👤 Agnieszka Anna Rawłuszko-Wieczorek

The occurrence of colorectal cancer (CRC) is inversely correlated with estrogen receptor beta (ERβ) presence. Additionally, multiple studies associate low ERβ expression with poorer overall survival of CRC patients. Molecular pathways involved in ERβ - related reduced tumorigenesis include enhanced apoptosis, decreased proliferation, or repression of oncogenes. Moreover, the development of solid tumors, such as CRC, is often associated with an increased tumor mass that results in decreased oxygen partial tension, known as hypoxia, clinically associated with decreased prognosis and therapeutic resistance. Our high-throughput study suggests that ERβ also represses a hypoxic response in CRC cells. We observed a significantly altered transcriptional profile in HCT116 ERβ overexpressing cells that was further stimulated by E2 treatment under hypoxic conditions. The achieved data for downregulation of VEGFA, PDGFA and ANGPTL4 were validated in a time course experiment in DLD-1 cells. In addition, using an ERβ construct with a mutated DNA binding domain we observed that the downregulation of selected genes is dependent on the direct binding of this receptor to regulatory region genes. In addition, we observed that ERβ may affect the expression of the main hypoxia regulator, HIF1A, at the transcriptional and translational levels. In summary, ERβ alters the hypoxic outcome in CRC cells. Show less

Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete. This study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Our approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers. Show less