Intronic Binding Site Research Articles

Abstract 2063: Endothelial Cell Scavenger Receptor Class B, Type I Expression Is Transcriptionally Controlled By HIF-1α

Background: Scavenger Receptor Class B, Type I (SR-BI), encoded by Scarb1, mediates LDL cholesterol transcytosis in endothelial cells (EC) to deliver circulating LDL into the subendothelial space to drive atherogenesis. Research Questions: It is unknown whether EC SR-BI expression is altered in atherosclerosis, and how EC SR-BI expression is regulated. Approach: Single cell RNAseq was performed on coronary arteries from 5 ischemic heart disease patients, in segments both with and without a grossly apparent atheroma. Transcription factor (TF) network analysis was done to reveal possible regulatory relationships between differentially-expressed genes (DEGs). The modulation of EC SR-BI was studied in primary human aortic EC (HAEC) and in vivo in mouse aortic EC. ChIP-seq, ChIP-qPCR and CRISPR-based mutagenesis were used to interrogate TF binding to the human Scarb1 gene. EC SR-BI expression was determined by immunoblotting and qPCR, and EC LDL transcytosis was studied in transwells using DiI-labeled LDL. Results: SR-BI expression was greater in EC in coronary segments with atheroma versus those without atheroma (p=0.003). TF networks for DEGs in atheroma versus non-atheroma EC, and for DEGs in EC with low versus high SR-BI revealed multiple TF with possible regulatory relationships with Scarb1. One TF in common was HIF-1α. In HAEC, an increase in HIF-1α with the HIF prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) caused upregulation of SR-BI mRNA and protein, and a parallel increase in LDL transcytosis that was entirely SR-BI-dependent. Expression of a constitutively-active HIF-1α variant also increased SR-BI and LDL transcytosis. ChIP-seq revealed a HIF-1α binding site in Scarb1 intron 1, and HIF-1α binding there increased 4-fold with DMOG. CRISPR deletion of the binding site fully prevented SR-BI upregulation by DMOG without affecting known HIF-1α target genes. In mice, 3d treatment with DMOG increased SR-BI in aortic EC. Conclusions: EC expression of SR-BI is upregulated with increasing atherosclerosis severity in human coronary artery. HIF-1α is upregulated in parallel, and HIF-1α causes the direct transcriptional upregulation of EC SR-BI leading to enhanced EC LDL transport. HIF-1α control of SR-BI in EC may be critical to atherogenesis.

The Alpha-Synuclein Gene (SNCA) is a Genomic Target of Methyl-CpG Binding Protein 2 (MeCP2)—Implications for Parkinson’s Disease and Rett Syndrome

Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson’s disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2.

BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites

BackgroundPervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters.ResultsHere we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements.ConclusionsThus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype.

Mixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. We used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti-A specific mixed-field agglutination. We discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes.

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

Deciphering the role of alternative splicing as modulators of defense response in the MYMIV- Vigna mungo pathosystem.

Alternative splicing (AS) is a crucial regulatory mechanism that impacts transcriptome and proteome complexity under stressful situations. Although its role in abiotic stresses is somewhat understood, our understanding of the mechanistic regulation of pre-mRNA splicing in plant-pathogen interaction is meagre. To comprehend this unexplored immune reprogramming mechanism, transcriptome profiles of Mungbean Yellow Mosaic India Virus (MYMIV)-resistant and susceptible Vigna mungo genotypes were analysed for AS genes that may underlie the resistance mechanism. Results revealed a repertoire of AS-isoforms accumulated during pathogenic infestation, with intron retention being the most common AS mechanism. Identification of 688 differential alternatively spliced (DAS) genes in the resistant host elucidates its robust antiviral response, whereas 322 DAS genes were identified in the susceptible host. Enrichment analyses confirmed DAS transcripts pertaining to stress, signalling, and immune system pathways have undergone maximal perturbations. Additionally, a strong regulation of the splicing factors has been observed both at transcriptional and post-transcriptional levels. qPCR validation of candidate DAS transcripts with induced expression upon MYMIV-infection demonstrated a competent immune response in the resistant background. The AS-impacted genes resulted either in partial/complete loss of functional domains or altered sensitivity to miRNA-mediated gene silencing. A complex regulatory module, miR7517-ATAF2, has been identified in an aberrantly spliced ATAF2 isoform that exposes an intronic miR7517 binding site, thereby suppressing the negative regulator to enhance defense reaction. The present study establishes AS as a non-canonical immune reprogramming mechanism that operates in parallel, thereby offering an alternative strategy for developing yellow mosaic-resistant V. mungo cultivars. This article is protected by copyright. All rights reserved.

Cooperative Binding of SRSF3 to Structured 3'ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA.

ZO-1α+ and ZO-1α- proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3'ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3-hairpin RNA interaction is crucial in the early recognition of 3'ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity.

Development of an In Vitro Chloroplast Splicing System: Sequences Required for Correct pre-mRNA Splicing.

Chloroplast genomes in land plants include approximately 20 intron-containing genes. Most of the introns are similar to the group II introns found in fungi, algae and some bacteria, but no self-splicing has been reported. To analyze splicing reactions in chloroplasts, we developed a tobacco (Nicotiana tabacum) chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor messenger RNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is not necessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 and 2) complementary to two intron-binding sites (IBS1 and 2) in the 3′ end of 5′ exons, which are involved in 5′ splice-site selection. Using our in vitro system and atpF pre-mRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1–IBS1 pairing is essential, while EBS2–IBS2 pairing is important but not crucial for splicing. The first 3′ exon nucleotide determines the 3′ splice sites of self-splicing introns. However, mutations to this nucleotide in atpF pre-mRNA did not affect splicing. This result suggests that the mechanism underlying chloroplast pre-mRNA splicing differs partly from that mediating the self-splicing of group II introns.

Regulation of S100A10 Gene Expression.

S100A10, a member of the S100 family of Ca2+-binding proteins, is a widely distributed protein involved in many cellular and extracellular processes. The best recognized role of S100A10 is the regulation, via interaction with annexin A2, of plasminogen conversion to plasmin. Plasmin, together with other proteases, induces degradation of the extracellular matrix (ECM), which is an important step in tumor progression. Additionally, S100A10 interacts with 5-hydroxytryptamine 1B (5-HT1B) receptor, which influences neurotransmitter binding and, through that, depressive symptoms. Taking this into account, it is evident that S100A10 expression in the cell should be under strict control. In this work, we summarize available literature data concerning the physiological stimuli and transcription factors that influence S100A10 expression. We also present our original results showing for the first time regulation of S100A10 expression by grainyhead-like 2 transcription factor (GRHL2). By applying in silico analysis, we have found two highly conserved GRHL2 binding sites in the 1st intron of the gene encoding S100A10 protein. Using chromatin immunoprecipitation (ChIP) and luciferase assays, we have shown that GRHL2 directly binds to these sites and that this DNA region can affect transcription of S100A10.

Diaphanous-Related Formin Protein mDia2 Regulates Hematopoietic Stem and Progenitor Cell Functions through Srf-Integrins Pathway

Distinct mature cell types in mammalian blood system are derived from the Hematopoietic Stem/Progenitor cells (HSPCs), which are multipotent progenitor cells with self-renewing and engraftment ability. Various intrinsic and extrinsic molecules modulate HSPCs' fates to either undergo self-renewal or differentiation. The Diaphanous-related formin protein mDia2, is an effector of Rho GTPases. Previously, we reported that hematopoietic-specific mDia2 knock out mice developed anemia due to ineffective erythropoiesis caused by bi-nucleated erythroblasts. However, how mDia2 is involved in HSPC functions are unknown. Here we show that mDia2 expression is enriched in HSPCs. Although mDia2 was largely dispensable for HSPCs' function at steady-state, hematopoietic-specific inactivation of mDia2 severely impaired the competitive repopulation capacity of HSPCs. After primary bone marrow transplantation, the absolute number of bone marrow HSPCs from mDia2 knockout (KO) transplants was significantly decreased. mDia2 deficient LSK cells also showed skewed differentiation with increased percentage of more differentiated multi-potent progenitor cells (MPP), which was further supported by loss of stem cell quiescence. Under serial transplantation, mice transplanted with mDia2 knockout (KO) bone marrow showed rapid lethality. Importantly, these defects can be rescued by the constitutively active mDia2 mutant mDia2-ΔDAD. Furthermore, mDia2 KO HSPCs showed a marked reduction in the motility ability from bone marrow to the peripheral blood upon G-CSF stimulation. Loss of mDia2 also severely compromised the recovery of the mice from 5-FU-induced myelosuppression.mDia2 functions to nucleate linear actin filaments. We found that loss of mDia2 expression in HSPCs reduced the F-actin levels. This further attenuated the adhesion of HSPCs that was confirmed by adhesion assay and decreased protrusion formation in vitro . We demonstrated that the defect in F-actin formation caused by mDia2 deficiency repressed the transcriptional activity of serum response factor (SRF), which caused down-regulation of SRF target genes such as Acta2, Actb and SRF itself. Ectopic expression of SRF rescued HSPC phenotypes with loss of mDia2. We further identified that the expression levels of several integrins, including Itga2, Itgam (encodes CD11b) , Itagal (encodes CD11a)and Itgb2 (encodes CD18) , were significantly attenuated in mDia2 KO c-Kit+ HSPCs. Chromatin immunoprecipitation (ChIP) sequencing data further revealed intronic SRF binding sites in the genomic loci of Itgam and Itgb2, therefore suggesting Itgam and Itgb2 as new downstream targets of SRF pathway. Our data revealed a novel mDia2-SRF-integrin axis that regulates HSPCs adhesion and engraftment. DisclosuresNo relevant conflicts of interest to declare.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Presenilin 1 mutation likely contributes to U1 small nuclear RNA dysregulation and Alzheimer's disease–like symptoms

Previous studies showed that U1 small nuclear RNA (snRNA) was selectively enriched in the brain of individuals with familial Alzheimer's disease (AD), resulting in widespread changes in RNA splicing. Our study further reported that presenilin-1 (PSEN1) induced an increase in U1 snRNA expression, accompanied by changed amyloid precursor protein expression, β-amyloid level, and cell death in SH-SY5Y cells. However, the effect of U1 snRNA overexpression on learning and memory is still unclear. In the present study, we found that neuronal U1 snRNA overexpression could generate U1 snRNA aggregates in the nuclear, accompanied by the widespread alteration of RNA splicing, resulting in the impairments of synaptic plasticity and spatial memory. In addition, more U1 snRNAs is bound to the intron binding sites accompanied by an increased intracellular U1 snRNA level. This suggests that U1 snRNA overexpression regulates RNA splicing and gene expression in neurons by manipulating the recruitment of the U1 snRNA to the nascent transcripts. Using in situ hybridization staining of human central nervous system–type neurons, we identified nuclear aggregates of U1 snRNA in neurons by upregulating the U1 snRNA level. Quantitative polymerase chain reaction analysis showed U1 snRNA accumulation in the insoluble fraction of neurons with PSEN1 mutation neurons rather than other types of U snRNAs. These results show an independent function of U1 snRNA in regulating RNA splicing, suggesting that aberrant RNA processing may mediate neurodegeneration induced by PSEN1 mutation.

Dm Ime4 depletion affects permeability barrier and Chic function in Drosophila spermatogenesis

Adenosine methylation of messenger RNA at the N6 position (m6A) is a non-editing modification that can affect several aspects of mRNA metabolism. Dm Ime4, also known as METTL3, MTA, and MTA-70 in other organisms, is the catalytic subunit of the methyltransferase complex that adds this modification. Dm ime4 is evolutionarily conserved and essential for development in metazoans and plants. Because of its pleiotropic effects, it has been difficult to establish the main reason why embryonic arrest occurs in plants, mice, and zebrafish. Using a strategy that depletes Dm Ime4 specifically in the somatic cyst cells of Drosophila testes without affecting essential functions in development, our lab has found that Dm Ime4 may potentially regulate splicing of profilin (chic) mRNA, the message for an essential and evolutionarily conserved protein mainly known for its function in actin polymerization. One of the lesser known roles for Chic is its requirement for establishment and maintenance of the somatic cyst-cell permeability barrier in Drosophila spermatogenesis. Chic and Dm Ime4 colocalize and are abundant in somatic cyst cells throughout spermatogenesis. Upon selective depletion of Dm Ime4, we observe significant reduction of Chic protein levels and malfunction of the permeability barrier. We have found that chic mRNA contains intronic Dm Ime4 binding sites that can form the hairpin structures required for recognition by the methyltransferase complex. Our data show that the reduced levels of Chic protein observed in Dm ime4 somatic cyst-cell knockdowns could be the result of aberrant splicing of its mRNA. In turn, low levels of Chic are known to affect the function of the somatic permeability barrier, leading to germline death and the reduced fertility observed in Dm ime4 knockdown males. We propose that Dm Ime4 may regulate chic in other developmental contexts and in other organisms, including mice and humans. Chic is an essential protein that is evolutionarily conserved, and establishment and maintenance of cell barriers and domains are important strategies used in metazoan development. Taken together, our findings define a framework to investigate specific functions of Dm Ime4 and its homologs in multicellular organisms by bypassing its pleiotropic requirement in early developmental stages.

Hepatitis B protein HBx binds the DLEU2 lncRNA to sustain cccDNA and host cancer-related gene transcription

ObjectiveThe HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin.DesignWe explored,...

BTG4 is A Novel p53 Target Gene That Inhibits Cell Growth and Induces Apoptosis.

BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.

Abstract A123: Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g. dexamethasone, prednisolone) form a critical component of chemotherapy regimens for pediatric ALL and initial resistance to glucocorticoid therapy is predictive of poor outcome. Acquired resistance to glucocorticoids is common at relapse and is disparate compared with other chemotherapeutic drugs used in the treatment of pediatric ALL. Glucocorticoids induce apoptosis in lymphoid cells which is mediated through the glucocorticoid receptor (GR). Resistance to glucocorticoids in pediatric ALL is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM. The purpose of this study was to identify small molecules that are able to reverse glucocorticoid resistance in pediatric ALL patient-derived xenografts (PDXs). Methods: A 40,000 compound high-throughput screening (HTS) campaign was carried out to identify compounds that potentiated the cytotoxicity of dexamethasone against a pediatric ALL PDX derived from a patient with aggressive and fatal relapse. Cytotoxicity assays were carried out by alamarBlue assay. Gene expression analysis was carried out using Illumina microarrays. mRNA and protein analysis was carried out by RT-PCR and immunoblotting, respectively. Results: The HTS campaign identified a novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3). The sensitizing effect was specific to glucocorticoids, with synergy observed when GCS-3 was combined with dexamethasone or prednisolone, but not with other chemotherapeutic drugs. Synergy was observed in a range of dexamethasone-resistant and -sensitive xenografts representative of B-ALL, T-ALL, early T-cell precursor ALL and Philadelphia chromosome positive ALL. GCS-3 required a functional GR to sensitise ALL xenografts to dexamethasone. Gene expression analysis showed that GCS-3 in the presence of dexamethasone upregulated a previously published gene expression signature induced by glucocorticoid treatment of B-ALL patients (Rhein et al. Leukemia, 2007, 21:897-905). Moreover, GCS-3 in combination with dexamethasone significantly downregulated C-MYC and upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. Treatment with the GR antagonist, RU486, significantly abrogated the cytotoxic effects of the GCS-3/dexamethasone combination. Although RU486 increased C-MYC expression in the dexamethasone treated cells, there was no increase in C-MYC expression in the GCS-3/dexamethasone combination. RU486 suppressed the marked induction of BIM following treatment with the GCS-3/dexamethasone combination, indicating that BIM upregulation is critical for the cytotoxicity of the GCS-3/dexamethasone combination. The GCS-3/dexamethasone combination significantly increased binding of the GR to an intronic binding site at the BIM locus which is associated with BIM transcription and glucocorticoid sensitivity in pediatric ALL. Conclusion: This study describes the potential of the novel glucocorticoid sensitizing agent, GCS-3, as a biological tool to understand glucocorticoid action and resistance. Citation Format: Richard B Lock, Cara E Toscan, Duohui Jing, Chelsea Mayoh. Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A123. doi:10.1158/1535-7163.TARG-19-A123

Biological and RNA regulatory function of MOV10 in mammalian germ cells

BackgroundRNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells.ResultsMOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3′-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites.ConclusionsThese data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.

Regulation of GLI1 by cis DNA elements and epigenetic marks

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

INKA2, a novel p53 target that interacts with the serine/threonine kinase PAK4.

The p53 protein is a tumour suppressor and transcription factor that regulates the expression of target genes involved in numerous stress responses systems. In this study, we designed a screening strategy using DNA damage-induced mouse and human transcriptome data to identify novel downstream targets of p53. Our method selected genes with an induced expression in multiple organs of X-ray-irradiated p53 wild-type mice. The expression of inka box actin regulator 2 gene, known as Inka2, was upregulated in 12 organs when p53 expression was induced. Similarly, INKA2 was induced in a p53-dependent manner at both the mRNA and protein level in human cells treated with adriamycin. Reporter assays confirmed that p53 directly regulated INKA2 through an intronic binding site. The overexpression of INKA2 produced a slight decrease in cancer cell growth in the colony formation assay. Moreover, the analysis of The Cancer Genome Atlas (TCGA) data revealed a decreased INKA2 expression in tumour samples carrying p53 mutations compared with p53 wild-type samples. In addition, significantly higher levels of DNA methylation were observed in the INKA2 promoter in tumour samples, concordant with the reduced INKA2 expression in tumour tissues. These results demonstrate the potential of INKA2 as a cancer cell growth inhibitor. Furthermore, INKA2 protein interacts with the serine/threonine-protein kinase, p21 (RAC1) activated kinase (PAK)4, which phosphorylates β-catenin to prevent ubiquitin-proteasomal degradation. As β-catenin was downregulated in a stable INKA2-expressing cell line, the findings of this study suggest that INKA2 is a novel, direct downstream target of p53 that potentially decreases cell growth by inhibiting the PAK4-β-catenin pathway.