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BackgroundThe histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences.ResultsRecently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1.ConclusionsAltogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.

BackgroundDespite today's advances in the treatment of cancer, breast cancer-related mortality remains high, in part due to the lack of effective targeted therapies against breast tumor types that do not respond to standard treatments. Therefore, identifying additional breast cancer molecular targets is urgently needed. Super-enhancers are large regions of open chromatin involved in the overactivation of oncogenes. Thus, inhibition of super-enhancers has become a focus in clinical trials for its therapeutic potential. Here, we aimed to identify novel super-enhancer dysregulated genes highly associated with breast cancer patients' poor prognosis and negative response to treatment.MethodsUsing existing datasets containing super-enhancer-associated genes identified in breast tumors and public databases comprising genomic and clinical information for breast cancer patients, we investigated whether highly expressed super-enhancer-associated genes correlate to breast cancer patients' poor prognosis and to patients' poor response to therapy. Our computational findings were experimentally confirmed in breast cancer cells by pharmacological SE disruption and gene silencing techniques.ResultsWe bioinformatically identified two novel super-enhancer-associated genes - NSMCE2 and MAL2 - highly upregulated in breast tumors, for which high RNA levels significantly and specifically correlate with breast cancer patients' poor prognosis. Through in-vitro pharmacological super-enhancer disruption assays, we confirmed that super-enhancers upregulate NSMCE2 and MAL2 transcriptionally, and, through bioinformatics, we found that high levels of NSMCE2 strongly associate with patients' poor response to chemotherapy, especially for patients diagnosed with aggressive triple negative and HER2 positive tumor types. Finally, we showed that decreasing NSMCE2 gene expression increases breast cancer cells' sensitivity to chemotherapy treatment.ConclusionsOur results indicate that moderating the transcript levels of NSMCE2 could improve patients' response to standard chemotherapy consequently improving disease outcome. Our approach offers a new avenue to identify a signature of tumor specific genes that are not frequently mutated but dysregulated by super-enhancers. As a result, this strategy can lead to the discovery of potential and novel pharmacological targets for improving targeted therapy and the treatment of breast cancer.

Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.