Role In Transcriptional Repression Research Articles

Design, synthesis, biological evaluation and molecular docking study of novel quinolone derivatives as potent HDAC1 (Histone Deacetylase) inhibitors and anticancer agents

Histone deacetylases (HDACs), especially HDAC1, are attractive targets for anticancer drug design because of their significant role in transcriptional repression and chromatin remodeling. Benzamide groups are widely reported as the most effective zinc binding groups (ZBGs) in design of HDAC inhibitors. Quinolone derivatives, exhibiting various therapeutic effects, are attractive scaffolds for developing novel HDAC inhibitors with potentially supposed therapeutic profiles. To explore the potential of 4-quinolones as a novel scaffold for a cap of HDACis, 14 novel structures were rationally designed and synthesized. Four cancer cell lines (HCT116, HT29, MCF7, and A549) and one normal cell (CHO) were applied to investigate the cytotoxicity of synthesized compounds. Furthermore, the inhibitory activity of the synthesized compounds was analyzed against pan-HDAC isozymes (HDAC1, HDAC2, HDAC3 and HDAC6). Five compounds (5a, 5b, 5c, 5e and 5h) showed strong cytotoxicity and HDAC inhibition even superior to entinostat as the reference drug. Collectively, these results suggest that compounds with an electron-donating group at the positions 6 or 8 of the quinolone ring (cap) appear to enhance the anti-proliferative potency. After performing induce-fit docking (on HDAC1, 2, 3, 6) for all the synthesized compounds, a significant correlation was identified between the MTT results, the inhibitory activity of pan-HDAC, and the docking scores of HDAC1. Therefore, compounds 5e and 5a (the most potent compounds) were identified as the most favorable leads for further investigation of HDAC1-specific enzyme inhibition. Compounds 5a and 5e showed impressive HDAC1 inhibitory potency with IC50 values of 0.48 μM and 0.36 μM, respectively, equal or more than that of Entinostat. Compound 5e (3 µM) also exhibited significant induction of apoptosis in the HCT116 cell line (the percentage of apoptotic cells was 81.87%), even stronger than the reference drug Entinostat (the percentage of apoptotic cells was 37.5%) at the same concentration which is consistent with their antiproliferative activities as well. 100 ns molecular dynamic (MD) simulation revealed that compound 5e can form stable interactions with HDAC1 through coordination with zinc ion, strong hydrophobic interactions and formation of hydrogen bonds. These results suggested that compound 5e can be promising lead for further development of potent anticancer and HDAC inhibitor drugs.

The PIF1/PIF3-MED25-HDA19 transcriptional repression complex regulates phytochrome signaling in Arabidopsis.

Light signals are perceived by photoreceptors, triggering the contrasting developmental transition in dark-germinated seedlings. Phytochrome-interacting factors (PIFs) are key regulators of this transition. Despite their prominent functions in transcriptional activation, little is known about PIFs' roles in transcriptional repression. Here, we provide evidence that histone acetylation is involved in regulating phytochrome-PIFs signaling in Arabidopsis. The histone deacetylase HDA19 interacts and forms a complex with PIF1 and PIF3 and the Mediator subunit MED25. The med25/hda19 double mutant mimics and enhances the phenotype of pif1/pif3 in both light and darkness. HDA19 and MED25 are recruited by PIF1/PIF3 to the target loci to reduce histone acetylation and chromatin accessibility, providing a mechanism for PIF1/PIF3-mediated transcriptional repression. Furthermore, MED25 forms liquid-like condensates, which can compartmentalize PIF1/PIF3 and HDA19 invitro and invivo, and the number of MED25 puncta increases in darkness. Collectively, our study establishes a mechanism wherein PIF1/PIF3 interact with HDA19 and MED25 to mediate transcriptional repression in the phytochrome signaling pathway and suggests that condensate formation with Mediator may explain the distinct and specific transcriptional activity of PIF proteins.

Structural basis of paralog-specific KDM2A/B nucleosome recognition.

The nucleosome acidic patch is a major interaction hub for chromatin, providing a platform for enzymes to dock and orient for nucleosome-targeted activities. To define the molecular basis of acidic patch recognition proteome wide, we performed an amino acid resolution acidic patch interactome screen. We discovered that the histone H3 lysine 36 (H3K36) demethylase KDM2A, but not its closely related paralog, KDM2B, requires the acidic patch for nucleosome binding. Despite fundamental roles in transcriptional repression in health and disease, the molecular mechanisms governing nucleosome substrate specificity of KDM2A/B, or any related JumonjiC (JmjC) domain lysine demethylase, remain unclear. We used a covalent conjugate between H3K36 and a demethylase inhibitor to solve cryogenic electron microscopy structures of KDM2A and KDM2B trapped in action on a nucleosome substrate. Our structures show that KDM2-nucleosome binding is paralog specific and facilitated by dynamic nucleosomal DNA unwrapping and histone charge shielding that mobilize the H3K36 sequence for demethylation.

Role of EZH2 in Uterine Gland Development.

Enhancer of zeste homolog 2 (EZH2) is a core component of polycomb repressive complex 2 that plays a vital role in transcriptional repression of gene expression. Conditional ablation of EZH2 using progesterone receptor (Pgr)-Cre in the mouse uterus has uncovered its roles in regulating uterine epithelial cell growth and stratification, suppressing decidual myofibroblast activation, and maintaining normal female fertility. However, it is unclear whether EZH2 plays a role in the development of uterine glands, which are required for pregnancy success. Herein, we created mice with conditional deletion of Ezh2 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase that is expressed in mesenchyme-derived cells of the female reproductive tract. Strikingly, these mice showed marked defects in uterine adenogenesis. Unlike Ezh2 Pgr-Cre conditional knockout mice, deletion of Ezh2 using Amhr2-Cre did not lead to the differentiation of basal-like cells in the uterus. The deficient uterine adenogenesis was accompanied by impaired uterine function and pregnancy loss. Transcriptomic profiling using next generation sequencing revealed dysregulation of genes associated with signaling pathways that play fundamental roles in development and disease. In summary, this study has identified an unrecognized role of EZH2 in uterine gland development, a postnatal event critical for pregnancy success and female fertility.

A reciprocal translocation involving Aspergillus nidulans snxAHrb1/Gbp2 and gyfA uncovers a new regulator of the G2-M transition and reveals a role in transcriptional repression for the setBSet2 histone H3-lysine-36 methyltransferase.

Aspergillus nidulans snxA, an ortholog of Saccharomyces cerevisiae Hrb1/Gbp2 messenger RNA shuttle proteins, is-in contrast to budding yeast-involved in cell cycle regulation, in which snxA1 and snxA2 mutations as well as a snxA deletion specifically suppress the heat sensitivity of mutations in regulators of the CDK1 mitotic induction pathway. snxA mutations are strongly cold sensitive, and at permissive temperature snxA mRNA and protein expression are strongly repressed. Initial attempts to identify the causative snxA mutations revealed no defects in the SNXA protein. Here, we show that snxA1/A2 mutations resulted from an identical chromosome I-II reciprocal translocation with breakpoints in the snxA first intron and the fourth exon of a GYF-domain gene, gyfA. Surprisingly, a gyfA deletion and a reconstructed gyfA translocation allele suppressed the heat sensitivity of CDK1 pathway mutants in a snxA+ background, demonstrating that 2 unrelated genes, snxA and gyfA, act through the CDK1-CyclinB axis to restrain the G2-M transition, and for the first time identifying a role in G2-M regulation for a GYF-domain protein. To better understand snxA1/A2-reduced expression, we generated suppressors of snxA cold sensitivity in 2 genes: (1) loss of the abundant nucleolar protein Nsr1/nucleolin bypassed the requirement for snxA and (2) loss of the Set2 histone H3 lysine36 (H3K36) methyltransferase or a nonmethylatable histone H3K36L mutant rescued hypomorphic snxA mutants by restoring full transcriptional proficiency, indicating that methylation of H3K36 acts normally to repress snxA transcription. These observations are in line with known Set2 functions in preventing excessive and cryptic transcription of active genes.

A Class of Protein-Coding RNAs Binds to Polycomb Repressive Complex 2 and Alters Histone Methylation

Polycomb repressive complex 2 (PRC2) is a multi-subunit protein complex mediating the methylation of lysine 27 on histone H3 and playing an important role in transcriptional repression during tumorigenesis and development. Previous studies revealed that both protein-coding and non-coding RNAs could bind to PRC2 complex. However, the functions of protein-coding RNAs that bind to PRC2 complex in tumor are still unknown. Through data mining and RNA immunoprecipitation (RIP) assay, our study found that there were a class of protein-coding RNAs bound to PRC2 complex and H3 with tri-methylation on lysine 27. The Bayesian gene regulatory network analysis pointed out that these RNAs regulated the expression of PRC2-regulated genes in cancer. In addition, gene set enrichment analysis (GSEA), gene ontology (GO) analysis, and weighted gene co-expression network analysis (WGCNA) also confirmed that these RNAs were associated with histone modification in cancer. We also confirmed that MYO1C, a PRC2-bound transcript, inhibited the modification level of H3K27me3. Further detailed study showed that TMEM117 regulated TSLP expression through EZH2-mediated H3K27me3 modification. Interestingly, the RNA recognition motif of PRC2 complex might help these RNAs bind to the PRC2 complex more easily. The same regulatory pattern was found in mice as well.

Shade avoidance syndrome in 4D: illuminating a role for transcriptional repression of protein biogenesis genes.

Shade avoidance syndrome in 4D: illuminating a role for transcriptional repression of protein biogenesis genes.

Loss of Wiz Function Affects Methylation Pattern in Palate Development and Leads to Cleft Palate.

WIZ (Widely Interspaced Zinc Finger) is associated with the G9a-GLP protein complex, a key H3K9 methyltransferase suggesting a role in transcriptional repression. However, its role in embryonic development is poorly described. In order to assess the loss of function of WIZ, we generated CRISPR/Cas9 WIZ knockout mouse model with 32 nucleotide deletion. Observing the lethality status, we identified the WIZ knockouts to be subviable during embryonic development and non-viable after birth. Morphology of developing embryo was analyzed at E14.5 and E18.5 and our findings were supported by microCT scans. Wiz KO showed improper development in multiple aspects, specifically in the craniofacial area. In particular, shorter snout, cleft palate, and cleft eyelids were present in mutant embryos. Palatal shelves were hypomorphic and though elevated to a horizontal position on top of the tongue, they failed to make contact and fuse. By comparison of proliferation pattern and histone methylation in developing palatal shelves we brought new evidence of importance WIZ dependent G9a-GLP methylation complex in craniofacial development, especially in palate shelf fusion.

Mediator subunit Med15 dictates the conserved \u201cfuzzy\u201d binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a “fuzzy” protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein–protein interaction.

Intestinal FGF15/19 physiologically repress hepatic lipogenesis\xa0in the late fed-state by activating SHP and DNMT3A

Hepatic lipogenesis is normally tightly regulated but is aberrantly elevated in obesity. Fibroblast Growth Factor-15/19 (mouse FGF15, human FGF19) are bile acid-induced late fed-state gut hormones that decrease hepatic lipid levels by unclear mechanisms. We show that FGF15/19 and FGF15/19-activated Small Heterodimer Partner (SHP/NR0B2) have a role in transcriptional repression of lipogenesis. Comparative genomic analyses reveal that most of the SHP cistrome, including lipogenic genes repressed by FGF19, have overlapping CpG islands. FGF19 treatment or SHP overexpression in mice inhibits lipogenesis in a DNA methyltransferase-3a (DNMT3A)-dependent manner. FGF19-mediated activation of SHP via phosphorylation recruits DNMT3A to lipogenic genes, leading to epigenetic repression via DNA methylation. In non-alcoholic fatty liver disease (NAFLD) patients and obese mice, occupancy of SHP and DNMT3A and DNA methylation at lipogenic genes are low, with elevated gene expression. In conclusion, FGF15/19 represses hepatic lipogenesis by activating SHP and DNMT3A physiologically, which is likely dysregulated in NAFLD.

KDM5c Promotes Colon Cancer Cell Proliferation Through the FBXW7-c-Jun Regulatory Axis.

KDM5c is a histone demethylase that specifically demethylates trimethylated and dimethylated H3 Lys-4 to play a central role in transcriptional repression. C-Jun is a proto-oncogene and promotes cell proliferation when ectopically accumulated, but can be ubiquitinated by SCF (FBXW7), leading to its degradation. FBXW7 is an E3 ubiquitin ligase of c-Jun, and exhibits carcinostasis in colon cancer. Here, we report that overexpression of KDM5c in human colon cancer cells results in attenuated FBXW7 transcription and accumulated c-Jun protein, leading to increased proliferation of colon cancer cells. We show that overexpression of KDM5c can result in increased c-Jun protein levels and decreased ubiquitin levels, with no significant change in mRNA levels of c-Jun. KDM5c overexpression blocks the ubiquitin-proteasome proteolytic pathway of c-Jun by down-regulating the expression of FBXW7. KDM5c down-regulation of FBXW7 occurs by demethylation of H3K4me3 at TSS and downstream of the FBXW7 gene. And interaction of KDM5c with H3K4me3 downstream of FBXW7 gene may be followed by recruitment of DNMT3b to methylate the spatially close CpG island located near the FBXW7 TSS. This methylation represses FBXW7 gene expression, which can reduce c-Jun degradation via the ubiquitin-proteasome pathway. TCGA database analysis revealed high expression of KDM5c in colon cancer tissues. KDM5c expression in colon cancer was correlated with poor overall survival of patients in the first 7 years. Data from TCGA showed that high expression of KDM5c was correlated with high DNA methylation of the FBXW7 gene, but was not positively correlated with methylation of the Jun gene. These results suggest that KDM5c regulation of colon cell proliferation is mainly mediated by the KDM5c-FBXW7-c-Jun axis.

BcRPD3-Mediated Histone Deacetylation Is Involved in Growth and Pathogenicity of Botrytis cinerea.

Histone deacetylase activity plays an important role in transcriptional repression. Botrytis cinerea is an important necrotrophic fungal pathogen distributed worldwide and parasites a wide range of hosts. However, the molecular mechanisms of how B. cinerea regulates growth and host infection remain largely unknown. Here, the function of BcRPD3, a histone deacetylase of B. cinerea, was investigated. Overexpression of the BcRPD3 gene resulted in significantly decreased acetylation levels of histone H3 and H4. The BcRPD3 overexpression strains showed slightly delayed vegetative growth, dramatically impaired infection structure formation, oxidative stress response, and virulence. RNA-Seq analysis revealed that enzymatic activity related genes, including 9 genes reported to function as virulence factors, were downregulated in BcRPD3 overexpression strain. Chromatin immunoprecipitation followed by qPCR confirmed the enrichment of BcRPD3 and H3Kac at the promoter regions of these nine genes. These observations indicated that BcRPD3 regulated the transcription of enzymatic activity related genes by controlling the acetylation level of histones, thereby affecting the vegetative growth, infection structure formation, oxidative stress response, and virulence of B. cinerea.

Polycomb Repression without Bristles: Facultative Heterochromatin and Genome Stability in Fungi.

Genome integrity is essential to maintain cellular function and viability. Consequently, genome instability is frequently associated with dysfunction in cells and associated with plant, animal, and human diseases. One consequence of relaxed genome maintenance that may be less appreciated is an increased potential for rapid adaptation to changing environments in all organisms. Here, we discuss evidence for the control and function of facultative heterochromatin, which is delineated by methylation of histone H3 lysine 27 (H3K27me) in many fungi. Aside from its relatively well understood role in transcriptional repression, accumulating evidence suggests that H3K27 methylation has an important role in controlling the balance between maintenance and generation of novelty in fungal genomes. We present a working model for a minimal repressive network mediated by H3K27 methylation in fungi and outline challenges for future research.

Evaluation of EED Inhibitors as a Class of PRC2-Targeted Small Molecules for HIV Latency Reversal.

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.

Epigenetic regulation of the human ATP2A3 gene promoter in gastric and colon cancer cell lines.

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.

Change in Nucleosome Dynamics During Stress Responses in Plants.

The dynamic nature of chromatin is the basis for the regulation of various biological processes in eukaryotic organisms. Nucleosome, the basic unit of chromatin in eukaryotes, undergo various reversible posttranslational modifications (PTM) in response to both external and internal cues. This PTM is recognized by different reader molecules, which facilitates the recruitment of various chromatin remodeling proteins that modulate the chromatin structure. In plants, the promoters of active genes are associated with higher lysine acetylation of histones H3 and H4, and these modifications are recognized by Bromo-domain (BRM) containing chromatin remodelers. This leads to the remodeling of the nucleosome at promoter regions, thereby increasing accessibility of the transcription machinery. It also plays a role in transcriptional repression when enriched in repressed genes. Lysine methylation recruits methyl-binding domain-containing proteins such as LIKE HETEROCHROMATIN PROTEIN1 (LHP1), which facilitates a more condensed chromatin structure that further inhibits access by the transcriptional machinery. In this article, protocols to study the regulation of chromatin conformations and nucleosome dynamics in plants in response to different stress signals are provided.

Results from a Phase Ib Evaluation of Tazemetostat (EPZ-6438) in Combination with R-CHOP in Poor Prognosis Newly Diagnosed Diffuse Large B Cell Lymphoma (DLBCL): a Lysa Study

Results from a Phase Ib Evaluation of Tazemetostat (EPZ-6438) in Combination with R-CHOP in Poor Prognosis Newly Diagnosed Diffuse Large B Cell Lymphoma (DLBCL): a Lysa Study

PRC2 Inactivation Induces Resistance to Chemotherapy-Induced Apoptosis By Upregulating the TRAP1 Mitochondrial Chaperone in T-ALL

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming.We analyzed pre-treatment T-ALL clinical specimens from a cohort of 47 patients (enriched for treatment failure, but with sufficient controls) treated on the COG AALL0434 or DFCI 05001 clinical trials using BH3 profiling analysis to assess mitochondrial apoptotic priming. We found that there was a strong association between resistance to mitochondrial apoptosis and a poor response to induction chemotherapy (P = 0.008). Furthermore, mitochondrial apoptosis resistance predicted significantly inferior event-free survival (65% vs. 91% at 5 years; P = 0.0376).To define the molecular determinants of this mitochondrial apoptosis resistance, we performed targeted exon sequencing and array CGH copy number analysis. This revealed that loss-of-function mutations in the polycomb repressive complex 2 (PRC2) core subunits (EZH2, EED or SUZ12) were associated with mitochondrial apoptosis resistance (P = 0.007) in clinical specimens. PRC2 is a chromatin modifying complex best known for its role in transcriptional repression, which functions as a tumor suppressor in T-ALL, but whether PRC2 regulates mitochondrial apoptosis is unknown.Using shRNA knockdown in human T-ALL cells, we found that depletion of PRC2 subunits in T-ALL cells induced mitochondrial apoptosis resistance, as assessed by BH3 profiling analysis (P < 0.001). PRC2 inactivation also induced resistance to chemotherapy-induced apoptosis (P < 0.0001), and increased T-ALL fitness following treatment with the antileukemic drug vincristine (P = 0.0001). Apoptosis resistance upon inactivation of EZH2 (a PRC2 catalytic subunit) was reversed by transduction of wild-type EZH2, but not by an EZH2 point mutant with impaired methyltransferase activity, indicating that this effect is mediated by the enzymatic activity of PRC2. In normal mouse thymocytes, heterozygous deletion of the PRC2 subunits Ezh2 or Eed was sufficient to induce apoptosis resistance in non-transformed double-negative T-cell progenitors (P < 0.010), indicating that apoptosis resistance can arise prior to oncogenic transformation.The best-known regulators of mitochondrial apoptosis are BCL2-family genes, but RNA-seq analysis of shRNA knockdown of the PRC2 subunits in a T-ALL cell line revealed that PRC2 did not regulate expression of any of the known BCL2 family members. Instead, PRC2 loss led to upregulation of TRAP1, a mitochondrially localized chaperone of the HSP90 family. TRAP1 upregulation was necessary for induction of apoptosis resistance following PRC2 inactivation, because shRNA knockdown of TRAP1 in the human CCRF-CEM cell line completely blocked induction of apoptosis resistance following PRC2 inactivation (P < 0.0001). Moreover, pharmacologic TRAP1 inhibition synergized with the antileukemic drugs dexamethasone and doxorubicin (combination index = 0.37 and 0.42, respectively).To define how PRC2 regulates TRAP1, we performed ChIP-seq analysis, which revealed that TRAP1 regulation by PRC2 is indirect. Combined ChIP-seq and RNA-seq analysis revealed a number of direct targets of PRC2, all of which were tested for their ability to upregulate TRAP1 and induce apoptosis resistance. This showed that the LIM domain transcription factor CRIP2 is a direct target of PRC2 that is necessary and sufficient for regulation of TRAP1, and for induction of apoptosis resistance downstream of PRC2 inactivation. To confirm the relevance of our findings, we used the EZH2 inhibitor GSK126 to inhibit enzymatic activity of PRC2, which revealed that EZH2 normally represses CRIP2 and TRAP1 expression in primary patient-derived xenografts. Finally, we found that increased TRAP1 expression correlates with treatment failure in T-ALL clinical specimens (P = 0.028).Taken together, our findings support a model in which loss of PRC2 induces transcriptional upregulation of its direct target CRIP2, which subsequently activates expression of TRAP1, leading to resistance to chemotherapy-induced mitochondrial apoptosis. DisclosuresAries:Pfizer: Employment. Teachey:Amgen: Consultancy; La Roche: Consultancy. Letai:AstraZeneca: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership.

NKAP functions as an oncogene and its expression is induced by CoCl2 treatment in breast cancer via AKT/mTOR signaling pathway.

PurposeNKAP plays an important role in transcriptional repression, T-cell development, maturation and function acquisition, maintenance and survival of hematopoietic stem cells, and RNA splicing. In this study, we tried to explore the physiological role of NKAP in breast cancer.MethodsWe investigated NKAP expression in breast cancer patients and normal controls and its correlation with survival in breast cancer patients by searching on GEPIA. We knocked down the expression of NKAP in MCF-7 cells by RNAi technique and studied its effect on cell proliferation, migration, invasion, and apoptosis. And we revealed the effect of NKAP on MCF-7 cells under hypoxic conditions in vitro.ResultsNKAP was differentially expressed in breast cancer and normal tissues and is a potential prognostic indicator of breast cancer. Subsequently, NKAP knockdown significantly inhibited the proliferation and clonality of MCF-7 cells and induced its apoptosis through caspase 3-dependent pathway. In addition, knockdown of NKAP could strongly inhibit the migration and invasion of MCF-7 cells. In MCF-7 cells, NKAP affected the AKT/mTOR signaling pathway and markedly reduced the phosphorylation of AKT and mTOR, as well as the downstream protein. What’s interesting is CoCl2 was found to induce NKAP expression in MCF-7 cells. Downregulation of NKAP hindered the impact of CoCl2 on the MCF-7 cells, including cell proliferation and invasion, by adjusting AKT/mTOR signaling.ConclusionNKAP functioned as an oncogene, and its expression was induced by hypoxia in breast cancer via AKT/mTOR signaling pathway.

Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.