GC-rich Motifs Research Articles

CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2,3-dioxygenase of Spodoptera frugiperda.

In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.

RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells.

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.

Abstract LT03: C/EBPα Acts as an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

Abstract The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα’s separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα’s DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα’s RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations. It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together though, we demonstrate that C/EBPα is also an RNA binding protein with unique functions distinct from its DNA binding activity that is essential for monocyte maturation, differentiation and mobilization. Citation Format: Mahmoud A. Bassal, Yanzhou Zhang, Yanjing V. Liu, Virginia Camacho, Simone Ummarino, Robert S. Welner, Daniel G Tenen. C/EBPα Acts as an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT03.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

C/EBPα Acts As an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

GDF15 deficiency hinders human trophoblast invasion to mediate pregnancy loss through downregulating Smad1/5 phosphorylation

Growth differentiation factor 15 (GDF15) belongs to the Transforming growth factor β(TGF-β) superfamily. The decrease of GDF15 in the serum of pregnant women was associated with miscarriage. Both IHC and ELISA assays showed that GDF15 in trophoblast tissue and serum of pregnant women who miscarried was significantly lower than in those who had a live birth. GDF15 deficiency was associated with embryo resorption in GDF15 knockout mice through CRIPSR editing. In addition, the migration and invasion ability of HTR-8/SVneo and JEG-3 cells were promoted by GDF15. Mechanistically, GDF15 increased Smad1/5 phosphorylation, resulting in upregulating SNAI1/2, VIMENTIN and downregulating E-CADHERIN. A dual-luciferase reporter assay confirmed that Smad-binding elements (SBE) and/or GC-rich motifs were activated and target genes such as SNAI1/2, SERPINE1, and TIMP3 were transcriptionally regulated by GDF15/Smad5 signaling. Therefore, our data revealed a crucial role of GDF15 on invasion of trophoblast by upregulating the activity of TGF-β/Smad1/5 pathway.

Expression of targets of the RNA-binding protein AUF-1 in human airway epithelium indicates its role in cellular senescence and inflammation.

The RNA-binding protein AU-rich-element factor-1 (AUF-1) participates to posttranscriptional regulation of genes involved in inflammation and cellular senescence, two pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). Decreased AUF-1 expression was described in bronchiolar epithelium of COPD patients versus controls and in vitro cytokine- and cigarette smoke-challenged human airway epithelial cells, prompting the identification of epithelial AUF-1-targeted transcripts and function, and investigation on the mechanism of its loss. RNA immunoprecipitation-sequencing (RIP-Seq) identified, in the human airway epithelial cell line BEAS-2B, 494 AUF-1-bound mRNAs enriched in their 3'-untranslated regions for a Guanine-Cytosine (GC)-rich binding motif. AUF-1 association with selected transcripts and with a synthetic GC-rich motif were validated by biotin pulldown. AUF-1-targets' steady-state levels were equally affected by partial or near-total AUF-1 loss induced by cytomix (TNFα/IL1β/IFNγ/10 nM each) and siRNA, respectively, with differential transcript decay rates. Cytomix-mediated decrease in AUF-1 levels in BEAS-2B and primary human small-airways epithelium (HSAEC) was replicated by treatment with the senescence- inducer compound etoposide and associated with readouts of cell-cycle arrest, increase in lysosomal damage and senescence-associated secretory phenotype (SASP) factors, and with AUF-1 transfer in extracellular vesicles, detected by transmission electron microscopy and immunoblotting. Extensive in-silico and genome ontology analysis found, consistent with AUF-1 functions, enriched RIP-Seq-derived AUF-1-targets in COPD-related pathways involved in inflammation, senescence, gene regulation and also in the public SASP proteome atlas; AUF-1 target signature was also significantly represented in multiple transcriptomic COPD databases generated from primary HSAEC, from lung tissue and from single-cell RNA-sequencing, displaying a predominant downregulation of expression. Loss of intracellular AUF-1 may alter posttranscriptional regulation of targets particularly relevant for protection of genomic integrity and gene regulation, thus concurring to airway epithelial inflammatory responses related to oxidative stress and accelerated aging. Exosomal-associated AUF-1 may in turn preserve bound RNA targets and sustain their function, participating to spreading of inflammation and senescence to neighbouring cells.

Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Sequence-Specific Structural Features and Solvation Properties of Transcription Factor Binding DNA Motifs: Insights from Molecular Dynamics Simulation.

Sequence-specific recognition of transcription factor (TF) binding motifs in the target site of DNA over the vast amount of non-target DNA is of primary importance for the transcriptional regulation of gene expression by the TFs. Binding of TFs to the target site of DNA relies not only on the direct contact formation but also on the structural and conformational features of DNA. Recognition of DNA structural features or shape readout by proteins is an important factor in the context of TF-DNA interaction. Based on the atomistic molecular simulation, here we report the sequence-dependent unique structural features, solvation, and ion-binding properties of biologically relevant AT- and GC-rich human TF binding motifs in DNA. Counterion and water distribution around the motif is found to be sensitive to the motif sequence, which is accompanied with the DNA shape features. The motif sequence affects the electrostatic potential along the grooves, and cytosine methylation alters the DNA shape features. Characteristic solvation properties of TF binding motif DNA fragments infer that an ionic environment and hydration influences are essential to describe TF-DNA interactions.

Appropriate Amounts and Activity of the Wilms' Tumor Suppressor Gene, wt1, Are Required for Normal Pronephros Development of Xenopus Embryos.

The Wilms' tumor suppressor gene, wt1, encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes. wt1 was first identified as a tumor suppressor gene in Wilms' tumor, a pediatric kidney tumor, and has been implicated in normal kidney development. The WT1 protein has transcriptional activation and repression domains and acts as a transcriptional activator or repressor, depending on the target gene and context. In Xenopus, an ortholog of wt1 has been isolated and shown to be expressed in the developing embryonic pronephros. To investigate the role of wt1 in pronephros development in Xenopus embryos, we mutated wt1 by CRISPR/Cas9 and found that the expression of pronephros marker genes was reduced. In reporter assays in which known WT1 binding sequences were placed upstream of the luciferase gene, WT1 activated transcription of the luciferase gene. The injection of wild-type or artificially altered transcriptional activity of wt1 mRNA disrupted the expression of pronephros marker genes in the embryos. These results suggest that the appropriate amounts and activity of WT1 protein are required for normal pronephros development in Xenopus embryos.

Transcription Factor Sp1 in the Expression of Genes Encoding Components of MAPK, JAK/STAT, and PI3K/Akt Signaling Pathways

Sp1 is a transcription factor of the Sp/KLF family that binds to GC-rich motifs in regulatory regions of genes. Sp1 is involved in the regulation of cell proliferation, apoptosis and differentiation, and angiogenesis. A high level of SP1 expression, as well as its aberrant transcriptional activity due to post-translational modifications, is found in cells in oncological diseases, such as lung, breast, pancreatic, thyroid, gastric cancer, and glioma; congenital heart disease, as well as neurodegenerative disorders, including Huntington's and Parkinson's diseases. Binding of Sp1 to GC-rich motifs of the regulatory regions of the genes encoding components of the MAPK, p38, JAK/STAT, PI3K/Akt signaling pathways, is involved in the control of cell proliferation, differentiation, and death. In addition, kinases of these signaling pathways are able to change the transcriptional activity of Sp1 by phosphorylation of certain amino acid residues, which leads to a change in the efficiency of its binding to cofactors and DNA regulatory regions. This review presents data on the relationship between the Sp1 transcription factor and the activity of the MAPK, p38, JAK/STAT, and PI3K/Akt signaling pathways in normal tissues and in various pathologies, including malignant diseases.

KLF15 cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms, Influenced by an Insertion in the 'Regulatory Loop'.

The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the ‘regulatory loop (involved in mRNA binding)’. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: ‘open (isoform_2; without insertion)’ and ‘closed (isoform_1; with insertion)’. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the ‘open conformation’. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA–UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein–mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

Maize decrease in DNA methylation 1 targets RNA-directed DNA methylation on active chromatin.

DNA methylation plays vital roles in repressing transposable element activity and regulating gene expression. The chromatin-remodeling factor Decrease in DNA methylation 1 (DDM1) is crucial for maintaining DNA methylation across diverse plant species, and is required for RNA-directed DNA methylation (RdDM) to maintain mCHH islands in maize (Zea mays). However, the mechanisms by which DDM1 is involved in RdDM are not well understood. In this work, we used chromatin immunoprecipitation coupled with high-throughput sequencing to ascertain the genome-wide occupancy of ZmDDM1 in the maize genome. The results revealed that ZmDDM1 recognized an 8-bp-long GC-rich degenerate DNA sequence motif, which is enriched in transcription start sites and other euchromatic regions. Meanwhile, 24-nucleotide siRNAs and CHH methylation were delineated at the edge of ZmDDM1-occupied sites. ZmDDM1 co-purified with Argonaute 4 (ZmAGO4) proteins, providing further evidence that ZmDDM1 is a component of RdDM complexes in planta. Consistent with this, the vast majority of ZmDDM1-targeted regions co-localized with ZmAGO4-bound genomic sites. Overall, our results suggest a model that ZmDDM1 may be recruited to euchromatic regions via recognition of a GC-rich motif, thereby remodeling chromatin to provide access for RdDM activities in maize.

C11orf95-RELA fusion drives aberrant gene expression through the unique epigenetic regulation for ependymoma formation

Recurrent C11orf95-RELA fusions (RELAFUS) are the hallmark of supratentorial ependymomas. The presence of RELA as the fusion partner indicates a close association of aberrant NF-κB activity with tumorigenesis. However, the oncogenic role of the C11orf95 has not been determined. Here, we performed ChIP-seq analyses to explore genomic regions bound by RELAFUS and H3K27ac proteins in human 293 T and mouse ependymoma cells. We then utilized published RNA-Seq data from human and mouse RELAFUS tumors and identified target genes that were directly regulated by RELAFUS in these tumors. Subsequent transcription factor motif analyses of RELAFUS target genes detected a unique GC-rich motif recognized by the C11orf95 moiety, that is present in approximately half of RELAFUS target genes. Luciferase assays confirmed that a promoter carrying this motif is sufficient to drive RELAFUS-dependent gene expression. Further, the RELAFUS target genes were found to be overlapped with Rela target genes primarily via non-canonical NF-κB binding sites. Using a series of truncation and substitution mutants of RELAFUS, we also show that the activation domain in the RELAFUS moiety is necessary for the regulation of gene expression of these RELAFUS target genes. Lastly, we performed an anti-cancer drug screening with mouse ependymoma cells and identified potential anti-ependymoma drugs that are related to the oncogenic mechanism of RELAFUS. These findings suggested that RELAFUS might induce ependymoma formation through oncogenic pathways orchestrated by both C11orf95 and RELA target genes. Thus, our study unveils a complex gene function of RELAFUS as an oncogenic transcription factor in RELAFUS positive ependymomas.

Landscape of meiotic crossovers in Hericium erinaceus

Meiotic crossover shows marked interspecific and intraspecific variation, and knowledge about the molecular mechanism of crossover variation remains limited. Herein, we described the genome-wide scanning of crossover in one mushroom-forming fungus Hericium erinaceus. Utilizing the whole-genome single-nucleotide polymorphism (SNP) data-sets of a 127 F1 haploid progeny, we localized a total of 1316 crossover events and found that they were more likely to occur in the genic than intergenic regions. More than 30 % of the crossovers were concentrated in 59 crossover hotspots that were preferentially located close to chromosome ends. We then examined the genomic features around crossover hotspots. Results showed that the crossover hotspots were associated with increased gene density and guanine-cytosine (GC) content. An 8-bp GC-rich motif (GCGTCAGC) was found to be significantly enriched in these hotspots. The presence of mating-type loci affected the crossover at local scale rather than the overall crossover number. In order to dissect the genetic mechanisms shaping crossover variation, we then conducted quantitative trait locus (QTL) mapping for the total crossovers (TCO) and the crossover events that solely occurred within hotspots (HCO). Genome-wide QTL scanning identified four TCO-QTLs and two HCO-QTLs, which all located within or next to the crossover-hotspots. Crossover variations were shaped by multiple small-effect loci, with individual QTL contributing 6.9 %–11.7 % of variation. A few recombination pathway genes, including Spo11, Msh5, and Smc5 were found to be co-localized with the mapped crossover QTLs. Taken together, findings of this study offer insights into the crossover distribution and genetic factors conferring crossover variation in H. erinaceus, and advance our understandings for meiotic recombination in mushroom-forming fungi.

Abstract 1304: FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance

Abstract Background: FGFR1 amplification occurs in ~ 15% of estrogen receptor-positive (ER+) breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its engagement in transcription regulation remains unclear. Thus, we investigated the mechanisms underpinning the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1-ChIP-Seq was performed in CAMA1 ER+/FGFR1-amplified human breast cancer cells to identify genomic distribution of FGFR1. IP with FLAG antibody followed by Mass Spectrometry (MS) was carried out on nuclear plus chromatin fractions of CAMA1 cells overexpressing 3XFLAG-FGFR1 to uncover the nuclear FGFR1 interactome. Results: FGFR1-ChIP-Seq detected 4408 peaks in CAMA1 cells cultured in estrogen-free conditions, with marked enrichment of GC-rich consensus motifs. 67% of peaks were enriched at promoter regions. ChIP-PCR confirmed FGFR1 binding to several genomic loci in ER+/FGFR1-amplified cell lines (CAMA1 and MDA-MB-134) and PDX (HCI-011). Further, MS uncovered RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P-ChIP-Seq revealed that 65% (2867/4408) of FGFR1 peaks were shared with Pol II S5P in CAMA1 cells. Also, ChIP-Seq revealed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p<0.0001), suggesting that FGFR1 binds to actively transcribed genes. The pioneer factor FOXA1 was detected by MS among FGFR1 interacting proteins and ChIP-Seq showed its enrichment at FGFR1-bound promoters. Notably, siRNA-mediated FOXA1 knockdown reduced FGFR1 distribution to several genomic loci in CAMA1 cells, as measured by FGFR1-ChIP-Seq. Finally, we found that 10% (360/3558) of FGFR1 peaks overlapped with genes that were differentially expressed in FGFR1-amplified (n=7) vs FGFR1 non-amplified (n=30) ER+ breast tumors treated with neoadjuvant letrozole. Using Gene Set Variation Analysis, we developed a signature score for the top 32/360 genes (LogFC>1), whose expression is likely regulated by nuclear FGFR1. A high signature score correlated with resistance to letrozole (p<0.0001) and high post-treatment Ki67 index (p=0.0004) in our cohort, and with worse disease-free and overall survival in the METABRIC cohort of ER+/HER2− patients treated with endocrine therapy. Conclusions: These findings support a prominent role for FGFR1 in the transcriptional machinery of breast cancer cells. Whether this transcriptional action is causal to antiestrogen resistance in ER+/FGFR1-amplified breast cancer is currently under investigation. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Albert Lin, Dhivya R. Sudhan, Ariella B. Hanker, Sumanta Chatterjee, Hiroaki Akamatsu, Arnaldo Marin, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1304.

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences.

BackgroundThe concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings.MethodsThe tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer.ResultsAlthough the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. DiscussionThe G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.

Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5 ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3 In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.

NanoSatellite: accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

Technological limitations have hindered the large-scale genetic investigation of tandem repeats in disease. We show that long-read sequencing with a single Oxford Nanopore Technologies PromethION flow cell per individual achieves 30× human genome coverage and enables accurate assessment of tandem repeats including the 10,000-bp Alzheimer’s disease-associated ABCA7 VNTR. The Guppy “flip-flop” base caller and tandem-genotypes tandem repeat caller are efficient for large-scale tandem repeat assessment, but base calling and alignment challenges persist. We present NanoSatellite, which analyzes tandem repeats directly on electric current data and improves calling of GC-rich tandem repeats, expanded alleles, and motif interruptions.