Periodic Reporting for period 4 - Epiherigans (Writing, reading and managing stress with H3K9me)
Berichtszeitraum: 2021-01-01 bis 2022-05-31
Our genomes are packaged into distinct types of chromatin, in which DNA is either accessible or less accessible, called euchromatin and heterochromatin, respectively. Heterochromatin is distinct in containing nucleosomes that bear methylation on lysine 9 of histone H3, which correlates generally with reduced transcription. Nonetheless, heterochromatin plays a key role in governing organismal development, somatic tissue differentiation, and germline immortality. These phenomena depend both on genome stability and a controlled spatiotemporal regulation of gene expression. All higher eukaryotic genomes contain a large fraction of repetitive DNA, much of which stems from viral genomes that invaded long ago. This repetitive DNA must be kept transcriptionally silent to prevent RNA:DNA hybrid formation, DNA damage and apoptosis. The transcription of repetitive sequences must be restricted, which is largely achieved by blocking access to transcription factors and RNA polymerases. Not surprisingly, repetitive sequence elements account for the largest fraction of H3K9me-marked heterochromatin in the worm. It was assumed but not proven that H3K9 methylation silences transcription by excluding transcription factors and the transcription machinery from these sequences.
During development and tissue differentiation in multicellular organisms, H3K9me-mediated hetero-chromatin also represses tissue-specific genes that are inappropriate for a given tissue. Understanding how heterochromatin targets and silences these genes is key for understanding tissue generation and re-generation, for countering the misregulation of tissue specific genes in cancer and aging, and for preserving the integrity of the genome. These challenges are even more threatening in an aging society and are ever more relevant to medical application as therapies becomes targeted to specific genes or gene products. To provide just one example: immune-therapy for cancer is a powerful method that requires the reprogramming of the patient’s own T-cells to recognize and attack the tumor cells. This is only is only effective if neoantigens can be detected on the tumor cells, to prime the lymphocytes. Recent studies indicate that derepressed or misexpressed endogenous retroviral elements in the human genome are an important source of novel antigens (neoantigens) for T-cell programming. Their expression in cancer cells arises from the derepression of repetitive elements that normally would be repressed as heterochromatin. Identifying the controls over neoantigen expression and controlling thus contributes to more effective immunotherapy of cancer.
The objectives of our ERC grant were to understand what targets the H3K9 Histone methyltransferases (HMTs) to repetitive elements and to genes, and to identify what happens to cell and/or tissue identity when H3K9me3 deposition is abolished after tissue differentiation While it was assumed that the correct segregation of inactive heterochromatin and active euchromatin is essential for healthy tissue or organismal development, this becomes particularly important under conditions of stress or when cells lose their differentiated identity. We have established and characterized the role of H3K9me2/3 in restricting the expression of genes to individual tissues at various developmental stages, and we have characterized controls over the H3K9 HMT MET-2 (SETDB1 in man). Progress was also made in understanding what targets the H3K9 HMTs, MET-2 and SET-25 (G9a and SUV39 in man), and how they are controlled, which also helps us understand the genomic response to stress such as heat shock, tissue de-differentiation and viral invasion.
During development and tissue differentiation in multicellular organisms, H3K9me-mediated hetero-chromatin also represses tissue-specific genes that are inappropriate for a given tissue. Understanding how heterochromatin targets and silences these genes is key for understanding tissue generation and re-generation, for countering the misregulation of tissue specific genes in cancer and aging, and for preserving the integrity of the genome. These challenges are even more threatening in an aging society and are ever more relevant to medical application as therapies becomes targeted to specific genes or gene products. To provide just one example: immune-therapy for cancer is a powerful method that requires the reprogramming of the patient’s own T-cells to recognize and attack the tumor cells. This is only is only effective if neoantigens can be detected on the tumor cells, to prime the lymphocytes. Recent studies indicate that derepressed or misexpressed endogenous retroviral elements in the human genome are an important source of novel antigens (neoantigens) for T-cell programming. Their expression in cancer cells arises from the derepression of repetitive elements that normally would be repressed as heterochromatin. Identifying the controls over neoantigen expression and controlling thus contributes to more effective immunotherapy of cancer.
The objectives of our ERC grant were to understand what targets the H3K9 Histone methyltransferases (HMTs) to repetitive elements and to genes, and to identify what happens to cell and/or tissue identity when H3K9me3 deposition is abolished after tissue differentiation While it was assumed that the correct segregation of inactive heterochromatin and active euchromatin is essential for healthy tissue or organismal development, this becomes particularly important under conditions of stress or when cells lose their differentiated identity. We have established and characterized the role of H3K9me2/3 in restricting the expression of genes to individual tissues at various developmental stages, and we have characterized controls over the H3K9 HMT MET-2 (SETDB1 in man). Progress was also made in understanding what targets the H3K9 HMTs, MET-2 and SET-25 (G9a and SUV39 in man), and how they are controlled, which also helps us understand the genomic response to stress such as heat shock, tissue de-differentiation and viral invasion.
The question of how our genomes are regulated to produce different cell types in a reproducible manner, is key to understanding both healthy life and disease. Our laboratory has shed light on the role of a specific ubiquitous histone modification, histone H3 lysine 9 methylation, in the establishment and maintenance of heterochromatin. We use the nematode C. elegans, which has only 2 histone methyltransferases (HMTs) that deposit this conserved modification, rather than the 6 found in man. These two HMTs are members of conserved enzyme families. We have shown that the two C. elegans H3K9 methyltransferases (MET-2 and SET-25) have essential but distinct functions in ensuring genomic stability, and we have shown that the C. elegans mutant lacking a functional H3K9me-HMT MET-2, requires tumor suppressors BRCA-1 and BRCA-2, and other factors that stabilize replication forks such as the ATR-checkpoint kinase and RAD-51, for viability (Padeken et al., 2019). Loss of MET-2 leads to early aging and sterility in worms. We hypothesize that both of these phenomena can be traced to the promiscuous transcription of repeat DNA; this leads to aberrant RNA:DNA hybrids or R-loops that cause irreparable damage when encountered by replication forks. Further molecular and biochemical studies of the MET-2 enzyme identified novel interaction partners, one of which ensures the proper nuclear localization and functional activity of MET-2 (Delaney et al., 2019). This work also highlighted a critical role for MET-2 during temperature stress. A subsequent microscopy-based screen identified the factors LIN-65 and ARLE-14 as being essential for allowing MET-2 to form functional condensates and be targeted to specific genes and repeats. We also identified additional factors involved in the targeting of the second HMT, which deposits H3K9me3, namely a nuclear Argonaut which binds RNA and recruits the Dicer enzyme. This allowed us to connect the small RNA pathway to SET-25 targeting. A second targeting pathway for SET-25 depends on the H3K9me2 reader LIN-61. The two factors contribute to the H3K9me3-mediated silencing of endogenous viral transposons embedded in the genome.
We then studied the role of spatial organization of heterochromatin in differentiated tissues – namely gut and muscle. We have shown that a lamin mutation causes deterioration of muscle in worms, recapitulating a human disease (Harr et al., 2020) and we have shown that in intestinal cells, the disruption of a strict sequestration of a euchromatic factor, CBP/p300, away from heterochromatin, leads to an activation of repressed tissue specific genes and loss of intestine-cell identity (Cabianca et al., 2019). Intriguingly, this depends on another histone H3 methylation mark, H3K36me2 or me3, and its reader MRG-1. Finally we have discovered a new principle of silencing that depends on selective nuclear degradation of transcripts. We showed that a complex called LSM2-8 – coupled with an RNA degrading machine (XRN-2) degrades specifically messages arising from genes repressed by the so-called Polycomb pathway, a system that maintains another histone mark on conditionally silent genes, histone H3K27me3. This mRNA degradation is the first proof that select mRNA turnover in the nucleus communicates to the compaction state of chromatin on genes. This has relevance for the innate immunity genes in man.
Our most important contribution from our ERC funded program of research was to show that continued expression of the H3K9 HMT, MET-2, is necessary for the maintenance of nondividing, differentiated tissues, such as muscle. We could show that MET-2 continuously restores H3K9me2 to repress germline and genes specific to other tissues, thereby allowing muscle cells to maintain their identity as muscle. This is achieved by preventing transcription factor access. Depending on the tissue type and stage of development, the loss of MET-2 provokes the derepression of different sets of genes, reflecting the transcription factor profile of that stage/cell type. This indicates that although H3K9me marks are dispensible for development in general, they are essential for long-term tissue maintenance.
We then studied the role of spatial organization of heterochromatin in differentiated tissues – namely gut and muscle. We have shown that a lamin mutation causes deterioration of muscle in worms, recapitulating a human disease (Harr et al., 2020) and we have shown that in intestinal cells, the disruption of a strict sequestration of a euchromatic factor, CBP/p300, away from heterochromatin, leads to an activation of repressed tissue specific genes and loss of intestine-cell identity (Cabianca et al., 2019). Intriguingly, this depends on another histone H3 methylation mark, H3K36me2 or me3, and its reader MRG-1. Finally we have discovered a new principle of silencing that depends on selective nuclear degradation of transcripts. We showed that a complex called LSM2-8 – coupled with an RNA degrading machine (XRN-2) degrades specifically messages arising from genes repressed by the so-called Polycomb pathway, a system that maintains another histone mark on conditionally silent genes, histone H3K27me3. This mRNA degradation is the first proof that select mRNA turnover in the nucleus communicates to the compaction state of chromatin on genes. This has relevance for the innate immunity genes in man.
Our most important contribution from our ERC funded program of research was to show that continued expression of the H3K9 HMT, MET-2, is necessary for the maintenance of nondividing, differentiated tissues, such as muscle. We could show that MET-2 continuously restores H3K9me2 to repress germline and genes specific to other tissues, thereby allowing muscle cells to maintain their identity as muscle. This is achieved by preventing transcription factor access. Depending on the tissue type and stage of development, the loss of MET-2 provokes the derepression of different sets of genes, reflecting the transcription factor profile of that stage/cell type. This indicates that although H3K9me marks are dispensible for development in general, they are essential for long-term tissue maintenance.
Advances in medicine have contributed to people living longer than ever before. However, for a large proportion of the population these extra years are not disease-free, but are burdened with complex and often chronic diseases associated with lifestyle and aging. Not only is an individual’s quality of life affected, but it places an enormous burden to European society for healthcare. Recent work suggests that almost all chronic and age-linked diseases – apart from those caused by infectious agents – are linked to a loss of cell type integrity. This occurs both on the level of genomic integrity, i.e. the loss of an intact, nonmutated genome, and on the level of proper transcription – the conversion of the genetic code into a given cell type. Both are ensured largely by epigenetic modifications of the proteins that organize our genomes. Thus a detailed understanding of the epigenetic maintenance of a differentiated cell in a natural tissue, is crucial to controlling and prolonging healthy lives.
To understand and intervene effectively in age-related diseases, we must detect degeneration early and be able to reverse or slow its progress. Our studies aim at understanding the molecular events that establish and maintain cell fate. With this, we have uncovered cellular markers that allow us to identify disease precociously whenever it is linked with a loss of tissue integrity. Not only early detection, but the use of biomarkers based on the underlying molecular mechanisms of age-related predisposition to disease, will help stratify patients for clinical trials, increasing trial efficacy. Finally, the same molecular epigenetic markers will help physicians select the most appropriate treatment or combination of treatments for an individual. The thorough implementation of genetic and epigenetic markers in patients will reduce the application of inappropriate treatments. Elucidation of cell status through epigenetic marks will also identify new drug targets. The outcome of all this is to transform medical practice such that it is no longer dependent on outdated diagnostics. In brief, understanding how euchromatin and heterochromatin states are maintained and segregated helps to advance medicine through a basic understanding of cell differentiation, cell fate determination, cell maintenance and reprogramming. Our work will profoundly impact regenerative medicine and disease modelling approaches.
To understand and intervene effectively in age-related diseases, we must detect degeneration early and be able to reverse or slow its progress. Our studies aim at understanding the molecular events that establish and maintain cell fate. With this, we have uncovered cellular markers that allow us to identify disease precociously whenever it is linked with a loss of tissue integrity. Not only early detection, but the use of biomarkers based on the underlying molecular mechanisms of age-related predisposition to disease, will help stratify patients for clinical trials, increasing trial efficacy. Finally, the same molecular epigenetic markers will help physicians select the most appropriate treatment or combination of treatments for an individual. The thorough implementation of genetic and epigenetic markers in patients will reduce the application of inappropriate treatments. Elucidation of cell status through epigenetic marks will also identify new drug targets. The outcome of all this is to transform medical practice such that it is no longer dependent on outdated diagnostics. In brief, understanding how euchromatin and heterochromatin states are maintained and segregated helps to advance medicine through a basic understanding of cell differentiation, cell fate determination, cell maintenance and reprogramming. Our work will profoundly impact regenerative medicine and disease modelling approaches.