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Epigenetic chemical probes are well established tools in oncology research but their application to other research fields is an area of emerging interest. This blog is a summary of recent literature describing the role of bromodomain-containing proteins (BRDs), protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs) in research outside oncology. The literature referenced is only a snapshot and is meant as an update to a recent review [PMID: 28080202]. In addition, while some of the cited studies employ chemical probes, others utilize genetic ‘inhibition’ or small molecule-mediated knock-down (e.g PROTACs) to validate hypotheses. The former are presented as examples where chemical probes may also be used.
The importance of epigenetic regulation in stem cell programming is increasingly being recognized. Here are interesting examples of stem cell studies using epigenetic probes. Lee et al. [PMID: 27625395] identified miR-221 gene-encoded miR-221-3p and miR-221-5p as potential anti-stemness miRNAs. In a follow-up study, Chen et al. have produced an elegant study showing that PRMT7-mediated down-regulation of microRNA-24-2 maintains Oct4, Nanog, and Sox2 in mouse embryonic stem cells (mESCs) [PMID: 29378844 ]. Using a reporter assay and mutation studies, Chen et al. found that miR-221 is able to target the 3’UTRs of Oct4, Nanog, SOX2, Klf4, and PRMT7. CRISPR-mediated deletion of the miR-221 gene, as well as specific anti-sense inhibitors of miR-221-3p and -5p inhibited the spontaneous differentiation of PRMT7-depleted mouse ESCs. Further, the repressive marks H4R3me1 and H4R3me2s were decreased at the miR-221 promoter when PRMT7 is depleted (using two different shRNAs).
Epigenetic chemical probes have been used to investigate skeletal muscle remodeling, revealing PRMT1 involvement not only in myoblast differentiation, but also in mitochondrial biogenesis and metabolism. Shen et al. studied the gene and protein expression of PRMT1, 4 and 5 during myogenesis over a 7-day period [PMID: 29208765 ]. Using established histological and molecular markers myogenesis, the authors first confirmed that C2C12 cells progressed as expected from mononucleated myoblasts to robust myotubes over the 7-day period. PRMT1 mRNA was found to be significantly increased (peaking at day 5) while PRMT4 and 5 transcript levels were unchanged. Further, protein expression matched the transcript levels. PRMT4 and PRMT5 were expressed across the time-course with no change, while PRMT1 expression was at least 2-fold at day 3 of myogenesis and remained elevated until day 7.
Shen et al. also measured global monomethylarginine (MMA), asymmetric and symmetric dimethylarginine (ADMA and SDMA), and methylation of specific histone substrates. ADMA increased by 1.7-fold on day 3 and remained elevated, while MMA and SDMA remained unchanged. The PRMT1 substrates H4R3me2a and H3R17me2a were ~2-fold higher at day 7 compared with myoblasts whereas H3R8me2s status was unchanged. Chemical inhibition of PRMT1 using TC-E at 0.1 μM, did not affect cellular H4, but reduced the level of H4R3me2a by 25%, and attenuated all morphological metrics associated with myogenesis from days 3-7. Over this period, mitochondrial electron transport chain CI, CIII, CV, and PGC-1α were attenuated by ~20-40% while CII was unchanged. Finally, oxygen consumption from days 3-7 decreased by 20-35% in the presence of the inhibitor relative to vehicle controls.
Osteoclasts (OCs) play a major role in bone remodeling, repair and maintenance of minerals. DOT1L expression and subsequent H3K79 methylation are increased in RANKL-induced OC differentiation of RAW264.7 macrophage cells [PMID: 29348610]. Chemical inhibition of DOT1L with EPZ 004777 or EPZ5676 resulted in a global decrease in H3K79me2 in RAW264.7-derived OCs relative to RAW264.7. In these latter cells, there is some indication that methylation of H3K27 and H3K36 actually increased whereas in OCs only methylation of H3K36 increased slightly. In addition, autophagy-related proteins – SNAREs, Sqstm1, VPS35, and Atg3 – were up-regulated in 40-hour pre-OC cells; indeed, an increase in autophagy activity was confirmed by a commercial assay and by western blot against autophagosome markers.
In a related study, Kota et al. [PMID: 30189247 ] inhibited PRMT5 by treating mesenchymal stromal cell lines in steady state or undergoing osteogenic differentiation with EPZ015666 (0.6-1.4 μM). Inhibition of PRMT5 increased osteoblast differentiation and down-regulated several Gbp (guanylate binding protein) family genes. This resulted in global, as well as promoter specific decrease in H3R8me2s and H4R3me2s at promoters of interferon stimulated gene loci.
Strength and integrity of the human skeleton depends on a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. The BRPF scaffolding proteins have been shown to be involved in RANKL-induced differentiation with primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by repressing programs required for transcription in osteoclastogenesis [PMID: 28849908].
DOT1L also plays a vital role in the differentiation of cardiac progenitor cells [PMID:29631608]. ChIP identified the DOT1L specific mark H3K79me2 on GATA4, HAND1, NR2F2, NKX2.5, MESP1, ISL1, and WNT5A during hESC differentiation into cardiac progenitor cells and cardiomyocytes. Differentiation from KIND1 and HES3 showed co-localization of DOT1L with the master cardiac transcription factor NKX2.5 (after the pluripotency stage and during differentiation to cardiac progenitor and cardiomyocytes). siRNA knockdown of DOT1L did not have an effect on the pluripotency of hES, however, differentiation to the cardiac lineage was affected. This was confirmed morphologically as well as at the transcript and protein levels. The authors acknowledge that DOT1L may act with other histone modifying enzymes as it has been shown that H3K4me3, H3K36me3 are also involved in differentiation to cardiac progenitors.
Deregulation of inflammatory mechanisms is detrimental and is the underlying cause for widespread chronic diseases such as asthma, arthritis, psoriasis, atopic eczema, periodontitis and inflammatory bowel disease. Several recent publications where chemical probes are used in inflammation-related research are summarized below.
BET inhibitors have shown positive effects in a variety of inflammatory models. As current BET antagonists do not distinguish between the different BET family members, many studies do not provide unambiguous results regarding the biological functions of BRD4 versus BRD2 or BRD3. Recent publications include an in vivo study where the BET bromodomain inhibitor JQ1 attenuated acute inflammation due to spinal cord injury [PMID: 30134146 ]. PCAF/GCN5 degradation by a GSK4027-related PROTAC but not antagonism by GSK4027 identified a role in differentiation of monocytes into macrophages and dendritic cells [PMID:30200762].
PRMTs and PKMTs also play roles in the inflammatory response. PRMT1, 4, 5, and 6 all have various roles in inflammation related to cancer and metastasis, asthma and antigen-induced pulmonary airway conditions, responses to infection, transplant rejection, diabetes, and inflammatory bowel diseases [PMID: 27860244, 28087667, 29119338, 27840030, 27860244, and 29973649]. PKMTs including DOT1L, PRC2 complex, G9a and SMYD3 are involved in innate immune response using the DOT1L inhibitors EPZ5676 and SGC0946 [PMID: 30275539, 29765028], T cell antigen receptor (TCR)-mediated signaling using the EZH2 inhibitors UNC1999 and GSK503 [PMID: 29523590], regulation of liver-specific genes for inflammation using genetic knockout of G9a [PMID: 29912608], and regulations of pathogenic T-cell responses during pulmonary viral infection [PMID: 25669152].
RNA from the influenza virus interacts with its nucleoprotein (NP), whose function is analogous to histones in eukaryotes. A publication by Hatakeyama et al. [PMID: 29555684] identifies viral NP acetylation by two host cell acetyltransferases, PCAF and GCN5 at K31 and K90, respectively. siRNA targeting PCAF and GCN5 did not affect NP acetylation levels but did have opposing effects on viral transcriptional activity with PCAF-specific siRNA increasing viral transcription. K31 and K90 are located on opposite sides of the RNA-binding groove. These two facts indicate that GCN5 acetylation of K90 has a different biological role than PCAF acetylation of K31. Based on earlier interaction studies for NP, the authors further implicate roles for the acetyllysine binding domains of SMARCA2 and SMARCA4.
A related study by Marcos-Villar et al. reported a more global view of DNA and histone levels during influenza infection [PMID: 29352168]. DNA methylation was unaffected but the level of histone H3 and H4 methylation, H3K4me3 were reduced, while H3K36(me0), H4K20me2, and H3K79me2 were increased. These changes correlate with transcriptional inactivation. Either a DOT1L inhibitor (used at 1 micromolar) or (specific) shRNA reduced H3K79me2 and increased replication of the influenza virus. A more detailed investigation of the role of DOT1L in the response to influenza infection, showed that DOT1L down-regulation resulted in decreased nuclear translocation of the NF-κB complex, expression of IFNβ and ISGs (Mx1 and ISG56). Furthermore, influenza infected cells deficient in the IFN pathway were not affected by inhibition of DOT1L.
An unbiased CRISPR screen was performed on human iPS cell-derived β-like cells and subsequent gene ontology (GO) analysis identified genes associated with chromatin modification, cell cycle and transcription [PMID: 29754817]. Vitamin D receptor (VDR) was one of the most enriched gene targets (6 of 7 sgRNAs found in the GFP- cells). iPS cell lines, containing an shRNA knockdown of VDR, were differentiated into β-like cells and treated with IL1β. These cells featured higher levels of cytokine-induced cell death. Further investigation into the role of VDR identified the interacting proteins BRD9 and BRD7. BRD9 co-immunoprecipitates with HA-tagged VDR and endogenous VDR. This interaction was found to be recapitulated with BRD9 bromodomain, and was weakened in the presence of VDR ligand (calcipotriol). A BRD9 antagonist I-BRD9, also reduced the strength of the interaction between BRD9 and VDR. LC/MS/MS data in addition to mutation studies identified VDR-K91ac as being important for the interaction with BRD9. Although HA-tagged VDR bound to Flag-tagged BRD7, the interaction was strengthened by the addition of VDR ligand or VDR ligand and I-BRD9. This suggests a competitive binding of BRD9 and BRD7 to VDR. Based on an earlier study, PCAF was short-listed as the VDR-K91ac acetyltransferase, and in line with this wild-type PCAF and not the D608A (enzymatically dead) mutant increased VDR-K91ac. Disrupting the interaction with BRD9, shifts the equilibrium to PBAF and binding to BRD7.
Glycogen synthase kinase 3γ (GSK3γ) regulates glycogen synthase, and has recently been shown to be involved in bone resorption of OC, and tumor suppression in prostate carcinoma cells. Increased activity of the kinase is also implicated in type 2 diabetes (T2D). The authors found that an increase in BRD7 protein expression leads to an increase in GSK3b phosphorylation (even in the absence of AKT1/2 kinase activity) in an mTOR-dependent mode [PMID: 29127434]. (BRD7 overexpression while blocking the activity of AKT and mTOR did not increase the phosphorylation of GSK3b.) BRD7 brokers the phosphorylation of GSK3γ-S9 in the absence of AKT activity, and initiates a cascade where ribosomal protein S6 kinase is phosphorylated, which leads to increased phosphorylation of 4E-BP1 and therefore relieves its inhibition of eIF4E. In the absence of AKT activity and BRD7 overexpression, 4E-BP1 phosphorylation was blunted. Using liver-specific BRD7 KO mice, the authors show that BRD7 is required for mTORC1 activity on its downstream interactors.
In summary, epigenetics is involved in regulating every process in the body and gaining a deeper understanding of the role of epigenetics in every branch of science will help us build a more comprehensive picture of how all systems work together. It is hoped that this insight will generate new therapeutic targets for previously untreatable diseases. A comprehensive list of the SGC’s epigenetic chemical probes is here, http://www.thesgc.org/chemical-probes/epigenetics.