Molecular Epigenetics

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.

On-site remodelling by ZRF1 in transcriptional activation during differentiation
On-site remodelling by ZRF1 in transcriptional activation during differentiation. In pluripotent mESCs, PRC1-repressed genes that code for differentiation factors are occupied by MED12-Mediator, which is recruited to chromatin in a PRC1-dependent manner. PRC1 is also essential for forming MED12-ncRNA associations which, in later stages of differentiation, enhance gene activation. Upon retinoic acid (RA) treatment-triggered differentiation, ZRF1 is recruited to chromatin via recognition of the PRC1-deposited H2A-ubiquitin (H2AUb) mark and promotes the recruitment of CDK8 from the kinase module of Mediator. After the displacement of PRC1, MED12 remains bound to the ncRNA and assembles with CDK8. This switch in the molecular architecture of Mediator is facilitated by ZRF1.

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.

Role of Zrf1 during embryonic development
Role of Zrf1 during embryonic development. Representative images of Hematoxylin and Eosin stainings of control and Zrf1 knockdown embryoid bodies were taken at 40X magnification after 16 days of differentiation. Black arrows in the upper panel indicate the neural rosette, fibrous connective tissue and gut-like epithelium respectively. Scale bars, 50 µm.

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

Spatiotemporal regulation of chromatin factors and ubiquitylation events during NER
Spatiotemporal regulation of chromatin factors and ubiquitylation events during NER. The assembly of the UV-RING1B complex at DNA damage sites causes mono-ubiquitylation of histone H2A (H2AUb). ZRF1 is recruited to the damage site via XPC, and tethers to H2A-ubiquitin, causing on-site remodelling of the UV RING1B E3 ligase complex. The newly-established DDB-CUL4A complex catalyses the poly-ubiquitylation of various substrates, and most importantly XPC, which is thereby stabilized at the damage site.

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.

Hypothetical model for ZRF1 and DICER function at the DNA damage sites
Hypothetical model for ZRF1 and DICER function at the DNA damage sites. ZRF1 recruits and associates with the endoribonuclease DICER to impact on the conformation of chromatin at the DNA damage site.

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. 

Hypothetical model for nucleolar tethering of damaged DNA via H2A-K119-ubiquitylation.
Hypothetical model for nucleolar tethering of damaged DNA via H2A-K119-ubiquitylation. Histone H2A is mono-ubiquitylated (H2AUb) by the UV-RING1b complex at the DNA damage site. Ubiquitylated chromatin is tethered to ZRF1, which resides at the rim of the nucleolus.

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.