Michal-Ruth Schweiger - A 10

Activation of the heat shock response as well as chemotherapeutic interventions induce the expression of noncoding satellite III RNA (SatIII), the formation of nuclear stress bodies (nSB) and the recruitment of the heat shock factor 1 (HSF1). We found that the bromodomain protein 4 (BRD4) interacts with HSF1 and induces SatIII expression. Furthermore, SatIII expression is induced by chemotherapeutic agents and can be inhibited though BET inhibitors. This opens new therapeutic strategies for tumor entities with high SatIII expression. 

Introduction 

It has long been thought that regions of constitutive heterochromatin are relatively devoid of genes and function as constitutive repressive regions. However, recent data indicates that constitutive heterochromatic regions are by far not silent and static. In contrast, during development and disease specific protein complexes are recruited and induce the expression of satellite RNA located at pericentromeric regions in various biological contexts such as differentiation, development, senescence, stress response and transformation. Furthermore, DNA hypomethylations of centomeric and pericentromeric DNA regions and satellite RNA expression are frequently found in conjunction with tumor development, genomic instability and therapy resistance in cancer. These epigenetic processes have been reported in numerous cancer entities (Wilms neuroblastoma tumours, epidermal carcinoma cells, lung, pancreatic, kidney, colon and prostate cancer) and in several genetic disorders including ICF syndrome and Hutchinson-Gilford progeria. 

The pericentromeric transcription is induced by several kinds of stress including heat shock, exposure to heavy metals (cadmium) and DNA damaging agents (MMS, etoposide). Upon heat shock or chemical stress, pericentromeric SatIII repeats are activated and large amounts of stable non-coding RNAs (SatIII) are generated and accumulate at their genomic locus forming so called nuclear stress bodies (nSB). NSB are sub-nuclear assemblies that participate in epigenetic and transcriptional control of gene expression and mRNA splicing during heat stress. However, the function of the satellite RNA transcripts is largely unknown. Besides aputative role as modulators of stress-induced splicing process, SatIII RNA expression is implicated in genomic instability.

BRD4 and HSF1 co-localize at nSBs and induce SatIII RNA

Using co-immunoprecipitation experiments as well as co-localizations we uncovered that SatIII RNA expression is transcriptionally activated by the bromodomain protein 4 (BRD4) upon heat stress. BRD4 has gained extensive attention – mainly due to its involvement in tumour growth and its widespread success as therapeutic target in leukaemia, lung cancer, melanoma, colon cancer, lymphoma and diverse other tumour entities.It is an epigenetic reader and transcriptional regulator and therefore links the transcription process to epigenetic patterns. We found that BRD4 interacts with the heat shock factor 1 (HSF1), co-localizes with HSF1 in nuclear stress bodies (nSB) and regulates and co-localizes with SatIII RNA expression in an HSF1 dependent manner.

Chemotherapies induce SatIII expression 

The hypomethylation of repetitive DNA and the expression of satellite RNA is associated with genomic instability (GI). As such we found an increase in SatIII RNA in advanced stages of CLL which is associated with increased genomic rearrangements (Figure 1C). GI is, besides being found in advanced tumor stages, associated with poor prognosis and drug resistance. On the other side, several chemotherapeutic drugs induce chromosomal instability, e.g. paclitaxel, gemcitabine and cisplatin. We observed after cisplatin treatment in HeLa cells an increase in SatIII expression as well as nuclear stress body formation (Figure 1A,B). 

BET inhibitors down-regulate SatIII expression

To find drugs which may overcome epigenetic remodeling and SatIII induction in cancer we performed a chemical compound screen. Within this screen we found that inhibition of proteins of the BET (bromodomain and extraterminal domain) protein family generally caused a consistent and substantial decrease in SatIII RNA and foci expression. Effects were detected for all subgroups taken into account. The strongest and most consistent effect was observed for compounds targeting BRD2, 3 and 4 at once.

Perspectives

This project gives first insight into the de-regulation of pericentromeric DNA in tumor development and therapeutic interventions. By understanding these mechanisms it will be possible to fine-tune chemo-therapies targeting epigenetic structures in cancer. Furthermore, it gives insight into epigenetic regulatory mechanisms of non-coding DNA regions which are significantly and early hylopmethylated in cancer and might thus provide means to intervene at early tumor stages.

1. Herberg, M., Siebert. S., ...Hussong, M., Altmüller, J., Kerner, C., Galle, J.*, Schweiger, M.R.*, Aust, G.*. Loss of Msh2 and a single-radiation hit induce common, genome-wide, and persistent epigenetic changes in the intestine. (2019) Clin Epigenetics.Apr 27;11(1):65. * equal contribution 

2. Grimm C, Fischer A, Farrelly AM, Kalachand R, Castiglione R, Wasserburger E, Hussong M, ... Reinhardt HC, ...Buettner R, Schweiger MR. (2019) Combined Targeted Resequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for Poly(ADP-Ribose) Polymerase 1 Inhibitor Sensitivity Testing.J Mol Diagn.Mar;21(2):198-213.

3. Grasse, S.*, Lienhard, M.*, Frese, S., Kerick, M., Grimm, C., Hussong, M., ... Odenthal, M., Büttner R., ...Herwig R.^#, Schweiger MR.^#(2018) Epigenomic profiling of Non-Small Cell Lung Cancer (NSCLC) xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance Genome Med.Jul 20;10(1):55. *^,#equal contribution as first- or last author

4. Hussong, M., Kaehler, C., Kerick, M., Grimm, C., Franz, A., Timmermann, B., Welzel, F., Isensee, J., Hucho, T., Krobitsch, S. and Schweiger, M.R. The bromodomain protein BRD4 regulates splicing during heat shock. (2017) Nucleic Acids Res. 9;45(1):382-394.