H3K27ac Modification Research Articles

Epigenomic and transcriptomic analyses reveal differences between low-grade inflammation and severe exhaustion in LPS-challenged murine monocytes

Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.

Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin.

Objectives: Metformin, a first-line drug that has been used for type 2 diabetes treatment, recently attracts broad attention for its therapeutic effects on diverse human cancers. However, its effect and the underlying mechanisms on oral squamous cell carcinoma (OSCC) are not well known.Materials and Methods: OSCC cells were used to evaluate the effect of metformin on cell proliferation and colony formation in vitro. Tumor formation assay in nude mice was conducted to assess the effect of metformin in vivo. Western blotting and immunohistochemistry stain were performed to investigate the effect of metformin on the expression of acetylation at lysine 27 of histone H3 (H3K27ac) and methylation at lysine 27 of histone H3 (H3K27me3) in vitro and in vivo. RNA-seq and ChIP-seq were performed to explore the genome profile to metformin treatment in OSCC cells.Results: Metformin inhibited OSCC cell proliferation and colony formation in vitro, as well as OSCC growth in vivo. Metformin increased the global H3K27ac modification in vitro. Transcriptome analysis suggested that metformin mainly downregulated pluripotency stem cell pathway, development involved pathways and upregulated cytokine and inflammatory pathways. Additionally, H3K27ac was involved in transcription, DNA repair and replication in metformin-treated OSCC cells.Conclusions: Metformin inhibits OSCC growth concomitant upregulated global level of H3K27ac in vitro. This study provides insights into the molecule and epigenome basis on application of metformin in OSCC treatment, and highlights the underlying mechanisms of reprogrammed cancer regulation and epigenetic histone modification.

Centromere Protein A (CENPA) Regulates Metabolic Reprogramming in the Colon Cancer Cells by Transcriptionally Activating Karyopherin Subunit Alpha 2 (KPNA2)

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

H3K27Ac modification and gene expression in psoriasis

BackgroundNumerous alterations in gene expression have been described in psoriatic lesions compared to uninvolved or healthy skin. However, the mechanisms which induce this altered expression remain unclear.Epigenetic modifications play a key role in regulating genes' expression. Only three studies compared the whole-genome DNA methylation of psoriasis versus healthy skin. The present is the first study of genome-wide comparison of histone modifications between psoriatic to healthy skins. ObjectiveOur objective was to explore the pattern of H3K27Ac modifications in psoriatic lesions compared to uninvolved psoriatic and healthy skin, in order to identify new genes involved in the pathogenesis of psoriasis. MethodUsing ChIP-seq with anti H3K27Ac we compared the acetylation of lysine 27 on histone 3 (H3K27Ac) modification between psoriatic to healthy skins, combined with mRNA array. ResultsWe found a differential H3K27Ac pattern between psoriatic compared to uninvolved or healthy skins. We found that many of the overexpressed and H3K27Ac enriched genes in psoriasis, harbor a putative GRHL transcription factor-binding site. ConclusionsIn the most overexpressed genes in psoriasis, there is an enrichment of H3K27Ac. However, the loss of H3K27 acetylation modification does not correlate with decreased gene expression.GRHL appears to play an important role in the pathogenesis of psoriasis and therefore, might be a new target for psoriasis therapeutics.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2848.

Characterization of Causal Variants at the GATA3 Loci Associated with Susceptibility to Ph-like Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and there is growing evidence of inherited susceptibility to this type of leukemia. Recent genome-wide association studies have definitively identified at least 6 genomic loci with common variants that influence ALL risk, often in an age- and/or subtype-dependent fashion. In particular, we had previously reported germline intronic variants at the GATA3 locus that specifically associated with the risk of developing Philadelphia chromosome-like ALL (Ph-like ALL) and also inferior outcomes of ALL therapy (Nature Genetics 45:1494). However, the identity of the causal variant(s) that functionally alters GATA3 activity and drives the association with the susceptibility to Ph-like ALL remained unknown.To comprehensively identify ALL risk variants at the GATA3 locus, we first performed targeted sequencing of a ~27 Kb genomic region encompassing GATA3 exons and introns in 5,008 children with ALL enrolled on Children's Oncology Group and St. Jude Children's Research Hospital frontline trials. Of 1048 variants with sufficient sequencing quality, rs3824662 showed the strongest association with susceptibility to Ph-like ALL (P=1.2x10-8) and another 2 variants also reached statistical significance after corrected for multiple testing (P <1x10-5). However, multivariate analysis conditioning on rs3824662 revealed no independent signals, thus pointing to a single causal variant responsible for the association signal at this locus. This linkage disequilibrium block also overlapped with a chromatin segment epigenetically active in the hematopoietic cells, as inferred from histone modification marks.Next, we set out to systemically characterize potential functions of this regulatory DNA element and how its activity is influenced by rs3824662 genotype. The ALL risk variant A allele at rs3824662 was associated with higher enhancer activity in the reporter gene assay, and elevated levels of H3K4Me1 and H3K27Ac marks (for active chromatin) relative to the wildtype C allele in lymphoblastoid cells. ATAC-seq data from a representative panel of B-ALL cell lines confirmed a more open chromatin conformation linked to the A allele at this locus, e.g., in Ph-like ALL. By CRISPR-cas9 genome editing, we introduced the A allele at rs3824662 into the wildtype lymphoblastoid cell line GM12878. Engineered GM12878 cells with variant genotype (AA or CA) showed 3.7- and 3.8-fold increase in GATA3 expression as compared to isogenic cells with wildtype genotype, with a concomitant increase in H3K27Ac and H3K4Me1 modification at this locus, indicating that the risk allele at rs3824662 positively regulates GATA3 transcription.Ectopic expression of GATA3 in a non-Ph-like ALL cell line (Nalm6), induced a global change in gene transcription profile that resembles the Ph-like expression signature (P=0.0004), with particularly notable upregulation of CRLF2 . Chromosomal rearrangement-mediated CRLF2 overexpression is a defining molecular abnormality in up to 50% of Ph-like ALL cases. In fact, using GATA3 ChIP-seq, we identified 5 regions upstream of CRLF2 enriched for binding to this transcription factor in ALL cell line models, with binding sites located proximal to CRLF2 rearrangement hotspots observed in patients with Ph-like ALL. Inducible overexpression of GATA3 led to upregulation of CRLF2 in a time-dependent manner; conversely, downregulation of GATA3 suppressed CRLF2 transcription, further suggesting that GATA3 may function as transcriptional regulator of CRLF2 . In mouse hematopoietic cell BaF3, GATA3 overexpression resulted in elevated phosphorylation of JAK2 and STAT5, downstream of CRLF2 . Co-expression of GATA3 and JAK2R683G were sufficient to induce cytokine-independent growth and leukemic transformation of BaF3 cells, in a fashion similar to co-expression of CRLF2 and JAK2R683G. Interestingly, the addition of CRLF2 ligand, TSLP, potentiated transformation of BaF7 cells expressing GATA3 and JAK2R683G . Taken together, these data suggested that the GATA3 -mediated activation of CRLF2 - JAK signaling is a plausible mechanism by which it contributes to the pathogenesis of Ph-like ALL.In conclusion, our results indicate that rs3824662 causally regulates a cis-acting enhancer for GATA3 transcription and influences Ph-like ALL leukemogenesis by regulating CRLF2-JAK signaling. DisclosuresHunger:Erytech Pharmaceuticals: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Honoraria; Amgen: Consultancy, Equity Ownership. Mullighan:Amgen: Consultancy; Loxo Oncology: Research Funding.

HIRA stabilizes skeletal muscle lineage identity

The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.

A noncanonical AR addiction drives enzalutamide resistance in prostate cancer

Resistance to next-generation anti-androgen enzalutamide (ENZ) constitutes a major challenge for the treatment of castration-resistant prostate cancer (CRPC). By performing genome-wide ChIP-seq profiling in ENZ-resistant CRPC cells we identify a set of androgen receptor (AR) binding sites with increased AR binding intensity (ARBS-gained). While ARBS-gained loci lack the canonical androgen response elements (ARE) and pioneer factor FOXA1 binding motifs, they are highly enriched with CpG islands and the binding sites of unmethylated CpG dinucleotide-binding protein CXXC5 and the partner TET2. RNA-seq analysis reveals that both CXXC5 and its regulated genes including ID1 are upregulated in ENZ-resistant cell lines and these results are further confirmed in patient-derived xenografts (PDXs) and patient specimens. Consistent with the finding that ARBS-gained loci are highly enriched with H3K27ac modification, ENZ-resistant PCa cells, organoids, xenografts and PDXs are hyper-sensitive to NEO2734, a dual inhibitor of BET and CBP/p300 proteins. These results not only reveal a noncanonical AR function in acquisition of ENZ resistance, but also posit a treatment strategy to target this vulnerability in ENZ-resistant CRPC.

Genome-wide analysis of chromatin structure changes upon MyoD binding in proliferative myoblasts during the cell cycle.

MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3 and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription.

Identification and Characterisation of Putative Enhancer Elements in Mouse Embryonic Stem Cells

Enhancer elements control mammalian transcription largely in a cell-type-specific manner. The genome-wide identification of enhancer elements and their activity status in a cellular context is therefore fundamental to understanding cell identity and function. We determined enhancer activity in mouse embryonic stem (ES) cells using chromatin modifications and characterised their global properties. Specifically, we first grouped enhancers into 5 groups using multiple H3K4me1, H3K27ac, and H3K27me3 modification data sets. Active enhancers (simultaneous presence of H3K4me1 and H3K27ac) were enriched for binding of pluripotency factors and were found near pluripotency-related genes. Although both H3K4me1-only and active enhancers were enriched for super-enhancers and a TATA box like motif, active enhancers were preferentially bound by RNA polII (s2) and were enriched for bidirectional transcription, while H3K4me1-only enhancers were enriched for RNA polII (8WG16) suggesting they were likely poised. Bivalent enhancers (simultaneous presence of H3K4me1 and H3K27me3) were preferentially in the vicinity of bivalent genes. They were enriched for binding of components of polycomb complex as well as Tcf3 and Oct4. Moreover, a ‘CTTTCTC’ de-novo motif was enriched at bivalent enhancers, previously identified at bivalent promoters in ES cells. Taken together, 3 histone modifications successfully demarcated active, bivalent, and poised enhancers with distinct sequence and binding features.

CircSOD2 induced epigenetic alteration drives hepatocellular carcinoma progression through activating JAK2/STAT3 signaling pathway

BackgroundEmerging evidence suggests that circular RNAs play critical roles in disease development especially in cancers. Previous genome-wide RNA-seq studies found that a circular RNA derived from SOD2 gene was highly upregulated in hepatocellular carcinoma (HCC), however, the role of circSOD2 in HCC remains largely unknown.MethodsThe expression profiling of circSOD2 and microRNA in HCC patients were assessed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). SiRNA or CRISPR-CAS9 were used to silence gene expression. The biological function of circSOD2 in HCC was investigated using in vitro and in vivo studies including, trans-well cell migration, cell apoptosis, cell cycle, CCK8, siRNA interference, western blots, and xenograft mouse model. The underlying molecular mechanism was determined by Chromatin Immunoprecipitation quantitative real time PCR (ChIP-qPCR), bioinformatic analysis, biotin-pull down, RNA immunoprecipitation, 5-mc DNA pulldown and luciferase assays.ResultsIn accordance with previous sequencing results, here, we demonstrated that circSOD2 was highly expressed in HCC tumor tissues compared with normal liver tissues. Mechanically, we showed that histone writer EP300 and WDR5 bind to circSOD2 promoter and trigger its promoter H3K27ac and H3K4me3 modification, respectively, which further activates circSOD2 expression. SiRNA mediated circSOD2 suppression impaired liver cancer cell growth, cell migration, prohibited cell cycle progression and in vivo tumor growth. By acting as a sponge, circSOD2 inhibits miR-502-5p expression and rescues miR-502-5p target gene DNMT3a expression. As a DNA methyltransferase, upregulated DNMA3a suppresses SOCS3 expression by increasing SOCS3 promoter DNA methylation. This event further accelerates SOCS3 downstream JAK2/STAT3 signaling pathway activation. In addition, we also found that activated STAT3 regulates circSOD2 expression in a feedback way.ConclusionThe novel signaling axis circSOD2/miR-502-5p/DNMT3a/JAK2/STAT3/circSOD2 provides a better understanding of HCC tumorigenesis. The molecular mechanism underlying this signaling axis offers new prevention and treatment of HCC.

Trans-Ancestral Fine-Mapping and Epigenetic Annotation as Tools to Delineate Functionally Relevant Risk Alleles at IKZF1 and IKZF3 in Systemic Lupus Erythematosus.

Background: Prioritizing tag-SNPs carried on extended risk haplotypes at susceptibility loci for common disease is a challenge. Methods: We utilized trans-ancestral exclusion mapping to reduce risk haplotypes at IKZF1 and IKZF3 identified in multiple ancestries from SLE GWAS and ImmunoChip datasets. We characterized functional annotation data across each risk haplotype from publicly available datasets including ENCODE, RoadMap Consortium, PC Hi-C data from 3D genome browser, NESDR NTR conditional eQTL database, GeneCards Genehancers and TF (transcription factor) binding sites from Haploregv4. Results: We refined the 60 kb associated haplotype upstream of IKZF1 to just 12 tag-SNPs tagging a 47.7 kb core risk haplotype. There was preferential enrichment of DNAse I hypersensitivity and H3K27ac modification across the 3′ end of the risk haplotype, with four tag-SNPs sharing allele-specific TF binding sites with promoter variants, which are eQTLs for IKZF1 in whole blood. At IKZF3, we refined a core risk haplotype of 101 kb (27 tag-SNPs) from an initial extended haplotype of 194 kb (282 tag-SNPs), which had widespread DNAse I hypersensitivity, H3K27ac modification and multiple allele-specific TF binding sites. Dimerization of Fox family TFs bound at the 3′ and promoter of IKZF3 may stabilize chromatin looping across the locus. Conclusions: We combined trans-ancestral exclusion mapping and epigenetic annotation to identify variants at both IKZF1 and IKZF3 with the highest likelihood of biological relevance. The approach will be of strong interest to other complex trait geneticists seeking to attribute biological relevance to risk alleles on extended risk haplotypes in their disease of interest.

Abstract 2931: VGF as a theranostic marker in neuroendocrine lung cancer

Abstract Activating EGFR mutations are predictive markers for response in lung cancer patients under the treatment of EGFR-tyrosine kinase inhibitors (TKIs), whereas acquired resistance to EGFR-TKIs inevitably occurs in patients. Neuroendocrine transformation is one of the mechanisms behind the development of acquired resistance to EGFR-TKIs. In this study, we report that VGF, a secreted factor highly expressed in neurons and neuroendocrine tissues, is a potential theranostic marker in neuroendocrine lung tumors. Gene expression profiling analysis revealed that VGF levels were elevated during the differentiation of neuronal stem cells to neurons with surged H3K27ac modification at the VGF locus. We observed that VGF was highly expressed in lung neuroendocrine cancer cells. Moreover, VGF expression was low in lung adenocarcinoma but was enriched during EGFR-TKI selection. Correlation analysis showed that VGF was positively associated with chromogranin A, chromogranin B, and synaptophysin expression in lung tumors. ChIP-qPCR assays showed that the signal of H3K27ac modification at the VGF locus was increased in EGFR-TKI resistant cells compared to their sensitive counterparts. Clonogenic analysis and annexin-V staining assays revealed that VGF silencing attenuated cellular grown with increased apoptosis in neuroendocrine lung cancer cells as well as in EGFR-TKI resistant cells, indicating the potential of VGF as a therapeutic target. Kaplan-Meier analysis showed that high VGF expression predicted poor survival outcome in patients with lung tumors. Our findings provide an insight into the oncogenic role of VGF and suggest the potential of VGF as a theranostic for lung cancer. Citation Format: Li-Hao Yang, Chia-Cherng Yu, Yu-Ting Chou. VGF as a theranostic marker in neuroendocrine lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2931.

LncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy

The regulatory mechanism of long non-coding RNAs (lncRNAs) in trastuzumab resistance is not well established to date. In this research, we identified differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. HiSeq sequencing and quantitative real-time PCR were performed to identify the dysregulated lncRNAs. Mass spectrometry, RNA fluorescence in situ hybridization (RNA-FISH), and immunoprecipitation assays were performed to identify the direct interactions between ZNF649-AS1 and other associated targets, such as polypyrimidine tract binding protein 1 (PTBP1) and autophagy related 5 (ATG5). Our results showed that ZNF649-AS1 was more highly expressed in trastuzumab-resistant cells compared to sensitive cells. Increased expression of ZNF649-AS1 was associated with a poorer response and shorter survival time of breast cancer patients. ZNF649-AS1 was upregulated by H3K27ac modification at the presence of trastuzumab treatment, and knockdown of ZNF649-AS1 reversed trastuzumab resistance via modulating ATG5 expression and autophagy. Mechanically, ZNF649-AS1 was associated with PTBP1 protein, which further promoted the transcription activity of the ATG5 gene. In conclusion, we demonstrated that H3K27ac modification-induced upregulation of ZNF649-AS1 could cause autophagy and trastuzumab resistance through associating with PTBP1 and promoting ATG5 transcription.

LINC00941 promotes oral squamous cell carcinoma progression via activating CAPRIN2 and canonical WNT/β-catenin signaling pathway.

Dysregulation of long non‐coding RNAs (lncRNAs) has been implicated in many cancer developments. Previous studies showed that lncRNA LINC00941 was aberrantly expressed in oral squamous cell carcinoma (OSCC). However, its role in OSCC development remains elusive. In this study, we demonstrated that in OSCC cells, EP300 activates LINC00941 transcription through up‐regulating its promoter H3K27ac modification. Up‐regulated LINC00941 in turn activates CAPRIN2 expression by looping to CAPRIN2 promoter. Functional assays suggest that both LINC00941 and CAPRIN2 play pivotal roles in promoting OSCC cell proliferation and colony formation. In vivo assay further confirmed the role of LINC00941 in promoting OSCC cell tumour formation. Lastly, we showed that the role of LINC00941 and CAPRIN2 in OSCC progression was mediated through activating the canonical WNT/β‐catenin signaling pathway. Thus, LINC00941/CAPRIN2/ WNT/β‐catenin signaling pathway provides new therapeutic targets for OSCC treatment.

Hippo-YAP signaling controls lineage differentiation of mouse embryonic stem cells through modulating the formation of super-enhancers.

Hippo-YAP signaling pathway functions in early lineage differentiation of pluripotent stem cells, but the detailed mechanisms remain elusive. We found that knockout (KO) of Mst1 and Mst2, two key components of the Hippo signaling in mouse embryonic stem cells (ESCs), resulted in a disruption of differentiation into mesendoderm lineage. To further uncover the underlying regulatory mechanisms, we performed a series of ChIP-seq experiments with antibodies against YAP, ESC master transcription factors and some characterized histone modification markers as well as RNA-seq assays using wild type and Mst KO samples at ES and day 4 embryoid body stage respectively. We demonstrate that YAP is preferentially co-localized with super-enhancer (SE) markers such as Nanog, Sox2, Oct4 and H3K27ac in ESCs. The hyper-activation of nuclear YAP in Mst KO ESCs facilitates the binding of Nanog, Sox2 and Oct4 as well as H3K27ac modification at the loci where YAP binds. Moreover, Mst depletion results in novel SE formation and enhanced liquid-liquid phase-separated Med1 condensates on lineage associated genes, leading to the upregulation of these genes and the distortion of ESC differentiation. Our study reveals a novel mechanism on how Hippo-YAP signaling pathway dictates ESC lineage differentiation.

Light and Abscisic Acid Coordinately Regulate Greening of Seedlings.

The greening of etiolated seedlings is crucial for the growth and survival of plants. After reaching the soil surface and sunlight, etiolated seedlings integrate numerous environmental signals and internal cues to control the initiation and rate of greening thus to improve their survival and adaption. However, the underlying regulatory mechanisms by which light and phytohormones, such as abscisic acid (ABA), coordinately regulate greening of the etiolated seedlings is still unknown. In this study, we showed that Arabidopsis (Arabidopsis thaliana) DE-ETIOLATED1 (DET1), a key negative regulator of photomorphogenesis, positively regulated light-induced greening by repressing ABA responses. Upon irradiating etiolated seedlings with light, DET1 physically interacts with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and subsequently associates to the promoter region of the FHY3 direct downstream target ABA INSENSITIVE5 (ABI5). Further, DET1 recruits HISTONE DEACETYLASE6 to the locus of the ABI5 promoter and reduces the enrichments of H3K27ac and H3K4me3 modification, thus subsequently repressing ABI5 expression and promoting the greening of etiolated seedlings. This study reveals the physiological and molecular function of DET1 and FHY3 in the greening of seedlings and provides insights into the regulatory mechanism by which plants integrate light and ABA signals to fine-tune early seedling establishment.

RETRACTED ARTICLE: Exosome-mediated lncRNA AFAP1-AS1 promotes trastuzumab resistance through binding with AUF1 and activating ERBB2 translation

BackgroundAlthough trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer.MethodsLncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance.ResultsAFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level.ConclusionExosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.

The failure of DAC to induce OCT2 expression and its remission by hemoglobin-based nanocarriers under hypoxia in renal cell carcinoma.

Background: Human organic cation transporter 2 (OCT2) is the most abundant and important uptake transporter involved in the renal excretion of cationic drugs. Abnormal hypermethylation- mediated silencing of OCT2 results in oxaliplatin resistance in renal cell carcinoma (RCC). The epigenetic activation of OCT2 by decitabine (DAC) reversed this resistance in normoxic conditions. Given the hypoxic characteristic of RCC, it is still unclear whether hypoxia promotes DAC resistance and is involved in the regulation of OCT2.Methods: The mRNA and protein expression of OCT2 was determined by qRT-PCR and Western blotting. MSRE-qPCR and BSP were used to examine methylation modifications at the OCT2 promoter. The ChIP-qPCR analysis was performed to detect the abundance of histone modification and HIF-1α. The accumulation of DAC and 5-mC were detected using LC-MS, and the amount of 5-hmC was determined by dot blot analysis. To understand the role of hypoxia in the regulation of equilibrative nucleoside transporter 1 (ENT1) expression, the HIF-1α KO cell model was constructed. The re-emulsion method was used for the construction of H-NPs, an oxygen nanocarrier based on hemoglobin, to alleviate the drug resistance of DAC under hypoxia.Results: DAC was unable to upregulate OCT2 expression in hypoxic conditions because of the hypermethylation and low H3K4me3 modification in its promoter region. Hypoxia-mediated repression of human ENT1, which was markedly suppressed in RCC, resulted in a decrease in the cellular accumulation of DAC. Besides, hypoxia-induced upregulation of histone deacetylase HDAC9, which impaired the enrichment of H3K27ac modification in the OCT2 promoter, led to the transcriptional repression of OCT2. H-NPs could attenuate the hypoxia-induced loss of DAC activity and sensitize RCC cells to the sequential combination therapy of DAC and oxaliplatin.Conclusions: Hypoxia-mediated repression of ENT1 led to the inability of DAC to upregulate the expression of OCT2 under hypoxia. H-NPs could alleviate resistance to oxaliplatin and DAC in RCC cells under hypoxia and may have potential clinical applications.

Yeats4 drives ILC lineage commitment via activation of Lmo4 transcription.

Innate lymphoid cells (ILCs) play critical roles in defending infections and maintaining mucosal homeostasis. All ILCs arise from common lymphoid progenitors (CLPs) in bone marrow. However, how CLPs stratify and differentiate into ILC lineages remains elusive. Here, we showed that Yeats4 is highly expressed in ILCs and their progenitors. Yeats4 conditional KO in the hematopoietic system causes decreased numbers of ILCs and impairs their effector functions. Moreover, Yeats4 regulates α4β7 + CLP differentiation toward common helper ILC progenitors (CHILPs). Mechanistically, Yeats4 recruits the Dot1l-RNA Pol II complex onto Lmo4 promoter through recognizing H3K27ac modification to initiate Lmo4 transcription in α4β7 + CLPs. Additionally, Lmo4 deficiency also impairs ILC lineage differentiation and their effector functions. Collectively, the Yeats4-Lmo4 axis is required for ILC lineage commitment.