Transcription Factor Binding Sites Research Articles

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA.

Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.

Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort.

Crohn's disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn's disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn's disease imply hidden diversity of pathological mechanisms. We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a signature involving TGFß signaling in two individuals with Crohn's disease. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications for a personalized approach to understanding disease pathology. This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

Molecular phenotyping of small cell lung cancer using targeted cfDNA profiling of transcriptional regulatory regions.

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.

Synthesis, Anti-Inflammatory Activities, and Molecular Docking Study of Novel Pyxinol Derivatives as Inhibitors of NF-κB Activation

Pyxinol, an active metabolite of ginsenosides in human hepatocytes, exhibits various pharmacological activities. Here, a series of C-3 modified pyxinol derivatives was designed and virtually screened by molecular docking with the key inflammation-related proteins of the nuclear factor kappa B (NF-κB) pathway. Some of the novel derivatives were synthesized to assess their effects in inhibiting the production of nitric oxide (NO) and mitochondrial reactive oxygen species (MtROS) in lipopolysaccharide-triggered RAW264.7 cells. Derivative 2c exhibited the highest NO and MtROS inhibitory activities with low cytotoxicity. Furthermore, 2c decreased the protein levels of interleukin 1β, tumor necrosis factor α, inducible nitric oxide synthase, and cyclooxygenase 2 and suppressed the activation of NF-κB signaling. Cellular thermal shift assays indicated that 2c could directly bind with p65 and p50 in situ. Molecular docking revealed that 2c’s binding to the p65–p50 heterodimer and p50 homodimer was close to their DNA binding sites. In summary, pyxinol derivatives possess potential for development as NF-κB inhibitors.

Physalin H, physalin B, and isophysalin B suppress the quorum-sensing function of Staphylococcus aureus by binding to AgrA

The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B—–phytochemicals belonging to physalins found in plants of the Solanaceae family—–as novel Agr-QS modulators. Biological analyses and in vitro protein–DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

A PadR family transcriptional repressor regulates the transcription of chromate efflux transporter in Enterobacter sp. Z1.

Chromium is a prevalent toxic heavy metal, and chromate [Cr(VI)] exhibits high mutagenicity and carcinogenicity. The presence of the Cr(VI) efflux protein ChrA has been identified in strains exhibiting resistance to Cr(VI). Nevertheless, certain strains of bacteria that are resistant to Cr(VI) lack the presence of ChrB, a known regulatory factor. Here, a PadR family transcriptional repressor, ChrN, has been identified as a regulator in the response of Enterobacter sp. Z1(CCTCC NO: M 2019147) to Cr(VI). The chrN gene is cotranscribed with the chrA gene, and the transcriptional expression of this operon is induced by Cr(VI). The binding capacity of the ChrN protein to Cr(VI) was demonstrated by both the tryptophan fluorescence assay and Ni-NTA purification assay. The interaction between ChrN and the chrAN operon promoter was validated by reporter gene assay and electrophoretic mobility shift assay. Mutation of the conserved histidine residues His14 and His50 resulted in loss of ChrN binding with the promoter of the chrAN operon. This observation implies that these residues are crucial for establishing a DNA-binding site. These findings demonstrate that ChrN functions as a transcriptional repressor, modulating the cellular response of strain Z1 to Cr(VI) exposure.

Abstract LB257: Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC)

Abstract Immune checkpoint inhibitor (ICI) therapy has shown unprecedented results in RCC treatment, but durable responses have been hampered by acquired resistance (AR, initially respond but eventually develop cancer progression). Mechanisms for AR remain ambiguous. Previously, we reported elevated blood GPNMB to strongly associate with AR development. To study molecular mechanisms for AR-linked GPNMB elevation, we created an ICI-resistant RenCa (R-RenCa) mouse model by repeated selection of largest tumors in mice treated with PD1 Ab ICI. Parental cells (Gpnmblow) respond to ICI (S-RenCa), while R-RenCa is completely unresponsive to ICI and acquired Gpnmb overexpression (but PDL1 remained unchanged), recapitulating strong association of GPNMB with AR development. R-RenCa tumors displayed more immunosuppressive and angiogenic phenotypes and enhanced response to anti-Gpnmb Ab. In probing for genetic alterations unique to R-RenCa, whole exome seq analysis showed R-RenCa to bear a greater tumor mutation burden (184.9 and 174.6 mut/Mb for R- and S-RenCa, respectively) but no somatic mutations in beta-2 microglobulin and IFN-signal molecule genes known to be involved in AR. RNA-seq analysis showed; more transcriptionally active genes in R-RenCa tumors (heatmap analysis); more responsive genes to ICI (Volcano analysis); and dysregulation in microphthalmia (Mitf) family gene expression. Mitf RNA was upregulated 3-fold, but Tfe3 and other members downregulated 2-3-fold. RNA start sites of Mitf transcripts are highly heterogenous in R-RenCa compared to S-RenCa (Sashimi plot), indicating generation of more isoforms. Since Gpnmb promoter has E-boxes (DNA binding sites) for Mitf factor, we examined whether Mitf is responsible for Gpnmb expression using promoter-Luc reporter assays and found 4-fold higher in R-RenCa, which was lost completely in E-box-mutated promoter. In seeking transcriptional activators for Mitf gene, we found Sox10 factor to be upregulated in R-RenCa. The molecular link of Sox10-Mitf-Gpnmb was shown by the data of gene-knock down experiments that Sox10-siRNA diminished Mitf and Gpnmb expression completely, and Mitf-siRNA had no effect on Sox10 expression but diminished Gpnmb expression. We then studied mechanisms for Sox10 activation and found PDL1 signaling (triggered by Ab-crosslinking) to induce Sox10 RNA expression in tumor cells, which was 80% abrogated by deleting the RMLDVEKC cytoplasmic motif known to protect tumor cells from IFN cytotoxicity. For human relevance of Sox10-Mitf axis activation, we employed nested qRT-PCR of cell-free RNA (cfRNA) isolated from plasma of RCC patients (n=3) immediately before and after AR. SOX10 and MITF cfRNAs were increased by 2.3 ± 0.85 and 1.77 ± 0.05-fold, respectively, between these two time points. Thus, PDL1 signaling is involved in AR by dysregulating the SOX10-MITF-GPNMB cascade, indicating the potential of the cascade-targeting to break AR hindrance for ICI therapy. Citation Format: Jin-Sung Chung, Lei Guo, Vinita Popat, Ponciano D. Cruz, Lin Xu, Hans Hammers, Kiyoshi Ariizumi. Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB257.

Probing molecular pathways: Illuminating the connection between COVID-19 and Alzheimer's disease through the endocannabinoid system dynamics.

Our study investigates the molecular link between COVID-19 and Alzheimer's disease (AD). Weaim to elucidate the mechanisms by which COVID-19 may influence the onset or progression of AD. Using bioinformatic tools, we analyzed gene expression datasets from the Gene Expression Omnibus (GEO) database, including GSE147507, GSE12685, and GSE26927. Intersection analysis was utilized to identify common differentially expressed genes (CDEGs)and their shared biological pathways. Consensus clustering was conducted to group AD patients based on gene expression, followed by an analysis of the immune microenvironment and variations in shared pathway activities between clusters. Additionally, we identified transcription factor-binding sites shared by CDEGs and genes in the common pathway. The activity of the pathway and the expression levels of the CDEGs were validated using GSE164805 and GSE48350 datasets. Six CDEGs (MAL2, NECAB1, SH3GL2, EPB41L3, MEF2C, and NRGN) were identified, along with a downregulated pathway, the endocannabinoid(ECS) signaling pathway, common to both AD and COVID-19. These CDEGs showed a significant correlation with ECS activity (p < 0.05) and immune functions. The ECS pathway was enriched in healthy individuals'brains and downregulated in AD patients. Validation using GSE164805 and GSE48350 datasets confirmed the differential expression of these genes in COVID-19 and AD tissues. Our findings reveal a potential pathogenetic link between COVID-19 and AD, mediated by CDEGs and the ECS pathway. However, further research and multicenter evidence are needed to translate these findings into clinical applications.

The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development.

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. Indeed, TRB4 binds to several thousand sites in the genome, mainly at TSS and promoter regions of transcriptionally active and H3K4me3-marked genes, but unlike TRB1 it is not enriched at H3K27me3-marked gene bodies. Yet, TRB4 can physically interact with the catalytic components of PRC2, SWINGER and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and accordingly function as transcriptional activators of several hundred of CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades.Collectively, this study uncovers that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and independent mechanisms.

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest.

Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.

SARS-CoV2 Nsp1 is a metal-dependent DNA and RNA endonuclease.

Over recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the first time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses.

Abstract 2850: Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma

Abstract Background and Aim: Neuroblastoma (NB) is the most aggressive solid tumor of infancy and arises from neural-crest-derived progenitor cells. It is composed by cell types reflecting developmental stages of the adrenergic lineage: mesenchymal (MES) and adrenergic (ADRN) cell types. These cell identities result from the activity of NB specific Core Regulatory Circuitries (CRCs), transcription factor (TF) sets that co-occupy regulatory regions together impacting the expression of their own genes and those of lineage-specific ones governing cell state transitions and cellular identities. Our hypothesis is that noncoding somatic single nucleotide variants (SNVs) located in NB CRCs TF binding sites (TFBSs) underlie NB CRCs activity, affecting cellular differentiation and promoting tumor onset. We aim to investigate such pattern of regulatory elements and identify putative driver SNVs, exploring their role and characterizing their action mechanism. Methods: Transcriptionally active TFBSs (aTFBSs) bound by ADRN CRC TFs (GATA3, HAND2, ISL1, TBX2, MYCN, PHOX2B, ASCL1 and LMO1) were identified integrating 27 ChIP-seq and 11 ATAC-seq experiments in 6 NB ADRN cell lines (SKNBE2C, Kelly, NGP, COGN415, LAN5 and NB1643). In particular, aTFBSs were defined as ChIP-seq peaks overlapping ATAC-seq signals in the same cell line. SNVs from 317 NB whole genomes were mapped on aTFBSs and the enrichment of mutations in core regions respect to flanking ones was tested using Fisher test. Observed mutation rate in aTFBSs was compared with background mutation rate due to local sequence context. SNVs in mutated aTFBSs were selected and their impact on TF binding affinity was evaluated with FABIAN-variant and motifbreakR tools. aTFBSs target-genes were identified through promoter capture HiC (CHiC) in ADRN NB cells (SKNBE2C and SHSY5Y). Results: We found a significant (FDR &amp;lt; 0.05) mutation enrichment in aTFBSs of ISL1, PHOX2B, TBX2, GATA3 and MYCN, suggesting a common somatic mutation burden for a subset of ADRN CRC TFBSs in ADRN NB cells. Starting from 839 SNVs falling in significantly mutated aTFBSs, we selected 269 mutations impacting the binding of ADRN CRC TFs. This mutations subset mapped in aTFBSs interacting with genes that significantly enriched pathways involved in neuronal development and migration (FDR &amp;lt; 0.05), thus suggesting their possible role in affecting cell identity definition. As an example, chr6:137753475:G&amp;gt;T mapped in aTFBSs interacting with the promoter of OLIG3, which takes part in neuronal differentiation. Interestingly, chr6:137753475:G&amp;gt;T was predicted to break the binding to ISL1 (FABIAN score: -0.24), a key regulator of ADRN CRC. Conclusions: These results demonstrate that somatic noncoding SNVs can act synergistically to promote NB onset, affecting CRCs activity and, in turn, the normal cell identity definition. Citation Format: Mario Capasso, Vincenzo Aievola, Vito Alessandro Lasorsa, Annalaura Montella, Ferdinando Bonfiglio, Marianna Avitabile, Teresa Maiorino, Matilde Tirelli, Giuseppe D'Alterio, Matthias Fischer, Frank Westermann, Achille Iolascon. Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2850.

Abstract 3505: Classification of cancer subtypes by cfDNA fragmentomics analysis

Abstract Introduction Fragmentation patterns of cell-free DNA (cfDNA) from whole genome sequencing offer insights into nucleosome occupancy, providing a tool to infer epigenomic and transcriptomic information. The nucleosome complex, the primary protector of cfDNA, is reflected in the size distribution of cfDNA fragments, with a mode fragment size of 167 bp corresponding to the wrapping of DNA around a single nucleosome. Nucleosome occupancy patterns at transcription factor binding sites (TFBS) exhibit differences between circulating tumor DNA in cancer patients and normal plasma controls, as well as among different tumor subtypes. Therefore, cfDNA fragmentomics can be employed for cancer subtyping analysis. Methods We utilized the Griffin framework to classify tumor subtypes based on nucleosome profiling of cancer-specific TFBS and tumor subtype-specific chromatin accessibility regions from low-pass whole genome sequencing data of cfDNA. For prostate cancer subtyping, AR and ASCL1 binding sites were used to distinguish between androgen receptor-dependent prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). ER and ERBB2 were employed for breast cancer subtyping to differentiate between ER-positive and ER-negative tumor subtypes. Results Nucleosome profiling patterns at AR binding sites (ARBS) were compared among over 500 prostate cancer plasma samples and 42 normal plasma backgrounds. The ARBS nucleosome profiling abnormality score (ARBS score) was quantified by comparing its GC-corrected fragment central coverage with the normal background using a standard Z-score.Majority of prostate cancer samples with a high ARBS score could be classified as ARPC. For 12 samples with high tumor fraction but low ARBS score and no AR copy number amplification, we found that 8 had a high ASCL1 nucleosome profiling abnormality score. All these samples also exhibited RB1 copy number loss, suggesting that these 8 samples likely belong to NEPC. Nucleosome profiling of 171 breast cancer plasma samples at breast cancer-specific ER binding sites revealed that majority of samples had a high ERBS abnormality score, indicating likely ER-positive breast cancer. Conclusion Our results demonstrate that nucleosome profiling of tumor cfDNA exhibits distinct patterns across different tumor types. Integrating nucleosome profiling at cancer-specific transcription factor binding sites can facilitate cancer subtyping, highlighting its potential clinical utility for patient stratification. Citation Format: Lisha Zhu, Chao Dai, Shuang Gan, Shidong Jia, Pan Du. Classification of cancer subtypes by cfDNA fragmentomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3505.

Abstract 4899: Interface-guided phenotyping of coding variants in the transcription factor RUNX1

Abstract Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions. Citation Format: Kivilcim Ozturk, Rebecca Panwala, Jeanna Sheen, Kyle Ford, Nathan Jayne, Dong-Er Zhang, Stephan Hutter, Torsten Haferlach, Trey Ideker, Prashant Mali, Hannah Carter. Interface-guided phenotyping of coding variants in the transcription factor RUNX1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4899.

Abstract 1644: Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk

Abstract The chr 5p15.33 genomic locus encoding telomerase reverse transcriptase (TERT) is structurally complex, with several highly polymorphic intronic variable number tandem repeats (VNTRs). A germline VNTR located in TERT intron 6 (VNTR6-1, 38 bp repeat unit) has previously been associated with TERT alternative splicing but its potential role in regulating cellular functions and disease risk is unclear. To functionally characterize VNTR6-1, we deleted this region (1-2 kb) using CRISPR/Cas9 in UMUC3 (bladder cancer cell line). Cell count growth tracking of parental and VNTR6-1 knockout (VNTR6-1 KO) cells via automated live-cell monitoring revealed different relative growth rates under serum starvation vs. normal conditions, suggesting that VNTR6-1 might be sensitive to growth factors present in culture media. Evaluation of growth in media with full vs. charcoal-stripped (CS) serum, showed significant differences, with higher cell counts in parental vs. VNTR6-1 KO in CS-serum (P=4.1e-3), parental vs. parental in full (P=1.57e-4) or CS-serum (P=4.76e-2), but not in parental vs. VNTR6-1 KO in full serum (P=0.378). After 5-6 days of growth, cells in CS-serum also rapidly began outgrowing cells in full serum. We observed that VNTR6-1 KO dampened the outgrowth response in cells lacking growth factors in CS-serum. Proliferation-specific growth assays tracking the dilution of an incorporated dye in cells by flow cytometry further showed that VNTR6-1 KO leads to more pronounced proliferation differences due to the presence or absence of serum-supplied factors. Overall, cell growth patterns appear to be sensitive to serum in interaction with VNTR6-1, suggesting a potential VNTR6-1-dependent response to extracellular signaling. In human populations, we have identified two VNTR6-1 groups: Short (24-27 copies) and Long (40.5-66.5 copies). In the PLCO dataset (n=100,445 cancer cases and controls), logistic regression showed that VNTR6-1-Long group was associated with a decreased risk in male-prevalent cancers such as bladder and prostate, but an increased risk in female-prevalent cancers such as breast, endometrial and ovarian. These results support a link between the TERT region, and potentially VNTR6-1, with hormone signaling and cancer risk. Motif analysis within VNTR6-1 predicted binding sites for several transcription factors, including those associated with development and cell growth. Together, these results suggest that VNTR6-1 may be involved in regulating cell growth, potentially by facilitating responses to extracellular signaling pathways. Additionally, given the location of VNTR6-1 within a multi-cancer GWAS region, our findings provide functional context for the role of this polymorphic repeat in differential risk and outcomes in certain cancers. Citation Format: Michelle Ho, Oscar Florez-Vargas, Maxwell Hogshead, Brenen Papenberg, Chia Han Lee, Chi Zhang, Kaitlin Forsythe, Wusheng Yan, Raj Chari, Ludmila Prokunina-Olsson. Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1644.

Abstract 7435: PATTY: A bias estimation and correction model for bulk and single-cell CUT&amp;Tag data

Abstract The accurate detection of transcription factor (TF) binding sites and histone modifications (HM) on the genome-wide scale is essential for studying functional epigenetics and gene regulation. Cleavage Under Targets &amp; Tagmentation (CUT&amp;Tag) is a low-cost and easy-to-implement epigenomic profiling method that can be performed on a low number of cells and on the single-cell level. CUT&amp;Tag experiments use the hyperactive transposase Tn5 for tagmentation. We find that Tn5 is subject to intrinsic sequence insertion bias (intrinsic bias). Additionally, the preference of Tn5 insertion toward accessible chromatin also affects the distribution of CUT&amp;Tag reads (open chromatin bias). Both types of biases can significantly confound bulk and single-cell CUT&amp;Tag data analysis, which requires careful assessment and new analytical methods. To address this challenge, we present PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), a computational method for systematic characterization and correction of biases in CUT&amp;Tag data. We demonstrate that histone modification signals (H3K27me3 and H3K27ac) detected from CUT&amp;Tag data after bias correction using PATTY are better associated with orthogonal biological features such as gene expression. PATTY-corrected single-cell CUT&amp;Tag signals for histone modification can better cluster cells into their true cell types. This new computational method can improve both bulk and single-cell CUT&amp;Tag data analysis. Citation Format: Sheng'en Shawn Hu, Qingying Chen, Megan Grieco, Chongzhi Zang. PATTY: A bias estimation and correction model for bulk and single-cell CUT&amp;Tag data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7435.

Abstract 1694: Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways

Abstract Earli is developing a highly sensitive, orthogonal approach that is designed to be delivered via IV injection and uses a genetic construct to usurp dysregulated pathways, and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. The key element of our cancer-activated construct that drives specificity and sensitivity of expression is a promoter sequence, synthetically engineered to leverage transcription factors (TFs) activated in cancer pathways. Acting as a molecular sensor for dysregulated TF levels, the DNA construct expresses a measurable biomarker in cancer cells, while remaining transcriptionally silent in normal adjacent tissues and benign lesions. Previously, we described a multiomics factor analysis (MOFA) to interrogate 200 non-small cell lung cancer (NSCLC) and matched normal adjacent tissue samples comprised of RNA-seq based transcriptional profiling, and MS-based proteomics and phospho-proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and generated a list of top TFs dysregulated in NSCLC. In this study, we show the capability to design, engineer, and test cancer specific activity of combinations of response elements using a Massively Parallel Reporter Assay (MPRA) high-throughput pooled screening (HTS) method. More than 2,000 unique sequences of homotypic or heterotypic TF binding sites corresponding to the top 100 TFs were arrayed with diverse spacing and individually barcoded for the high-throughput screens in NSCLC cell lines. To validate our top hits, we individually tested 10 sequences from the top 25% and 8 sequences from the bottom 25%. Nine of the top sequences produced signals 5-10X higher than the background, whereas only one of the eight bottom sequences produced signal higher than the background. We have individually validated 30 different response elements and found that 28 of 30 drove the expected expression, with the majority of response elements (n=20) showing expression 5-10X higher than background. Remarkably, two of the engineered response elements were able to drive expression 20-30X higher in a specific cell line activated by WNT-signaling, suggesting that these response elements can sense specific cancer dysregulated pathways. Our lead response elements are being tested across a broad range of in vitro primary tumor lines with diverse genetic backgrounds and transcriptional profiles prior to in vivo testing using our imaging platform to distinguish malignant lung nodules. In summary, we have established a robust screening platform to identify response elements that can be activated by cancer dysregulated pathways. We are currently expanding this platform to other cancer models and indications, and leveraging this to explore more complex response elements that use combinations of TFs and natural spacing. Citation Format: Elizabeth Stroebele, Yue (Wendy) Zhang, Ishan Podar, Chloe Xia, Ajda Rojc, Dariusz Wodziak, Maggie Louie, David Suhy. Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1694.

Abstract 1695: Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers

Abstract Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems including cancer. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regarding which TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 528 DNA sequence motifs across the human genome and 6,650 TF binding profiles across different cell types to identify the molecular features contributing to the formation of transcriptional condensates. We found that the genomic distributions of sequence motifs for different TFs exhibit distinct clustering tendencies. Notably, TF motifs with a high genomic clustering tendency are significantly associated with super-enhancers. TF binding profiles showing a high genomic clustering tendency are further enriched at cell-type-specific super-enhancers. TFs with a high binding clustering tendency also possess high liquid-liquid phase separation abilities. Compared to non-clustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interaction, and stronger association with cancer outcomes. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates at cancer super-enhancers. Citation Format: Zhenjia Wang, Shengyuan Wang, Chongzhi Zang. Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1695.