The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFß, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

Understanding the Complex Circuitry of lncRNAs at the X-inactivation Center and Its Implications in Disease Conditions.

Balanced gene expression is a high priority in order to maintain optimal functioning since alterations and variations could result in acute consequences. X chromosome inactivation (X-inactivation) is one such strategy utilized by mammalian species to silence the extra X chromosome in females to uphold a similar level of expression between the two sexes. A functionally versatile class of molecules called long noncoding RNA (lncRNA) has emerged as key regulators of gene expression and plays important roles during development. An lncRNA that is indispensable for X-inactivation is X-inactive specific transcript (Xist), which induces a repressive epigenetic landscape and creates the inactive X chromosome (Xi). With recent advents in the field of X-inactivation, novel positive and negative lncRNA regulators of Xist such as Jpx and Tsix, respectively, have broadened the regulatory network of X-inactivation. Xist expression failure or dysregulation has been implicated in producing developmental anomalies and disease states. Subsequently, reactivation of the Xi at a later stage of development has also been associated with certain tumors. With the recent influx of information about lncRNA biology and advancements in methods to probe lncRNA, we can now attempt to understand this complex network of Xist regulation in development and disease. It has become clear that the presence of an extra set of genes could be fatal for the organism. Only by understanding the precise ways in which lncRNAs function can treatments be developed to bring aberrations under control. This chapter summarizes our current understanding and knowledge with regard to how lncRNAs are orchestrated at the X-inactivation center (Xic), with a special focus on how genetic diseases come about as a consequence of lncRNA dysregulation.

Single-cell multiplex chromatin and RNA interactions in aging human brain.

The dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA (caRNA) 1-3. Chromatin complex compositions change during cellular differentiation and aging, and are expected to be highly heterogeneous among terminally differentiated single cells 4-7. Here we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within individual nuclei. Applied to 14 human frontal cortex samples from elderly donors, MUSIC delineates diverse cortical cell types and states. We observed the nuclei exhibiting fewer short-range chromatin interactions are correlated with an "older" transcriptomic signature and with Alzheimer's pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci (cis eQTLs) and a promoter tends to be the cell type where these cis eQTLs specifically affect their target gene's expression. Additionally, the female cortical cells exhibit highly heterogeneous interactions between the XIST non-coding RNA and Chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploring chromatin architecture and transcription at cellular resolution in complex tissues.

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

Deciphering the roles of lncRNAs in breast development and disease.

Breast cancer is the second leading cause of cancer related deaths in women. It is therefore important to understand the mechanisms underlying breast cancer development as well as raises the need for enhanced, non-invasive strategies for novel prognostic and diagnostic methods. The emergence of long non-coding RNAs (lncRNAs) as potential key players in neoplastic disease has received considerable attention over the past few years. This relatively new class of molecular regulators has been shown from ongoing research to act as critical players for key biological processes. Deregulated expression levels of lncRNAs have been observed in a number of cancers including breast cancer. Furthermore, lncRNAs have been linked to breast cancer initiation, progression, metastases and to limit sensitivity to certain targeted therapeutics. In this review we provide an update on the lncRNAs associated with breast cancer and mammary gland development and illustrate the versatility of such lncRNAs in gene control, differentiation and development both in normal physiological conditions and in diseased states. We also highlight the therapeutic and diagnostic potential of lncRNAs in cancer.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.