Nuclear Corepressor Research Articles

7260 Nuclear Corepressor 1 (NCoR1) plays important role in the regulation of insulin gene transcription in mouse Min6 pancreatic beta cells

Abstract Disclosure: H. Shimizu: None. Y. Horibata: None. I. Amano: None. M.J. Ritter: None. M. Ohyama: None. M. Domae: None. H. Sugimoto: None. A.N. Hollenberg: None. Background: Nuclear Corepressor 1 (NCoR1) is a transcriptional corepressor which has been recognized as an important player in the regulation of hepatic lipogenesis and hematopoiesis in mouse embryo. NCoR1 is also widely expressed in mouse insulin sensitive organs, for instance adipocyte, skeletal muscle, and hepatocytes. Mouse genetic deletion strategies of either NCoR1 or SMRT(NCoR2; an ortholog corepressor of NCoR1) demonstrated that each corepressor effects the insulin sensitivity in the tissues. However, the role of each corepressor in pancreatic beta cell is not still elucidated. Methods: To clarify this, we took the gene-targeting strategy for NCoR1 using CRISPR Cas-9 and made the pancreatic beta Min6 clone which lacks NCoR1 protein (Min6-NCoR1KO; NKO cells). For this study, wild type Min6 cells (WT) and NKO cells were cultured in the Dulbecco's DMEM medium with 10% fetal bovine serum for 4-5 days, and the expression analyses compared between two cell types were performed. Results: Both comprehensive proteomics and following expression analyses compared between WT and NKO cells demonstrated that NKO cells have significantly lower expression level of insulin (INS-1 and INS-2), accompanied with high expression of histone deacetylase 6 (HDAC6). For further analyses, we also made the Min6 clone which lacks SMRT(Min6-SMRTKO; SKO cells). Interestingly, SKO cell had higher insulin and lower HDAC6 levels, accompanied with significant up-regulation of NCoR1. Conclusion: Taken together, these data indicate that NCoR1 has dominant effects in the regulation of both histone deacetylation and insulin gene transcription in Min6 cells. Further investigations for the function of NCoR1 in the insulin gene transcription observed in Min6 cells will be needed. Presentation: 6/2/2024

Nuclear receptor corepressor 1 levels differentially impact the intracellular dynamics of mutant thyroid hormone receptors associated with resistance to thyroid hormone syndrome

Thyroid hormone receptor α1 (TRα1) undergoes nucleocytoplasmic shuttling and mediates gene expression in response to thyroid hormone (T3). In Resistance to Thyroid Hormone Syndrome α (RTHα), certain TRα1 mutants have higher affinity for nuclear corepressor 1 (NCoR1) and may form stable complexes that are not released in the presence of T3. Here, we examined whether NCoR1 modulates intranuclear mobility and nuclear retention of TRα1 or RTHα-associated mutants in transfected human cells, as a way of analyzing critical structural components of TRα1 and to further explore the correlation between mutations in TRα1 and aberrant intracellular trafficking. We found no significant difference in intranuclear mobility, as measured by fluorescence recovery after photobleaching, between TRα1 and select RTHα mutants, irrespective of NCoR1 expression. Nuclear-to-cytoplasmic fluorescence ratios of RTHα mutants, however, varied from TRα1 when NCoR1 was overexpressed, with a significant increase in nuclear retention for A263V and a significant decrease for A263S and R384H. In NCoR1-knockout cells, nuclear retention of A263S, A263V, P389R, A382P, C392X, and F397fs406X was significantly decreased compared to control (wild-type) cells. Luciferase reporter gene transcription mediated by TRα1 was significantly repressed by both NCoR1 overexpression and NCoR1 knockout. Most RTHα mutants showed minimal induction regardless of NCoR1 levels, but T3-mediated transcriptional activity was decreased for R384C and F397fs406X when NCoR1 was overexpressed, and also decreased for N359Y in NCoR1-knockout cells. Our results suggest a complex interaction between NCoR1 and RTHα mutants characterized by aberrant intracellular localization patterns and transcriptional activity that potentially arise from variable repressor complex stability, and may provide insight into RTHα pathogenesis on a molecular and cellular level.

Abstract Tu149: FDA-Approved Antidepressant Trazodone Potentially Increases the Risk of Dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

Cannabidiol promotes intestinal cholesterol uptake mediated by Pregnane X receptor.

Cannabidiol (CBD), a non-psychoactive phytocannabinoid of cannabis, is therapeutically used as an analgesic, anti-convulsant, anti-inflammatory, and anti-psychotic drug. There is a growing concern about the adverse side effects posed by CBD usage. Pregnane X receptor (PXR) is a nuclear receptor activated by a variety of dietary steroids, pharmaceutical agents, and environmental chemicals. In addition to the role in xenobiotic metabolism, the atherogenic and dyslipidemic effects of PXR have been revealed in animal models. CBD has a low affinity for cannabinoid receptors, thus it is important to elucidate the molecular mechanisms by which CBD activates cellular signaling and to assess the possible adverse impacts of CBD on pro-atherosclerotic events in cardiovascular system, such as dyslipidemia. Our study aims to explore the cellular and molecular mechanisms by which exposure to CBD activates human PXR and increases the risk of dyslipidemia. Both human hepatic and intestinal cells were used to test if CBD was a PXR agonist via cell-based transfection assay. The key residues within PXR's ligand-binding pocket that CBD interacted with were investigated using computational docking study together with site-directed mutagenesis assay. The C57BL/6 wildtype mice were orally fed CBD in the presence of PXR antagonist resveratrol (RES) to determine how CBD exposure could change the plasma lipid profiles in a PXR-dependent manner. Human intestinal cells were treated with CBD and/or RES to estimate the functions of CBD in cholesterol uptake. CBD was a selective agonist of PXR with higher activities on human PXR than rodents PXRs and promoted the dissociation of human PXR from nuclear co-repressors. The key amino acid residues Met246, Ser247, Phe251, Phe288, Trp299, and Tyr306 within PXR's ligand binding pocket were identified to be necessary for the agonistic effects of CBD. Exposure to CBD increased the circulating total cholesterol levels in mice which was partially caused by the induced expression levels of the key intestinal PXR-regulated lipogenic genes. Mechanistically, CBD induced the gene expression of key intestinal cholesterol transporters, which led to the increased cholesterol uptake by intestinal cells. CBD was identified as a selective PXR agonist. Exposure to CBD activated PXR signaling and increased the atherogenic cholesterol levels in plasma, which partially resulted from the ascended cholesterol uptake by intestinal cells. Our study provides potential evidence for the future risk assessment of CBD on cardiovascular disease, such as dyslipidemia.

Investigating molecular interactions between Kaiso and nuclear co-repressor using molecular simulations

Investigating molecular interactions between Kaiso and nuclear co-repressor using molecular simulations

A9 LOSS OF INTESTINAL EPITHELIAL NCOR1 INCREASES OXIDATIVE METABOLISM

Abstract Background The nuclear co-repressor NCOR1 acts as a central platform of a complex that represses gene expression associated with inflammatory response. Literature also recently uncovered its possible role in oxidative metabolism in some tissues and organs. Although intestinal epithelial NCOR1 is crucial to restrict IBD symptoms during experimental colitis, its precise role in controlling intestinal epithelial cell biology has not been established. Aims We aimed to investigate the role of intestinal epithelial NCOR1 in oxidative metabolism. Methods Crossing Villin-Cre and Ncor1loxP/loxP mice was performed to delete Ncor1 in the whole intestinal epithelium conditionally. Crypts from control and NCOR1ΔIEC littermates were isolated to generate colonoids 3D cultures. Phenotypic, genotypic, and transcriptomic analyses were performed on those colonoids. Results Colonoids from NCOR1ΔIEC mice were phenotypically smaller but were found to display drastic higher intestinal stem cell propagation features during organoid clonogenic assays. A Gene Set Enrichment Analysis from RNA sequencing predicted an increase in oxidative metabolism in NCOR1ΔIEC colonoids. Several genes associated with oxidative metabolism were confirmed to be modulated in both NCOR1ΔIEC colonoids and the colon of NCOR1ΔIEC mice. Mitochondrial mass was also significantly increased in colonoids when NCOR1 was absent. Loss of the co-repressor did not affect the expression of PGC1-α, a known antagonist of NCOR1. However, transcripts of cytochrome c somatic (Cycs), a target gene of PGC1-α, were found higher in NCOR1ΔIEC colonoids, suggesting that the coactivator activity is increased in the absence of NCOR1. Conclusions Our results highlight a new role for NCOR1 in the oxidative metabolism of intestinal epithelial cells. Its action via PGC1-α activity could potentially be targeted as a therapeutic measure during inflammatory bowel diseases. Funding Agencies CIHR

Znf687 recruits Brd4-Smrt complex to regulate gfi1aa during neutrophil development.

Neutrophils are key component of the innate immune system in vertebrates. Diverse transcription factors and cofactors act in a well-coordinated manner to ensure proper neutrophil development. Dysregulation of the transcriptional program triggering neutrophil differentiation is associated with various human hematologic disorders such as neutropenia, neutrophilia, and leukemia. In the current study we show the zinc finger protein Znf687 is a lineage-preferential transcription factor, whose deficiency leads to an impaired neutrophil development in zebrafish. Mechanistically, Znf687 functions as a negative regulator of gfi1aa, a pivotal modulator in terminal granulopoiesis, to regulate neutrophil maturation. Moreover, we found BRD4, an important epigenetic regulator, directly interacts with ZNF687 in neutrophils. Deficiency of brd4 results in similar defective neutrophil development as observed in znf687 mutant zebrafish. Biochemical and genetic analyses further reveal that instead of serving as a canonical transcriptional coactivator, Brd4 directly interacts and bridges Znf687 and Smrt nuclear corepressor on gfi1aa gene's promoter to exert transcription repression. In addition, the ZNF687-BRD4-SMRT-GFI1 transcriptional regulatory network is evolutionary conserved in higher vertebrate. Overall, our work indicates Znf687 and Brd4 are two novel master regulators in promoting terminal granulopoiesis.

Overexpression of BQ323636.1 contributes to anastrozole resistance in AR+ve/ER+ve breast cancer.

Aromatase inhibitors (Ais) are used as adjuvant endocrine therapy for oestrogen receptor-positive (ER+ve) post-menopausal breast cancer patients. Ais, by inhibiting the enzyme aromatase, block the conversion of androgen to oestrogen, reducing oestrogen levels. Resistance to Ais limits their clinical utilisation. Here, we show that overexpression of BQ323636.1 (BQ), a novel splice variant of nuclear co-repressor NCOR2, is associated with resistance to the non-steroidal aromatase inhibitor anastrozole in ER+ve post-menopausal breast cancer. Mechanistic study indicates that BQ overexpression enhances androgen receptor (AR) activity and in the presence of anastrozole, causes hyper-activation of AR signalling, which unexpectedly enhanced cell proliferation, through increased expression of CDK2, CDK4, and CCNE1. BQ overexpression reverses the effect of anastrozole in ER+ve breast cancer in an AR-dependent manner, whilst co-treatment with the AR antagonist bicalutamide recovered its therapeutic effect both in vitro and in vivo. Thus, for BQ-overexpressing breast cancer, targeting AR can combat anastrozole resistance. Clinical study of 268 primary breast cancer samples of ER+ve patients who had been treated with non-steroidal Ais showed 32.5% (38/117) of cases with combined high nuclear expression of BQ and AR, which were found to be significantly associated with Ai resistance. Non-steroidal Ai-treated patients with high nuclear expression of both BQ and AR had poorer overall, disease-specific, and disease-free survival. These findings suggest the importance of assessing BQ and AR expression status in the primary ER+ve breast tumour prior to Ai treatment. This may save patients from inappropriate treatment and enable effective therapy to be given at an early stage. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nuclear receptor co-repressor NCOR2 and its relation to GPER with prognostic impact in ovarian cancer.

The significance of the non-classical G-protein-coupled estrogen receptor (GPER) as positive or negative prognostic factor for ovarian cancer patients remains still controversial. Recent results indicate that an imbalance of both co-factors and co-repressors of nuclear receptors regulates ovarian carcinogenesis by altering the transcriptional activity through chromatin remodeling. The present study aims to investigate whether the expression of the nuclear co-repressor NCOR2 plays a role in GPER signaling which thereby could positively impact overall survival rates of ovarian cancer patients. NCOR2 expression was evaluated by immunohistochemistry in a cohort of 156 epithelial ovarian cancer (EOC) tumor samples and correlated with GPER expression. The correlation and differences in clinical and histopathological variables as well as their effect on prognosis were analyzed by Spearman's correlation, Kruskal-Wallis test and Kaplan-Meier estimates. Histologic subtypes were associated with different NCOR2 expression patterns. More specifically, serous and mucinous EOC demonstrated a higher NCOR2 expression (P = 0.008). In addition, high nuclear NCOR2 expression correlated significantly with high GPER expression (cc = 0.245, P = 0.008). A combined evaluation of both high NCOR2 (IRS > 6) and high GPER (IRS > 8) expression revealed an association of a significantly improved overall survival (median OS 50.9 versus 105.1months, P = 0.048). Our results support the hypothesis that nuclear co-repressors such as NCOR2 may influence the transcription of target genes in EOC such as GPER. Understanding the role of nuclear co-repressors on signaling pathways will allow a better understanding of the factors involved in prognosis and clinical outcome of EOC patients.

Spindle Cell Lipoma and Pleomorphic Lipoma: An Update and Review.

Spindle cell lipoma (SCL) is a benign adipocytic tumor that primarily occurs in the subcutis of the posterior neck, upper back, and shoulder, particularly of middle-aged males. SCL and pleomorphic lipoma (PL) represent a morphological spectrum of one disease process. The lesion typically presents as a relatively small (<5 cm), mobile, slow-growing, painless mass. Magnetic resonance imaging reveals the lesion to be a well-defined subcutaneous mass with a mixture of adipose and non-adipose components. Intense enhancement of the non-adipose component is seen after contrast administration. Histologically, SCL is composed of variable distributions of mature adipocytes, bland spindle cells and ropey collagen bundles and PL also contains pleomorphic and multinucleated floret-like giant cells. By immunohistochemistry, the spindle and pleomorphic/floret-like giant cells of SCL/PL are diffusely positive for CD34 and show loss of nuclear RB transcriptional corepressor 1 (RB1) expression. Recent cytogenetic and molecular genetic studies have shown heterozygous deletions of 13q14, including the RB1 gene. SCL/PL can be successfully treated with simple excision, with a very low recurrence rate. Knowledge of these peculiar tumors is important because it can mimic a variety of benign and malignant soft-tissue tumors. This review provides an updated overview of the clinical, radiological, histopathological, cytogenetic, and molecular genetic features of SCL/PL.

Roles of the nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) in the developing brain

The nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoic acid and thyroid hormone (SMRT) are critical coregulators of the nuclear receptor (e.g., thyroid hormone receptors and retinoic acid receptors), mediating transcriptional repression via histone deacetylation. Although they are highly homologous and have similar nuclear receptor interaction domains, they have different roles in different organs. Recently, de novo genetic variants in nuclear corepressors were found in pediatric patients with neurodevelopmental disorders. Thus, we generated the mouse models to understand the role of NCOR1 and SMRT in the central nervous system. We used the mice with conditional NCoR1 or SMRT (NCoR1 lox/lox or SMRT lox/lox ) alleles in combination with the mice that express Cre recombinase in a neuronal specific fashion (Snap25-Ires2-Cre). First, we performed a battery of behavioral tests to screen behavioral phenotype of mice. We found that hypoactivity, social deficits, and mild anxiety in neuronal specific NCoR1 or SMRT KO mice. In addition, NCoR1 KO mice showed high learning abilities in pairwise visual discrimination task. Next, we performed RNA-sequencing analysis with amygdala from postnatal day 21 to investigate gene expression mediated by NCoR1 and SMRT. We found that 449 genes were upregulated by SMRT deletion, whereas only 8 genes were upregulated by NCoR1 deletion. Overall, our data demonstrate for the first time that NCoR1 and SMRT have separate functions in the central nervous system. JSPS Grant-in-Aid for Early-Career Scientists 19K16486, 21K15340 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A screen for MeCP2-TBL1 interaction inhibitors using a luminescence-based assay

Understanding the molecular pathology of neurodevelopmental disorders should aid the development of therapies for these conditions. In MeCP2 duplication syndrome (MDS)—a severe autism spectrum disorder—neuronal dysfunction is caused by increased levels of MeCP2. MeCP2 is a nuclear protein that binds to methylated DNA and recruits the nuclear co-repressor (NCoR) complex to chromatin via an interaction with the WD repeat-containing proteins TBL1 and TBLR1. The peptide motif in MeCP2 that binds to TBL1/TBLR1 is essential for the toxicity of excess MeCP2 in animal models of MDS, suggesting that small molecules capable of disrupting this interaction might be useful therapeutically. To facilitate the search for such compounds, we devised a simple and scalable NanoLuc luciferase complementation assay for measuring the interaction of MeCP2 with TBL1/TBLR1. The assay allowed excellent separation between positive and negative controls, and had low signal variance (Z-factor = 0.85). We interrogated compound libraries using this assay in combination with a counter-screen based on luciferase complementation by the two subunits of protein kinase A (PKA). Using this dual screening approach, we identified candidate inhibitors of the interaction between MeCP2 and TBL1/TBLR1. This work demonstrates the feasibility of future screens of large compound collections, which we anticipate will enable the development of small molecule therapeutics to ameliorate MDS.

THE NCoR CO-REPRESSOR INTERACTS WITH THE KAISO TRANSCRIPTION FACTOR THROUGH A MECHANISM DIFFERENT FROM THAT OF BCL6 INTERACTION

The vertebrate transcription factor Kaiso binds specifically to methylated DNA sequences using C2H2-type zinc fingers. In addition to C2H2-domains, the BTB/POZ domain, which forms homodimers, is located at the N-terminus of Kaiso. Kaiso, like several other well-studied BTB/POZ proteins, including BCL6, interacts with the NCoR (nuclear co-repressor) protein, which determines the landing of transcriptional repressive complexes on chromatin. Using the yeast two-hybrid system, we have shown that the N-terminal domain of NCoR interacts with the C-terminal zinc fingers of Kaiso, and not with its BTB/POZ domain, as previously assumed. The results obtained demonstrate that NCoR interacts with various transcription factor domains, which can increase the efficiency of attracting NCoR-dependent repressor complexes to regulatory regions of the genome.

The NCoR Co-repressor Interacts with the Kaiso Transcription Factor through a Mechanism Different from That of BCL6 Interaction.

The vertebrate transcription factor Kaiso binds specifically to methylated DNA sequences using C2H2-type zinc fingers. In addition to C2H2-domains, the BTB/POZ domain, which forms homodimers, is located at the N-terminus of Kaiso. Kaiso, like several other well-studied BTB/POZ proteins, including BCL6, interacts with the NCoR (nuclear co-repressor) protein, which determines the landing of transcriptional repressive complexes on chromatin. Using the yeast two-hybrid system, we have shown that the N-terminal domain of NCoR interacts with the C-terminal zinc fingers of Kaiso, and not with its BTB/POZ domain, as previously assumed. The results obtained demonstrate that NCoR interacts with various transcription factor domains, which can increase the efficiency of attracting NCoR-dependent repressor complexes to regulatory regions of the genome.

Nuclear corepressor SMRT acts as a strong regulator of both β-oxidation and suppressor of fibrosis in the differentiation process of mouse skeletal muscle cells.

Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. We found significant up-regulation of muscle specific β-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-β (TGF-β) receptor signaling. Consistent with this, treatment with a specific TGF-β receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both β-oxidation and the prevention for the fibrosis via TGF-β receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.

Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a “basal-like” state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed “basal-like” genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

CTCF: A novel fusion partner of ETO2 in a multiple relapsed acute myeloid leukemia patient

ETO2 is a nuclear co-repressor, which plays a critical role in the regulation of the cell cycle, self-renewal capacity, and differentiation of hematopoietic progenitor cells. We identified novel fusion transcripts involving ETO2 and CTCF by RNA-seq in a multiple relapsed AML case. The CTCF-ETO2 and ETO2-CTCF chimeric genes were validated by RT-PCR and Sanger sequencing. In addition, both transcripts apparently promoted cell proliferation via JAK/STAT3 pathway that is sensitive to STAT3 inhibitors. The novel fusions may have prognostic value and pathogenic mechanisms in acute myeloid leukemia.

Fusion Transcripts without Corresponding Cytogenetic Abnormalities in Acute Myeloid Leukemia: Implications for AML Pathogenesis

Chromosomal translocations and inversions that create fusion genes are important causal events in AML. 7 recurrent fusion genes define AML entities in the 2016 revision of the WHO classification of hematologic malignancies. We have developed a comprehensive diagnostic AML platform based on next-generation mRNA sequencing (RNAseq). The adaptable integrated bioinformatic pipeline HAMLET (Human AML Expedited Transcriptomics) analyses the raw data simultaneously for fusion transcripts and for sequence variants and expression levels of genes with relevance to AML pathogenesis, prognosis, or actionability.Poly(A)+ RNA from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%) from 96 patients (3 diagnosis-relapse pairs, 1 de novo AML and subsequent tAML) was sequenced at a depth of 59x106 paired-end reads/sample with median read length of 126 bp in an ISO17025-accredited Illumina HiSeq 2500 pipeline. Fusion transcripts were called by STAR-Fusion and FusionCatcher. HAMLET detected the recurrent fusion transcripts RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, ERG-FUS, DEK-NUP214, and KMT2A-MLLT3 plus the variants KMT2A-MLLT4, KMT2A-MLLT1, and KMT2A-MLLT6 with 100% sensitivity and 100% specificity compared to metaphase cytogenetics, FISH, and RT-PCR (Griffioen, ASH 2016).In addition, HAMLET identified fusion transcripts from 54 combinations of genes in 57 AML samples that lacked corresponding chromosomal aberrations by metaphase cytogenetics. 14 “unusual” fusions involved genes located on different chromosomes; 40 fusions represented intrachromosomal events. 8 fusions were detected recurrently, i.e. in at least 2 cases (Fig. A). 32 AML carried at least 2 unusual fusions, with a maximum of 9 different fusions in an AML with a complex karyotype (Fig. B). Unusual fusions were not correlated with classical recurrent AML fusion genes. The 5 most prevalent fusions and an ETV6-LYN fusion were successfully validated in a total of 18 AML by RT-PCR. The only fusion that could not be validated was a YWHAZ-KLF10 fusion (2 cases).The most prevalent RT-PCR-validated fusion (seen in 30 AML cases) has been described in ALL (Roberts, 2012; Safavi, 2015) and was an intrachromosomal TPM4-KLF2 fusion between the tropomyosin 4 and an alternative reading frame (ARF) of the transcription factor Kruppel-like factor 2 genes. Unexpectedly, we detected this fusion in all 3 tested normal bone marrows (BM) but not in EBV-transformed B cells. The hitherto unvalidated C15orf57-CBX3 fusion may be generated by retrotransposition (Schrider, 2013) and was present in 18 AML as well as in 2/3 normal BM and 2/3 EBV B-cells with CBX3 in ARF. The intrachromosomal NCOR2-UBC fusion (9 AML) is listed in the database ChimerDB without further characterization and involves the genes of Nuclear corepressor 2 and Ubiquitin C in the normal reading frame (NRF). The NUP98-NSD1 fusion (3 AML) with NSD1 in NRF is frequently found in hematological malignancies including AML and originates from a cryptic t(5;11) due to telomeric positions of both genes. The PIM3-SCO2 fusion between the serine/threonine kinase PIM3 and an ARF of Cytochrome C oxidase assembly protein can originate from a 0.6 Mb inv(22) in chronic neutrophilic leukemia (Menezes, 2013) and occurs frequently in pediatric AML (Kuhn, 2013). The well-characterized ETV6-LYN fusion (1 AML) fusing the TEL1 oncogene to the SRC family kinase LYN in NRF originates from a complex translocation with inversion involving chromosomes 8 and 12 (Telford, 2016) and has oncogenic activity by activation of STAT5 (Takeda, 2011).Fusion transcripts without recognizable chromosomal abnormalities on metaphase cytogenetics occur frequently in AML and involve actionable targets. The upstream sequence can be fused to the normal or alternative reading frame of the downstream coding sequences and involve potential or validated proto-oncogenes. Apart from cryptic translocations or inversions, trans-splicing is suggested as an alternative mechanism by the presence of some fusion genes in non-malignant hematopoietic cells. Trans-splicing without DNA rearrangement can occur physiologically and alter cellular development towards potential premalignant differentiation (Yuan, 2013). Increasing use of RNAseq technology in routine AML diagnostics, e.g. by HAMLET, will permit to unravel the prognostic significance of unexpected fusion transcripts and to guide novel targeted therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nuclear Receptor Corepressors NCoR1 and SMRT Plays Unique Roles in Central Nervous System

The nuclear corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) are critical coregulators of the thyroid hormone receptor (TR), mediating transcriptional repression via histone deacetylation. Thyroid hormone (TH) plays an essential role in many physiological processes via the TR. How the corepressors regulate TR signaling is not fully understood, especially in central nervous system (CNS). To determine the role of NCoR1 and SMRT in the CNS, we used mice with conditional NCoR1 (NCoR1lox/lox) and SMRT (SMRTlox/lox) alleles in combination with mice that express Cre recombinase in a neuronal specific fashion (Snap25-Cre). Global deletion of NCoR1 or SMRT during embryogenesis results in lethality. We also showed that NCoR1/SMRT double knock-out mice die within two weeks after induction of Cre activity in adult mice. Now, we found that neuronal specific NCoR1 or SMRT KO mice survive without obvious impairment of neuronal development. However, NCoR1/SMRT double knock-out mice die within postnatal 1-2 weeks and have impaired body growth. Thus, both NCoR1 and SMRT have important roles in maintaining normal neuronal function. Recently, cased of mutations in NCoR1 and SMRT in humans have been reported. These cases report phenotypes including Autism Spectrum Disorder (ASD) and intellectual disability. The cerebellum has been thought to contribute to motor control and learning. Surprisingly, it has also been shown to be a key brain structure involved in social cognition and its dysfunction may play a role in ASD. The Purkinje cell is the main neuron in the cerebellum. Thus, we generated cerebellar Purkinje cell specific NCoR1/SMRT knock-out mice using L7/Pcp2-Cre mice. In contrast to neuronal specific KO mice, both NCoR1 or SMRT single or double knock-out mice survive until adulthood. SMRT Purkinje cell knock-out mice showed abnormalities in 3ch social interaction test indicating impaired social functioning, similar to some ASD symptoms. Electrophysiological testing showed current injection evoked more action potentials in SMRT KO mice. These results suggest Purkinje cell dysfunction caused by SMRT deletion may result in social disability. Our data demonstrate for the first time that NCoR1 and SMRT have separate functions in different areas of the brain but also have some redundant function when knocked out together in all neurons.