In eukaryotic cells, transcription factors and polymerases must access DNA in the context of nucleosomes and chromatin. The accessibility of DNA sequences to such trans-acting factors is an important feature of gene regulatory elements, including promoters. In vertebrates, the majority of gene promoters coincide with CpG islands (CGIs), which remain free from DNA methylation and exhibit elevated CpG densities. This hypomethylated and CpG-rich state at CGI promoters is associated not only with transcriptional activity, but also with high levels of chromatin accessibility. However, the causes and consequences of such chromatin accessibility remain unclear. To address this, I have profiled chromatin accessibility in mouse embryonic stem cells (ESCs). In addition to confirming that CGI accessibility is independent of transcriptional activity, I was able to demonstrate that the loss of DNA methylation in ESCs resulted in increased chromatin accessibility at a subset of CpG-rich repetitive elements, suggesting that non-methylated CpG-rich sequences may, at least partially, facilitate open chromatin states. This was supported by preliminary work targeting bacterial CpG-rich sequences into the mouse genome, where they were sufficient to establish novel regions of chromatin accessibility. To examine potential mechanisms by which hypomethylated DNA could serve to promote chromatin accessibility, I profiled chromatin accessibility in mouse ESCs lacking various chromatin-modifying proteins which are normally enriched at CGIs, with the histone demethylases KDM2A/B linked to maintaining open chromatin at CGIs. As an alternative approach to understanding the causes of chromatin accessibility in mouse ESCs, I examined the mechanism by which the pioneer transcription factor OCT4 is able to access previously inaccessible chromatin, and reveal that it requires the chromatin remodeller BRG1 to remodel chromatin and facilitate transcription factor binding at distal regulatory elements. Ultimately, this work provides an insight into some of the molecular determinants of chromatin accessibility in mouse ESCs, although many of the consequences of such chromatin states remain unclear.

<p dir="ltr">Lung cancer is the leading cause of cancer-related deaths, and although important therapy advancements have been achieved, ~1.6 million people die from lung cancer annually. Non-small cell lung cancer (NSCLC), which makes up ~85% of lung cancer cases, is mainly treated with radiotherapy, chemotherapies, and targeted agents. Targeted agents are selected based on the mutation spectrum of the tumor. In NSCLC the epidermal growth factor receptor (EGFR) is commonly mutated and, leads to increased proliferation and cell survival. The standard-of-care treatment for patients with activating mutations in EGFR is treatment with tyrosine kinase inhibitors (TKI), such as erlotinib. While tumors initially respond to TKIs, after 1-2 years most patients develop resistance. In ~60% of TKI resistant tumors, resistance is the result of a secondary mutation in EGFR, whereas in the remaining 20%, tumors turn on bypass track-signals to overcome inhibition of the EGFR pathway. However, 15-20% of the cases the mechanisms underlying resistance are unknown. Most studies focus on the gain of function of oncogenes as mediators of resistance; however, little is known about the role that tumor suppressors play in TKI resistance. Hence, we performed a genome-wide CRISPR Cas9 knock-out screen to identify genes that when knocked-out would drive erlotinib resistance, and KMT5C was identified as the top candidate. KMT5C is a histone methyltransferase that trimethylates H4K20 (H4K20me3), enabling the establishment of constitutive and facultative heterochromatin. Data from human samples suggests that the <i>KMT5C</i> transcript is globally downregulated in NSCLC and in tumor samples resistant to the third generation TKI osimertinib. Additionally, loss of the modification H4K20me3, influences prognosis of NSCLC, indicating that loss of KMT5C function is a crucial mechanism in carcinogenesis. Here we describe how loss of KMT5C leads to increased transcription of the oncogene MET, due to a loss in H4K20me3-mediated repression of a long non-coding RNA transcription (LINC01510) upstream of MET. This mechanism was found to be partially responsible in driving TKI resistance in EGFR mutant cells. Historically, KMT5C has been associated with generation of constitutive heterochromatin (cHC); however, recent reports, including our own, indicate that KMT5C also regulates transcription in regions outside of cHC. Our preliminary evidence suggests that deposition of H42K0me3 via KMT5C in regions outside of cHC, is less stable than in cHC regions. This novel finding led us to hypothesize that regulation of KMT5C and H42K0me3 at different regions of heterochromatin is a dynamic process.</p>