The research undertaken in my laboratory aims at deciphering molecular pathways that underlie chromatin signalling networks that regulate physiological processes such as cellular differentiation, DNA repair and organismal ageing. Our scientific approach relies largely on dissecting the functions of diverse chromatin components, for example epigenetic players, in the cell culture system biochemically and by applying histology and high resolution microscopy. We complement our in vitro approach by employing genetics and RNAi screening techniques in C. elegans.
Epigenetic mechanisms of mESC differentiation
Self-renewal and cellular differentiation of embryonic stem cells is controlled by a variety of distinct epigenetic modifications and multi-protein complexes. Silencing of genes during pluripotency is linked to methylation of histone H3K27 (H3K27me3) and ubiquitylation of histone H2A, which is brought about by Polycomb-Repressive Complexes 1 and 2 (PRC1 and PRC2), respectively. Polycomb complexes not only contribute to gene silencing but also impact on the architecture of chromatin, thereby contributing to chromatin condensation. We have recently reported a novel function for the Mediator complex in gene silencing during pluripotency (Papadopoulou et al, 2016, Cell Cycle). Our research demonstrates that Mediator complexes together with PRC1 and ncRNAs form an RNA-multi-protein assembly that is essential to silence key developmental genes. During differentiation the epigenetic landscape changes dramatically and to transcriptionally activate genes the PRC complexes and their respective repressive marks have to be removed from chromatin. We had previously demonstrated that the protein ZRF1 acts at the onset of differentiation to remove PRC1 from chromatin (Richly et al, 2010, Nature). Upon recruitment to chromatin Zrf1 binds ncRNAs, dislocates PRC1 and assembles the Cdk8 submodule converting Mediator into a transcriptional activator (Papadopoulou et al, 2016, Cell Cycle ; Papadopoulou and Richly, 2016, Bioessays). Hence, we have provided new insights into the ncRNA-mediated activation of developmental genes during differentiation of stem cells.
We have moreover shown that the switch protein Zrf1 regulates the generation of the three germ layers during development in vivo (Kaymak et al, 2016, Cell Cycle). Importantly, Zrf1 is essential for the proper establishment of mesodermal tissues and controls chondrogenesis, adipogenesis and most importantly cardiogenesis.
Further, our research puts Zrf1 centre stage in the establishment and metastasis of breast cancer. We are currently looking into ZRF1 functions in breast cancer employing human cancer cells and organoid cell culture.
Ubiquitin signalling and crosstalk at chromatin during DNA repair
Epigenetic networks govern most cellular processes that take place in a chromatin environment, for example differentiation, DNA repair and replication. Our research provides evidence for how epigenetic factors act in concert with DNA repair factors. In our investigations into DNA repair we have largely concentrated on one particular histone mark, the mono-ubiquitylation of histone H2A at lysine 119 (H2A-ubiquitin). H2A-ubiquitylation is a hallmark of signalling cascades as part of the DNA damage response. We have recently demonstrated that timing of DNA repair specific E3 ligases is an important feature of nucleotide excision repair (NER) and we have discussed a new concept of remodelling E3 ligase complexes at chromatin during DNA lesion recognition. In brief, we have discovered that H2A-ubiquitin is catalysed predominantly by a novel E3 ligase complex (UV-RING1B complex) that operates early during lesion recognition (Gracheva et al, 2016, JCB; Richly and Papadopoulou and Richly, 2016, Bioessays). ZRF1 tethers to the H2A-ubiquitin mark at the damage site and mediates the remodelling of the UV-RING1B complex, a process that we have coined on-site remodelling.
Apart from remodelling multi-protein complexes, ZRF1 also plays a pivotal role in chromatin decondensation. We have recently shown that ZRF1 together with the endoribonuclease DICER causes decondensation of chromatin as part of the DNA damage response (Chitale et al, 2017, Nucleic Acids Research). Interestingly, the recruitment of DICER to chromatin is dependent on ZRF1 linking its action to the ubiquitin signalling pathway. Importantly, this action of DICER at chromatin is independent of its ribonuclease activity, highlighting a novel RNA-independent function for DICER at chromatin.
Another interest of my lab is the sub-nuclear localisation of NER, which is controlled through DNA damage-dependent setting of H2A-ubiquitin by the UV-RING1B complex. We have demonstrated that mono-ubiquitylated chromatin is tethered to the rim of the nucleolus where a subset of ZRF1 resides to generate DNA repair foci (Chitale and Richly, 2017, Oncotarget). Thus, we have provided the first evidence for compartmentalisation of DNA repair in the NER pathway.
Currently we are investigating the role of the epigenetic landscape and in particular histone marks, which are deposited at the DNA damage site. We are further engaged in deciphering the functions of ubiquitylation events at the DNA damage site. Our research shows that specific histone marks and ubiquitylation events are essential to specifically recruit DNA repair factors to chromatin. Taken together our research aims at understanding the complex chromatin-associated network that regulates the DNA damage response in the NER pathway.
Epigenetic regulation during organismal aging
Further, we are interested in understanding gene regulation during organismal aging. To this end we investigate aging in the nematode C. elegans, employing sophisticated RNAi screening techniques, genetics and high-resolution microscopy. We have generated a semi-automated RNAi screening technique and we have isolated chromatin-associated factors involved in the aging of the worms. We are now analysing in detail the functions and molecular mechanisms of the factors identified by the screen. To this end, we assess whether the identified factors have a tissue-specific function and whether they are linked to any of the characterized aging pathways. Moreover, we examine if the identified factors have an impact on the health span, using a broad array of assays. Our research shows that the timing of gene regulation is an important determinant of longevity. In the near future, we will start to investigate how they reprogramme the epigenome in the course of aging.
Holger is an Associate Member of the EpiGeneSys network of scientists.