Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

The role of telomeres in sustained tumor growth is well understood. However, mechanisms of how telomeres might impact the tumor microenvironment (TME) are not clear. Upon examining tumor associated macrophages (TAMs) in 94 hormone-negative (triple-negative) breast cancer (TNBC) cases we found infiltration of TAMs to be telomere sensitive: Tumors with relatively short telomeres had higher abundance of TAM and vice versa. This observation was replicated across TNBC clinical tissue, patient-derived organoids, tumor xenografts and cancer cells with long/short telomeres. Mechanistically, we demonstrate that non-telomeric binding of TRF2, a telomere-repeat-binding-factor; at the interleukin receptor IL1R1 promoter directly activates IL1R1 through recruitment of the histone-acetyl-transferase p300 and consequent H3K27 acetylation. Interleukin-1signaling could be induced in TRF2-high cells through ligands IL1A/B, but not TNFα, and abrogated by the receptor antagonist IL1RA, supporting specificity of the TRF2-IL1R1axis. TRF2 binding at the IL1R1 promoter was mediated by G-quadruplex motifs and was sensitive to telomere length – thereby establishing telomere-length-dependent regulation of IL1R1 and IL1-mediated TAM infiltration in cancers. Our results reveal a heretofore unknown function of telomeres in interleukin signaling and anti- tumor immune response, through non-telomeric TRF2. Therefore, we propose telomere length as a novel biomarker underlying patient-specific response to cancer immunotherapy.

Formation and recycling of an active epigenetic mark mediated by cell cycle-specific RNAs

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain poorly understood. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin.

Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis

The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.

Reduction in gene expression noise by targeted increase in accessibility at gene loci

Many eukaryotic genes are expressed in randomly initiated bursts that are punctuated by periods of quiescence. Here, we show that the intermittent access of the promoters to transcription factors through relatively impervious chromatin contributes to this “noisy” transcription. We tethered a nuclease-deficient Cas9 fused to a histone acetyl transferase at the promoters of two endogenous genes in HeLa cells. An assay for transposase-accessible chromatin using sequencing showed that the activity of the histone acetyl transferase altered the chromatin architecture locally without introducing global changes in the nucleus and rendered the targeted promoters constitutively accessible. We measured the gene expression variability from the gene loci by performing single-molecule fluorescence in situ hybridization against mature messenger RNAs (mRNAs) and by imaging nascent mRNA molecules present at active gene loci in single cells. Because of the increased accessibility of the promoter to transcription factors, the transcription from two genes became less noisy, even when the average levels of expression did not change. In addition to providing evidence for chromatin accessibility as a determinant of the noise in gene expression, our study offers a mechanism for controlling gene expression noise which is otherwise unavoidable.

JADE family proteins regulate proteasome abundance and activity

JADE family proteins (JADE1/2/3) have been implicated in diverse cellular functions and signaling pathways ranging from WNT signaling and cell cycle control to cell death and complex transcriptional regulation through histone acetyl-transferase complexes. JADE proteins show a high degree of sequence similarity and share two PHD zinc finger domains. JADE1 interacts with cilia-associated proteins and has been implicated in cilia-related genetic disorders with kidney phenotypes. However, the function of the widely expressed JADE proteins at the molecular level is still elusive. Here we show that JADE proteins regulate proteasome abundance and activity. Using kidney cells as a model, we demonstrate that loss of either JADE protein resulted in increased expression of almost all components of the 26S proteasome. Regulation occurred at the post-translational level and was not the consequence of transcriptional activation. Consistent with a role for JADE proteins in regulating overall proteasomal abundance, proteasomal activity was elevated in Jade-deficient cells, while exogenous expression of JADE1/2/3 decreased the level of proteasome activity. Coimmunoprecipitation experiments confirmed the interaction of proteasomal subunits with Jade1 suggesting a direct role of JADE proteins in regulating turnover, stability and abundance of the 26s proteasome. These data may now explain the plethora of cellular roles that have been attributed to JADE proteins.

Multi-Omics Approach to Dissect the Mechanisms of Rexinoid Signaling in Myoblast Differentiation

Stem cells represent a key resource in regenerative medicine, however, there is a critical need for pharmacological modulators to promote efficient conversion of stem cells into a myogenic lineage. We have previously shown that bexarotene, an agonist of retinoid X receptor (RXR) approved for cancer therapy, promotes the specification and differentiation of skeletal muscle progenitors. To decipher the molecular regulation of rexinoid signaling in myogenic differentiation, we have integrated RNA-seq transcription profiles with ChIP-seq of H4K8, H3K9, H3K18, H3K27 acetylation, and H3K27 methylation in addition to that of histone acetyl-transferase p300 in rexinoid-promoted myoblast differentiation. Here, we provide details regarding data collection, validation and omics integration analyses to offer strategies for future data application and replication. Our analyses also reveal molecular pathways underlying different patterns of gene expression and p300-associated histone acetylation at distinct chromatin states in rexinoid-enhanced myoblast differentiation. These datasets can be repurposed for future studies to examine the relationship between signaling molecules, chromatin modifiers and histone acetylation in myogenic regulation, providing a framework for discovery and functional characterization of muscle-specific loci.

Abstract P415: Etv2 Interacts With Vezf1 And Co-regulate Endothelial Gene Expression

Background: Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Results: Using a yeast two-hybrid assay, we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatics analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as candidate genes and have previously been shown to function as regulators of endothelial development. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histones. Additionally, we have undertaken ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) and Vezf1 ChIP-Seq studies to profile the epigenetic modifications and genome-wide occupancy of Vezf1 in the presence or absence of Etv2 in endothelial progenitor cells. Conclusion: In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and we further demonstrate their combined role in the regulation of downstream target genes, Amotl2 and Hdac7, during endothelial development.

Formation and recycling of an active epigenetic mark mediated by cell cycle-specific RNAs

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain poorly understood. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin.

Epigenetic immunomodulatory effect of eugenol and astaxanthin on doxorubicin cytotoxicity in hormonal positive breast Cancer cells

Background Hormonal receptor positive (HR+) breast cancer is the most commonly diagnosed molecular subtype of breast cancer; which showed good response to doxorubicin (DOX)-based chemotherapy. Eugenol (EUG) and astaxanthin (AST) are natural compounds with proved epigenetic and immunomodulatory effects in several cancer cell lines. This study has been initiated to investigate the molecular mechanism (s) whereby EUG and AST could enhance DOX cytotoxicity in MCF7 cells. Methods Cytotoxic activity of DOX alone and combined with either 1 mM EUG or 40 μM AST was performed using sulphorhodamine-B assay in MCF7 cells. Global histones acetylation and some immunological markers were investigated using ELISA, western blotting and quantitative RT-PCR techniques. Functional assay of multidrug resistance was performed using rhodamine 123 and Hoechst 3342 dyes. Flow cytometry with annexin V and propidium iodide were used to assess the change in cell cycle and apoptosis along with the expression of some differentiation, apoptosis and autophagy proteins. Results DOX alone resulted in concentration-dependent cytotoxicity with IC50 of 0.5 μM. Both EUG and AST significantly increased DOX cytotoxicity which is manifested as a significant decrease in DOX IC50 from 0.5 μM to 0.088 μM with EUG and to 0.06 μM with AST. Combinations of DOX with 1 mM EUG or 40 μM AST significantly increased the level of histones acetylation and histone acetyl transferase expression, while reduced the expression of aromatase and epidermal growth factor receptor (EGFR) when compared with 0.25 μM DOX alone. Also both combinations showed higher uptake of rhodamine but lower of Hoechst stains, along with increased the percentage of caspase 3, and decreased the expression of CK7 and LC3BI/II ratio. EUG combination induced IFγ but reduced TNFα causing shifting of cells from G2/M to S and G0/ G1 phases. Combination of DOX with EUG induced apoptosis through the higher BAX/ BCl2 ratio, while with AST was through the increase in caspase 8 expressions. Conclusion EUG and AST potentiated the anticancer activity of DOX through epigenetic histones acetylation along with the immunonomodulation of different apoptotic approaches in MCF7 cells.