCS1, NEUROG1, IGF2, and CACNA1G; female patients and proximal tumor location.
DNA methylation plays a significant role in leukemic transformation with silencing of TSG alleles via this mechanism event in immature MDS cells.
A key study performed analysis of methylation signatures across AML patients and demonstrated that methylation patterns could be used to divide patients into 16 different subgroups. 각각의 서브그룹은 다른 분자와 진화 경로로 부터 나타나게 된다. These included distinct methylation signatures for common chromosomal rearrangements associated with AML such as t8:21 and inv16.
This also raised the question as to whether the genetic change seeds the corresponding methylation pattern, or whether perhaps an epigenetic lesion contributed to the related genetic instability and mutation.
추가적으로 AML 관련 54개 유전자 메틸화 시그니처가 서브그룹간 공유되고 있음을 확인. This methylation pattern impacted TSGs along with genes involved in transcription and nuclear import.
Distinct DNA methylation signatures have been associated with various hallmark features of cancer such as inflammation, cell cycle progression, angiogenesis, and metastasis.
Alterations in DNA methylation are associated with chronic inflammation. - 위암의 경우 Helicobacter pylori와 연관됨. rodent 모델에서 5-aza-2-deoxycytidine 치료가 효과있음 - This result indicated that preventing methylation changes induced by inflammation can inhibit the progression from the environmental signal, chronic inflammation, to cancer.
DNA methylation plays a pathogenetic role in tumor cell evolution. A subpopulation of tumor cells has stem-like properties driven by epigenetic reprogramming.
Alteration in DNA methylation can induce pluripotency with cancer stem/progenitor cells driven by abnormal methylation programs.
Epigenetic plasticity is crucial for cancer cells, with undergo an epithelial to mesenchymal transition (EMT) to metastasize, followed by a mesenchymal to epithelial transtion to enable colonization at distal sites.
It is crucial to remember that tumor evolution and the resultant heterogeneity means that within a population of cancer cells many subclones and therefore cancer epigenomes exist. The variation within a single tumor complicates treatment considerably with resistant subclones contributing to disease recurrence and progression.
Posttranslational modification of histone proteins, or indeed, of proteins with the capacity to modify chromatin, can be associated with both active and repressive regions of the genome.
The exact residue and type of modification that decorates histones is often correlated with a panel of complementary marks that are easily identified as "active" or "repressive" combinations.
Methylation can be both active and repressive, and histones can also be acetylated, ubiquitinated, phosphorylated, and sumoylated, amongst others.
The "histone code" explins the coexistence of some marks but disparities do occur. 예를들어, 배아줄기세포의 어떤 프로모터에서 H3K4me3과 H3K27me3는 동시에 두드러지게 나타나고, 이들 프로모터들은 낮은 발현을 보인다. 정상세포에서의 서열기반 히스톤 변형 정보의 축적을 통해, 암에서 어떻게 달라지는지 알 수 있다.
Histone modification associated with both active and repressive chromatin are implicated in cancers and these can occur simultaneously and amongst the other epigenetic changes.
It is equally significant that
- a mark is lost from a particular site along genome as much as it gained (e.g., loss of H2K27ac from an active enhancer can lead to inactivity and acquisition of a posed state)
- or the combination with which it exists (e.g., H2K4me1 can coexist with DNA methylation at enhancers repressed by a nucleosome in cancer).
The altered histone code of cancer cells is associated with direct and indirect subsequent changes such as additional reprogramming of the histone landscape and concomitant changes of other epigenetic marks (e.g., DNA methylation).
All of these changes can be ultimately attribured to the atypical activity of chromatin "writers" or abnormal interactions with histone modification "readers" and "erasers".
It should also be considered that in normal cells, healthy competitition between "writers" must be decided since the same histone tail residue can be modified in multiple ways; for example, methylation or acetylation can mark H3K27me or H2K27ac, respectively.
The result of this particular competition has been partly explained in embryonic stem cells, with the repressive H2K27me3 mark found to be dominant when all components of the methyltransferase machinery (i.e., Polycomb repressive complex) are intact.
Therefore, the levels of expression of the catalytic enzyme complexes and their availability probably dictate the amount and distribution of histone marks. This is applicable to normal and cancer cells but can be further complicated by mutations that directly impair the "writers", "readers", and "erasers", as we discuss below.
The H2K4me3 and H3K27me3 modifications are the most widely studied and demonstrate the damage, though ironically, and the potential utility of histone modification disturbances in cancers.
For example, both H3K4me3 and H3K427me3 characterize sets of genes that allow the subtyping of maligant, "tumor-sustaining" and chemoresistant ovarian tumors.
The H2K27me3 modification is typically but now always exclusive of DNA methylation, though both are markers of gene inactivity and interestingly, genes that carry H3K27me3 in normal cells are more prone to aberrant DNA methylation events in prostate and colon cancer cells.
The catalytic subunits of complexes catalyzing H3K27me2 and H2K4me3 are amongst the most frequently mutated in many cancers, suggesting that these could be putative driver mutations leading to widespread epigenetic aberrations in cancers.
2.4 Nucleosome Positions and Higher-Order Structures #
Tools to map open chromatin and nucleosome positions are constantly evolving and most have now been adapted so that the entire genome can be interrogated, much like DNA methylation with whole genome bisulfite sequencing and histone modifications through chromatin immunoprecipitation sequencing.
These techniques include
- DNaseI hypersensitivity sequencing (DNaseI-seq)
- micrococcal nuclease sequencing (MNase-seq)
- formaldehyde-assisted interrogation of regulatory elements sequencing (FAIRE-seq)
- assay for transposase-accessible chromatin sequencing (ATAC-seq)
- and nucleosome occupancy and methylation sequencing (NOMe-seq)
, which directly measures nucleosome positions and simultaneously assays DNA methylation.
Many general features of cancer cells could be explained by atypical nucleosome dynamics. For example, an overall loss of DNA methylation in cancer cells is associated with genome instability, which could be due to concomitant loss of nucleosomes.
This would also be consistent with fewer histones as we age and the knowledge that DNMT enzymes require nucleosomes for selective anchoring and activity.
A potential order of events could be hypothesized from these and observations that nucleosome occupancy is the mode of silencing key pluripotent genes prior to DNA methylation of these loci during stem cell diferrentiation.
Nucleosome dynamics are also observed in cancer cell lines treated with 5-aza-2'-deoxycytidine, reflecting the fact that a reverse cooperation occurs between these two epigenetic mechanisms during gene reactivation.
Outside of promoter regions hypermethylation is accompanied by nucleosome occupancy of insulators and enhancers in breast and prostate cancer cell lines demonstrating that in cancers chromatin is deliberately organized, or reorganized, at a genome-wide scale.
These changes could have important therapeutic implications, as indicated by a comparison of endocrine-responsive versus endocrine-resistant breast cancer cell lines that revealed distinct differences in chromatin accessibility linked to pathways involving NOTCH in resistant breast cancer cells.
Nucleosome depletion is an important feature of the epigenome and one that must be precisely regulated so that transcription factors and other chromatin-modulating proteins bind appropreately.
In breast cancer, the long-range loop linking IGFBP3 promoter to its enhancer is disrupted but new interactions are formed, including those that may active EGFR and BCAS, which is particulary prone to chromosomal translocation.
2.5 Noncoding RNAs #
Noncoding DNA comprises a significant proportion of the human genome and until relatively recently these sequences were considered junk. It is now evident that, not only are these sequences extensively transcribed, they have critical roles to play in gene regulation and in turn cancer progression.
Among these nonprotein-coding sequences are microRNAs (miRNAs) and long noncoding RNAs (lncRNAs).
ncRNAs play a role in regulating gene expression via translational repression and protein binding. An ever-growing list of ncRNAs has been implicated in tumor initiation and progression.
Two mechanisms can trigger tumorigenic ncRNA alterations, either genetic mutation resulting in sequence changes or epigenetic mechanisms which can impact the level of ncRNA transcribed.
Altered expression or sequence mutation in ncRNA impacts target binding and therefore function.
2.5.1 microRNAs #
miRNAs are 18-25 bases in length and are involved in posttranscriptional regulation via mRNA degradation and translational repression. mrRNAs bind the 3' UTR of the target mRNA and require near perfect base complementarity.
mrRNAs are involved in all aspects of cancer initiation and progression including apoptosis, cell cycle progression, invasion and metastasis, and angiogenesis.
It is important to note that some miRNAs can be both oncogenic or tumor-suppressive in their function, depending on cellular context and the targets expressed in a particlar cell type.
miR-21, mrR-92, mrR-155 are overexpressed in cancer.
miR-200 levels is associated with EMT in cancer.
The ability of miRNA to reflect genotype, DNA methylation, and change expression over time demonstrates the ever-increasing layers of epigenetic complexity in cancer cell evolution.
2.5.2 Long noncoding RNAs #
Many cancer-associated SNPs lie beyond the protein-coding sequences indicating that the noncoding sequences play a significant role in tumorigenesis.
lncRNAs are 200 nucleotides or more in length. These ncRNAs have been shown to interact with proteins to alter inding and function via conformational change.
In contrast to miRNAs, most lncRNAs appear to be poorly conserved and while selective pressures have enabled lncRNAs to evolve it is clear they have an important role in regulating numerous cellular pathways.
2.5.3 Other noncoding RNAs #
Other ncRNAs emerging as regulators of tumorigenesis include the human ortholog of Piwi-interacting RNAs (piRNAs). piRNAs are involved in germline stem cell maintenance and maintain genome integrity by siilencing transposons.
Small nucleolar RNAs (snoRNAs) have also been implicated in cancer. snoRNAs are responsible for processing rRNA, however their role in the cell has been expanded.
2.6 Mutation of Epigenetic Enzymes #
Epigenetic enzymes play a wide range of roles in modifying the epigenome and therefore gene expression patterns in cells.
In cancer these epigenetic modifiers are often altered and the change in activity has a significant impact on the resulting epigenetic landscape.
Epigenetic enzymes can be divided into several broad categories including chromatin-remodeling enzymes, histone modifiers, DNMT, and enzymes involved in processin ncRNAs.
Sequence mutation or dysregulated expression of these enzymes can dramatically alter the transcriptome of the cell.
Epigenetic enzymes are frequently mutated in pediatric cancers highlighting their ability to fast-track tumorigenesis.
2.6.1 Chromatin-remodeling enzymes #
Chromatin-remodeling enzymes help to shape the epigenetic landscape necessary for day-to-day cellular activities such as transcription and DNA replication.
Dysregulation of these enzymes leads to the accumulation of epigenetic abnormalities necessary for cancer initiation and progression.
Five major families of ATP-dependent chromatin-remodeling enzymes exist: SWI/SNF, ISWI, Nurd/Mi/CHD, SWR1, and INO80.
SWI/SNF mutation s are found in up to 20% of cancers and the majority of these are inactivating mutations, indicating a tumor-suppressive function for this ATPase.
While the major role of SWI/SNF is gene activation it is also involved in double-strand DNA repair and lineage determination, therefore functional loss of this ATPase can increase sensitivity to DNA damage and disrupt differentiation programs.
2.6.2 Histone modifiers #
Enzymes responsible for catalyzing the addition or removal of groups from histone tails help to establish chromatin structure and accessibility.
Altered expression or function of histone modifying enzymes results in aberrant chromatin architecture and gene expression programs.
Histone deacetylases (HDACs) are often overexpressed in cancer, resulting in increased acetylation levels and transcriptional activity. HDAC1, HDAC2, and HDAC3 are frequently upregulated in colorectal cancer and increased expression is linked to poor prognosis.
Altered histone acetyltransferase (HAT) activity has also been reported in cancer. Dysregulation of HATs triggers aberrant histone acetylation signatures and transcriptional profiles.
The histone methyltransferase PRC2 is involved in promoting H3K27 trimethylation and repression of genes involved in differentiation, thereby maintaining epigenetic silencing during development and cancer.
2.6.3 DNA methyltransferases #
Abnormal methylation profiles are driven by defects in the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. These enzymes catalyze the addition of methyl groups to hemimethylated and unmethylated CpG dinucleotides.
With a role in methylation maintenance and de novo methylation events these enzymes are key contributors to the aberrant methylation profiles observed in cancer.
It also appears to play a role in DNA repair pathways with DNMT1 recruited to sites of DNA damage. Consequently, aberrations in DNMT1 levels or activity can have a significant impact on the methylation profile of the cell and accrual of mutations.
2.6.4 ncRNA machinery #
2.7 Conclusion #