We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function.African American (AA) prostate cancer associates with vitamin DOur study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer. TMPRRS2 and ETS genetic fusion in AA prostate cancer (Among African American (AA) men, prostate cancer occurs in a more aggressive form, and at a younger age compared with European American (EA) counterparts . Genomice cancer , is juste cancer .3 signaling 2D3. This secosteroid hormone is able to bind with high affinity to its target receptor, the vitamin D receptor (VDR) and thereby regulate gene expression  reduction in cancer risk, which is suggestive of a functional relationship and justification for increased AA participation in future studies n rhythm . Again, n rhythm . Togethee cancer .2D3-regulated VDR protein interactome (RIME), the VDR cistrome [Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq)], and the VDR transcriptome [RNA sequencing (RNA-seq)]. These interactome-cistrome-transcriptome relationships were associated with outcomes in three clinical cohorts; (i) in AA and EA men with high-grade prostatic intraepithelial neoplasia (HGPIN) who progressed to prostate cancer; (ii) a prostate cancer chemoprevention trial where AA and EA men were supplemented with vitamin D3; and (iii) a prostate cancer cohort of AA and EA men with gene expression data, clinical data, and measured serum vitamin D3 levels. We also mined three publicly available datasets to identify and subsequently test a mechanism for BAZ1A, a member of the ATP-dependent ACF-1/5 ISWI chromatin remodeling complex, to suppress VDR signaling in AA prostate cancer; an overview of the workflow is shown in In the current study, we aimed to establish VDR genomic functions in AA and EA prostate cancer with the goal to assess how this may contribute to health disparities. We utilized EA and AA nonmalignant prostate and prostate cancer cell models with confirmed genomic ancestry and defined the basal and 1α,25(OH)2 (Sanyo); HPr1AR, RC43N, RC43T, RC77N, RC77T cells were maintained in keratinocyte serum-free media ; LNCaP cells were maintained in RPMI1640 containing 10% FBS. All media contained 100 U/mL Penicillin-Streptomycin. 1α,25(OH)2D3 was kept as 10 mmol/L EtOH stocks. Cell lines were authenticated by short tandem repeat profiling and confirmed Mycoplasma free.Cells utilized were the nonmalignant EA prostate cells HPr1AR and malignant LNCaP, and nonmalignant AA prostate cells  and isogenic AA prostate cancer . HPr1AR cells were a generous gift of Dr. C K Choo  for mRNA detection by the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). cDNA was prepared using iScriptTM cDNA Synthesis Kit (Bio-Rad) and relative gene expression quantified via Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems), for both TaqMan and SYBR Green (Thermo Fisher Scientific) applications. All SYBR Green primers were tested for specificity by melting curve analysis. All qRT-PCR experiments were performed in biological triplicates, with at least technical duplicates, and fold changes (FC) determined using the 2eviously .Total cellular protein was harvested from exponentially growing cells, washed in ice-cold PBS. Cell lysis was in ice-cold RIPA buffer  containing 1× cOmplete Mini Protease Inhibitor Tablets (Roche). Protein concentrations were quantified using DC Protein Assay (Bio-Rad). Equal amounts of proteins (30–60 μg) were resolved via SDS-PAGE using precast polyacrylamide gradient gels  and transferred onto polyvinylidene fluoride membrane (Roche) for 30 V for 16 hours. Post transfer, membranes were blocked with 5% nonfat dry milk for 1 hour at room temperature. Blocked membranes were probed with primary antibody against BAZ1A, SMARCA5, VDR, GAPDH either overnight at 4°C or for 3 hours at room temperature. Primary antibody was detected with horseradish peroxidase–linked rabbit anti-mouse IgG  or goat anti-rabbit IgG  secondary antibody at room temperature using enhanced chemiluminescence Western Blotting substrate (Pierce). Signal quantification was performed using the ProteinSimple Fluorochem M Imager, as described previously .Bioluminescent detection of cellular ATP, as a measure of cell viability, was undertaken using CellTiter-Glo (Promega) reagents. Cells at optimal seeding density to ensure exponential growth were plated in 96-well, white-walled plates. Wells were dosed with agents to a final volume of 100 μL. Dosing occurred at the beginning of the experiment, and cells were incubated for up to 120 hours. Luminescence was detected with Synergy 2 multimode microplate reader (BioTek Instruments). Each experiment was performed in at least triplicate wells in triplicate experiments, as described previously .2D3 every 3 days for a period of 14 days, and then washed and fixed with neutral buffered formalin and stained with crystal violet stain and quantified antified .GFP-BAZ1A was purchase from Addgene (plasmid # 65371) and cells were stable transducted by selection and maintenance in media supplemented with puromycin (2 μg/mL).2D3. A total of 20 × 106 cells were cross-linked with 1% formaldehyde solution, quenched with glycine , and harvested in cold PBS. Nuclei were separated eparated  and sonieparated . LC/MS-M2D3 2D3  or EtOH in triplicate independent experiments. Briefly, 50 × 103 cells were resuspended in 50 μL of ATAC-resuspension buffer  containing  and pipetted up and down three times. Furthermore, 1 mL of ATAC-wash-resuspension buffer (ATAC-RSB + 0.1% tween 20) was used to pellet down the nuclei. The nucleic were further resuspended in transposition mix . Mixing, cleanup and library preparation, quantification, and sequencing were performed using NovaSeq6000 S1 PE150 bp sequencing as per protocol col ref. . ATAC-se2D3 2D3  or EtOH in triplicate independent experiments. Briefly, approximately 20 × 106 cells were cross-linked with 1% formaldehyde solution, quenched with glycine (0.125 mol/L), and harvested in cold PBS. Sonication of cross-linked chromatin was performed using a Bioruptor UCD-200 Sonicator (Diagenode) with optimized cycles for each cell type. Immunoprecipitation of sonicated material was performed with antibodies against VDR  or IgG (Rabbit IgG sc2729—Santa Cruz Biotechnology) for 16 hours, and antibody/bead complexes isolated with Magna ChIP Protein A+G magnetic beads (Millipore). Complexes were washed, reverse cross-linked, and treated sequentially with RNase and proteinase K prior to DNA isolation, as described previously eviously .Rsubread/csaw ((MotifDb). To find potential transcription factor (TF) binding enrichment within cistromes, GIGGLE was utilized to query the complete human TF ChIP-seq dataset collection  in Cistrome DB  or EtOH in biological triplicate samples and analyzed by RNA-seq. Sequencing libraries prepared with the TruSeq Stranded Total RNA kit (Illumina Inc), from 1 μg total RNA. Alignment of raw sequence reads to the human transcriptome (hg38) was performed via Rsubread  were performed using gene sets from the Molecular signatures database (MSigDB). For transcript-aware analyses, the FASTQ files were aligned with salmon Rsubread  and tranh salmon  and diff. Expression counts were called against the miRbase consensus miRnome using featureCounts and a standard edgeR pipeline determined differentially expressed miRNA as described previously  for mature miRNA and other small RNA products (140–150 bp). Successful constructs were purified using a Pippen prep (Sage Inc.), using 125 to 160 bp product size settings with separation on a 3% agarose gel. The purified samples were validated for size, purity, and concentration using a DNA-HS Bioanalyzer chip. Validated libraries were pooled at equal molar to a final concentration of 10 nmol/L in Tris-HCI 10 mmol/L, pH 8.5, before 50 cycle sequencing on a MiSeq . FASTQ files were aligned to the genome (hg38) using eviously .Sequencing was performed at the Nationwide Children's Hospital Institute for Genomic Medicine, Columbus, OH.NanoStringDiff and significantly different miRNA identified (logPV > 1 and absFC > 0.58).In serum samples from SWOG S9917 (HGPIN to prostate cancer), nanostring PCR was used to identify miRNA associated with progression in AA patients, within race and across race by progression status. The data were processed with 3  prior to radical prostatectomy. Significantly differentially regulated genes in the AA prostate cancer group (there were no DEGs in the EA prostate cancer group) were overlapped with genes annotated to ATAC-seq or ChIP-seq regions within 100 kb. The percentage overlap of DEGs with the total number of the indicated cistrome genes was calculated.Transcriptomic data from tumors were obtained from a cohort of 7 AA patients with prostate cancer with confirmed African genomic ancestry and 16 EA patients with prostate cancer who were treated with vitamin D3  or obesity . In each case, BMI or 25(OH)D3 levels were kept as a continuous variable, respectively. The DEGs were overlapped with genes annotated to ATAC-seq or ChIP-seq regions within 100 kb and the percentage overlap of DEGs and cistrome genes was calculated.Transcriptomic data from were obtained from a cohort of 57 AA and 18 EA patients with prostate cancer who underwent radical prostatectomy. Tumor-specific significant DEGs were identified for deficient serum 25(OH)DAll analyses were undertaken using the R platform for statistical computing (R version 4.1.3) and the indicated library packages implemented in Bioconductor.RNA-, ChIP- and ATAC-seq data are available (GSE223406).3 prior to radical prostatectomy were collected in accordance with recognized ethical guidelines and under local IRB approval  of their respective clinical institutions . The serapproval . The radapproval .The datasets generated and/or analyzed during the current study will be available on Gene Expression Omnibus.In the first instance, we confirmed that the RC43N, RC43T, RC77N, and RC77T cell lines all had more than 90% African genomic ancestry, and as expected LNCaP were predominantly European genomic ancestry . Recentl2D3 treatment, which was most pronounced in nonmalignant AA RC43N cells; RC77T cells did not change VDR expression in response to 1α,25(OH)2D3  in HPr1AR, LNCaP, RC43N, and RC43T.VDR protein levels were detected in all cells, and generally were elevated following 1α,25(OH)5(OH)2D3 . RIME wa2D3 treatment, VDR-enriched proteins were increased in RC43N (∼200), and decreased in RC43T (n = 15), and modestly so in LNCaP , DNA repair enzymes  and DNA helicases .Broadly, the number of enriched proteins in the basal state were equivalent across cells (∼100), although highest in RC43T. Following 1α,25(OH)in LNCaP . Classifin LNCaP  of protein LNCaP . Across 2D3 treatment enriched with HDAC2, the CoA, SMARCA4 and the TF, STAT1. In RC43T compared with LNCaP, the basal VDR was enriched for the TF, SAFB2 and following 1α,25(OH)2D3 treatment there was a gain of the NFκB CoR PPP1R13L, TRIM29 and TGFB1, and loss of PARP1 of PARP1 .2D3-regulated switches in CoA and CoR; for example, CoA gains included RBMXL1, a transcriptional regulator identified in leukemia 2D3 in RC43T. Compared with LNCaP, RC43T is modestly less sensitive in liquid culture and completely resistant to inhibition of colony formation ormation  and S2E.2D3 . The greatest 1α,25(OH)2D3-dependent impact on NF and mononucleosome (mono) regions was in RC43N and RC43T . There were no significant 1α,25(OH)2D3-regulated changes in mono regions in HPr1AR and RC43N, but there was a gain of approximately 1,800 mono regions in LNCaP cells. In contrast, the response to 1α,25(OH)2D3 in RC43T was a loss approximately 1,900 NF and approximately 800 mono regions, suggesting 1α,25(OH)2D3 induced a loss of chromatin accessibility, and was a striking difference to the isogenic RC43N cells. Furthermore, 1α,25(OH)2D3-regulated NF regions significantly overlapped (sharing at least 1 bp) (P < 4.1e-230) in RC43N, RC43T, and HPr1AR, whereas the LNCaP regions were distinct. Given that the shared NF regions between RC43N and RC43T are induced by 1α,25(OH)2D3 in RC43N but lost in RC43T, this further supports the concept that VDR function is distinct between these cells.To understand the impact of the VDR complex on nucleosome positioning, we undertook ATAC-seq following 1α,25(OH)2D3-stimulated NF and mono regions were enriched for ChromHMM-defined epigenetic states . Ranking TF family motif enrichment c states  and TF m regions . Reflectrichment  revealedLNCaP cells displayed the fewest significant associations although one of the most enriched motifs was the AR/Half-site , which w2D3-treatment 2D3-stimulated sites in RC43T overlapped with 1α,25(OH)2D3-stimulated sites in RC43N  basal VDR binding was identified in LNCaP, RC43N, and RC43T cells but not HPr1AR, and in all cells following 1α,25(OH)cistrome . Althougcistrome , there wcistrome , left. Iin RC43N , right. ondition . Thus, t2D3 treatment. For example, basal VDR enrichment in bivalent promoters was most significant in LNCaP and RC43N, and reduced by 1α,25(OH)2D3; in LNCaP log10(Padj) = 46, and reduced to 6 in the presence of 1α,25(OH)2D3. In contrast, the score in RC43N (∼20) was broadly equivalent in the basal and 1α,25(OH)2D3-stimulated states, suggesting one aspect of cancer cells, regardless of genomic ancestry is for 1α,25(OH)2D3 to reduce VDR binding at these regions, whereas it is sustained in AA prostate cells. Binding at an enhancer region illustrates that RC43N binding is comparable in both basal and 1α,25(OH)2D3-stimulated treatment, but reduced in RC43T 2D3 treatment only increased motif enrichment in RC43N cells, notably for ZNF189, TEAD1, and TEAD3 and the circadian rhythm–associated TF, NFAT.The enrichment of motifs suggested the VDR cistromes were comparable in nonmalignant cells, and distinct from prostate cancer cells. The predicted VDR motif was enriched in LNCaP and RC43T but the predicted RARα and TRβ motifs was enriched in HPr1AR and RC43N. Others nuclear receptor motifs such as NR4A1/NUR77 and PGR were common . Again, 10] in the basal and 1α,25(OH)2D3-treated cells, respectively. These were much reduced other cells. Similarly, there were 1α,25(OH)2D3-dependent increases in enrichment for other nuclear receptors including AR, ERα, and RARγ.Comprehensive cistrome enrichment analyses with GIGGLE  identifi2D3-treated RC43N cells 3N cells . Togethe3N cells , right; 2D3-stimulated state within each cell (2D3-dependent NF regions in RC43N were enriched (logPV = 14.9) for 1α,25(OH)2D3-dependent VDR-interacting proteins identified in the same cell, including the VDR itself, the splicing factor PTBP1, and the DNA helicase XRCC6. The 1α,25(OH)2D3-dependent NF regions in RC43T annotated to multiple proteins that were enriched in the basal VDR complex (logPV = 26.8) including VDR and XRCC5 and given that these represent loss of NF regions it may reflect why the number of proteins in the 1α,25(OH)2D3-regulated VDR complex in RC43T is much reduced compared with the basal state 2D3 2D3-dependent NF region (red symbol) and/or a VDR binding peak (symbol with dark border) in the same cell background were more apparent in AA cells. This was even more noticeable comparing DEGs between RC43N compared with HPr1AR, or in RC43T compared with LNCaP 2D3-dependent NF regions and in RC43T miR-99a was a prominent example.Standard and small RNA library RNA-seq  was undertaken and similarity and principal component analyses revealed that experimental conditions explained most of the variation in expression . Reflectth LNCaP . Small Rn = 96; LNCaP, n = 124; RC43N, n = 136; RC43T, n = 77) and related to cistrome date  were also identified ll types . HPr1AR ll types . For exariptomes . Supplemriptomes  also sup2D3 transcriptional signaling is more impactful in AA compared with EA prostate models, and that within the isogenic AA cells, there were frequent divergent enrichment of pathways suggesting that 1α,25(OH)2D3 signaling is significantly altered in RC43T compared with RC43N, which reflects the ATAC- and ChIP-seq.Together, these findings reveal that 1α,25(OH)2D3-stimulated VDR ChIP-seq and ATAC-seq data was infrequent. That is, individual genes had both a significant VDR binding site and a significant 1α,25(OH)2D3-stimulated NF region, but these sites did not frequently overlap. In contrast, in RC43N the basal and 1α,25(OH)2D3-stimulated VDR ChIP-seq data significantly overlapped with the basal NF regions 2D3-dependent NF regions). Similarly, in LNCaP the basal VDR ChIP-seq overlapped with the basal NF regions  between 1α,25(OH) regions . In thes2D3-induced NF regions, and gene expression; namely, peak:gene relationships. To refine these relationships further, the VDR cistromes were annotated to within a 100 kb of the nearest gene, including those that were members of the VDR interactome as defined by BioGRID.Next, relationships were identified between either VDR binding or 1α,25(OH)VDR itself and NCOR1 associated most frequently (n∼90) with the 1α,25(OH)2D3-stimulated NF regions in RC43N, but this was less frequent in the other cell lines. The basal and 1α,25(OH)2D3-stimulated VDR ChIP-seq data displayed a similar pattern 2D3; AA.T.D3 2D3 compared LNCaP/1α,25(OH)2D3]. 1α,25(OH)2D3-stimulated ATAC-seq cistromes were similar across cells and frequent in Promoters and Enhancers 2D3 in HPr1AR cells.Next we filtered the ChromHMM-classified cistrome gene:peak relationships to 1α,25(OH)nhancers ; left. Vnhancers ; right, 2D3-stimulated states and appeared most frequent downstream of the transcription start site. In contrast, in RC43T, the gene:peak relationships were only detected in the basal state. That is, the 30 1α,25(OH)2D3-stimulated VDR-binding sites in RC43T 2D3.Next, we calculated the frequency of peak:gene relationships in 10 kb bins around from DEGs , and SMARCA5/WSTF , which functions cooperatively with BAZ1A n Rhythm . SupportTMPRSS2 fusion positive from negative tumors in TCGA-PRAD cohort (χ2P = 0.002), suggesting that genomic ancestry impacted expression of these genes was most common in the absence of TMPRSS2 fusion (n = 596) compared with EA tumors .We also analyzed the RNA-seq data from the Berchuck and colleagues cohort of AA and EA tumors , and ide2D3 stimulation  in BAZ1A transfected cells compared with vector controls controls ; Fig. 5Cnd RC43T . Howevernd RC43T  found no2D3-induced expression changes in RC43N and RC43T, and calculating the enrichment terms that change the most, in either the same or a divergent manner, revealed that BAZ1A exerted a potent and cell-specific impact on gene expression patterns induced by 1α,25(OH)2D3. Indeed, GSEA terms are illustrated that changed to convergent from a divergent response in parental cells asticity . This rell fates .Finally, we examined how these VDR cistrome-transcriptome relationships that were identified in cell line models were detected in three clinical prostate cancer cohorts of AA and EA patients.2D3/VDR cistrome regions including MIR23B and RTCA  clinical trial  prior to radical prostatectomy. Ancestry informative markers confirmed the African genomic ancestry of the AA patients. As we reported previously . In both cases, these DEGs were enriched for VDR ChIP-seq or 1α,25(OH)2D3-stimulated NF ATAC-seq genes 2D3 5(OH)2D3 ; both ce2D3 2D3-stimulated gain of NF regions in RC43N occurred more frequently than in other models, whereas these regions were lost in RC43T. Motif enrichment identified commonly enriched TFs, including AP-1 factors, such as KLF5, but differential enrichment of circadian rhythm TF motifs, such as CLOCK. Similarly, ChIP-seq revealed shared VDR sites in the absence of 1α,25(OH)2D3 across LNCaP, RC43N, and RC43T but addition of 1α,25(OH)2D3 resulted in highly distinct VDR binding patterns within a cell, including a dramatic reduction in VDR binding in RC43T and somewhat in LNCaP. At the transcriptional level, the RC43N and RC43T cells were also most responsive but the responses were frequently divergent again, with many GSEA terms being switched in direction between the cells. VDR cistrome-transcriptome relationships were significantly greater in RC43N compared with other cells, although basal proximal VDR binding in RC43T was strongly associated with DEGs.The VDR complex differed by genomic ancestry of the cell. For example, in associations with CoAs , CoRs , splicing factors , as well as proteins that impact regulation of circadian rhythm, . This suggests that VDR is primed to produce a different response in AA cells such as RC43N and RC43T. Reflecting the divergent enrichment between RC43T and RC43N of these factors in the VDR complex, 1α,25(OH)2D3 signaling in RC43T cells compared with RC43N cells. 1α,25(OH)2D3 treatment induced chromatin accessibility around the BAZ1A locus only in RC43N cells, and 1α,25(OH)2D3 treatment upregulated BAZ1A and SMARCA5 in RC43N, but downregulated these proteins in RC43T. Transcript-aware analyses revealed that 1α,25(OH)2D3 induced different transcripts of SMARCA4 only in RC43N; the BAZ1A complex contains multiple SMARCA components. Reflecting this, RIME analyses revealed that SMARCA5 was only significantly enriched in the VDR complex in RC43N and RC43T, but significantly reduced by 1α,25(OH)2D3-treatment in RC43T. It is noteworthy that in RC43N the VDR ChIP-seq significantly overlapped with publicly available SMARCA4 ChIP-seq from LNCaP cells. To test the clinical significance of these relationships, we used partial correlation analyses to demonstrate that BAZ1A and SMARCA5 expression, as well as other VDR-interacting proteins identified in cell lines by RIME, were able to significantly strengthen the correlations between VDR and AA cistrome genes in AA prostate cancer.We sought to identify mechanisms that may drive the divergent responses between EA and AA cells, and between RC43N and RC43T. TCGA analyses identified a potential role for altered expression of the BAZ1A/SMARCA5 SWI/SNF complex, and this was replicated in another AA EA prostate cancer cohort . These d2D3-autoregulatory mechanism for BAZ1A/SMARCA5 expression and this function that is corrupted in RC43T, and AA prostate cancer. Finally, it is interesting to note that and genome-wide association study SNPs in BAZ1A associate significantly with heel bone strength, also supporting a role in regulating VDR function function .2D3-regulated transcriptome, but these genes were only significantly enriched for VDR bound and NF regions in RC43N and RC43T suggesting that BAZ1A expression had the most significant impact on VDR function in AA cells. The most strongly upregulated gene in RC43N that is annotated to RC43N 1α,25(OH)2D3-regulated NF region was PIK3R6, which recently was identified as a novel antigen in a clinical immunotherapy trial in advanced prostate cancer e cancer .3 levels, and indeed this interacted significantly with obesity (BMI > 30.0 kg/m2) status. The strength of the correlation between VDR and AA ChIP-seq target genes was also significantly impacted by coregulators interacting with the VDR such as PARP1 and NONO, as well as BAZ1A.Validation in three clinical cohorts revealed that the footprint of VDR signaling was most apparent in AA prostate cancer. For example, miRNA that predicted progression from HGPIN to prostate cancer in AA men were highly enriched for VDR cistrome data, as were genes that responded in prostate tumors from men receiving vitamin D3 supplementation prior to radical prostatectomy. This was also strikingly apparent in prostate tumors from men who had deficient serum 25(OH)D3 levels, changes in membrane transport D3 deficiency among AAs, which is highly prevalent, impacts cancer and these other diseases. Our genomic data suggest that the powerful example of changing melanin content in the skin through ancestral adaptation has occurred in parallel with a range of genomic mechanisms to govern VDR functions in noncalcemic tissues.It is reasonable to suggest that the VDR stands at the crossroads of biopsychosocial signaling that impacts prostate cancer by a distinct pattern of VDR genomic binding in a manner that is governed by African genomic ancestry. Although guidance is available for serum 25(OH)DThe current study suggests that the functions of the VDR may have adapted with significant distinctions between people of different genomic ancestry. More specifically, we reason that adaptation to environments of lower UVB exposure that could potentially be associated with vitamin D insufficiency and alter the prominence of how VDR signaling occurs in a wide variety of tissues. Furthermore, we propose that the prostate is an important gland with which to test this possibility given it is the site of significant syndromes and diseases that are highly impactful on U.S. men, and furthermore many of these conditions display significant health disparities.Supplementary Table 1Supplementary Table_1 RIMEClick here for additional data file.Supplementary Table 2Supplementary Table _2 - RIME IIClick here for additional data file.Supplementary Table 3ST_3 ATAC-SeqClick here for additional data file.Supplementary Table 4ST_4 ChIP-SeqClick here for additional data file.Supplementary Table 5ST_5 ChromHMMClick here for additional data file.Supplementary Table 6ST_6 ChIP-Seq MotifClick here for additional data file.Supplementary Table 7ST_7 ATAC-Seq to RIMEClick here for additional data file.Supplementary Table 8ST_8 ATAC/ChIP-Seq to splicingClick here for additional data file.Supplementary Table 9ST_9 LISA analyses of RNA-SeqClick here for additional data file.Supplementary Table 10ST_10 ATAC overlap ChIPClick here for additional data file.Supplementary Table 11ST_11 ATAC- to RNA-SeqClick here for additional data file.Supplementary Table 12ST_12 ChIP-Seq to RNA-SeqClick here for additional data file.Supplementary Table 13ST_13 BAZ1A and lineage plasticityClick here for additional data file.Supplementary Table 14ST_14 Serum miRNA and PCaClick here for additional data file.Supplementary Table 15ST_15 Cistrome and miRNA expressionClick here for additional data file.Supplementary Table 16ST_16 Cistrome and RNA-Seq in tumorsClick here for additional data file.Supplementary Figure 1SF_1 workflowClick here for additional data file.Supplementary Figure 2SF_2 Ancestry cell linesClick here for additional data file.Supplementary Figure 3SF_3 motifs in ATAC-SeqClick here for additional data file.Supplementary Figure 4SF_4 VDR cistromeClick here for additional data file.Supplementary Figure 5SF_5 ChIP-Seq motifClick here for additional data file.Supplementary Figure 6SF_6 RNA-Seq PCAClick here for additional data file.Supplementary Figure 7SF_7 RNA-Seq volcanosClick here for additional data file.Supplementary Figure 8SF_8 RNA-Seq GSEAClick here for additional data file.Supplementary Figure 9SF_9 overlap miRNAClick here for additional data file.Supplementary Figure 10SF_10 cistrome-transcriptomeClick here for additional data file.Supplementary Figure 11SF_11 BAZ containing complexes expressionClick here for additional data file.Supplementary Figure 12SF_12 BAZ1A-GSEAClick here for additional data file.Supplementary Figure 13SF_13 GSEA tumorsClick here for additional data file.


EWSR1 RNA-binding protein and ETS transcription factor gene (EWS-FLI being the most common). Interestingly, murine ES models have failed to produce tumors phenotypically representative of ES. Genomic alterations (GA) in ES are infrequent and may work synergistically with EWS-ETS translocations to promote oncogenesis. Aberrations in fibroblast growth factor receptor (FGFR4), a receptor tyrosine kinase (RTK) have been shown to contribute to carcinogenesis. Mouse embryonic fibroblasts (MEFs) derived from knock-in strain of homologous Fgfr4G385R mice display a transformed phenotype with enhanced TGF-induced mammary carcinogenesis. The association between the FGFRG388R SNV in high-grade soft tissue sarcomas has previously been demonstrated conferring a statistically significant association with poorer OS. How the FGFR4G388R SNV specifically relates to ES has not previously been delineated. To further define the genomic landscape and corresponding pathway alterations in ES, comprehensive genomic profiling (CGP) was performed on the tumors of 189 ES patients. The FGFR4G388R SNV was identified in a significant proportion of the evaluable cases . In line with previous analyses, TP53 , CDK2NA/B , and STAG2  represented the most frequent alterations in our cohort. Co-occurrence of CDK2NA and STAG2 alterations was observed . Notably, we identified a higher proportion of TP53 mutations than previously observed. The most frequent pathway alterations affected MAPK , HRR , Notch1 , Histone/Chromatin remodeling , and PI3K . These findings help to further elucidate the genomic landscape of ES with a novel investigation of the FGFR4G388R SNV revealing frequent aberration.Ewing Sarcoma (ES) is an aggressive, mesenchymal malignancy associated with a poor prognosis in the recurrent or metastatic setting with an estimated overall survival (OS) of <30% at 5 years. ES is characterized by a balanced, reciprocal chromosomal translocation involving the  EWS) gene, EWSR1, and members of the E26 transformation-specific (ETS) gene family. This results in an in-frame fusion of the EWS gene to ETS to generate a hybrid fusion gene, EWS-FLI1 [FLI1) gene is the most common involved gene, other members of the ETS gene family include ERG, ETV1, E1AF, and FEV [Ewing Sarcoma (ES) is the second most common bone cancer in children and young adults with approximately 1.5 cases per million . MultimoEWS-FLI1 . While F and FEV ,10,11,12EWS-FL1 have successfully formed tumors morphologically resembling ES [EWSR1-FLI1 translocation positive cells. Of note, viable clones were observed only after co-occurring alteration of CDKN2A. Furthermore, additional chromosomal translocations reminiscent of chromoplexy, a loop-like rearrangement previously noted in ES tumors, were observed [Reproducing the EWS-ETS chimeric transcript in genetically engineered mouse models (GEMM) has presented several challenges. In a comprehensive analysis of transgenic and non-transgenic mouse models, researchers were unable to reproduce ES, instead resulting lack of phenotypic expression or embryonic death . Only imbling ES . More reobserved . These dSTAG2 gene, which encodes a subunit of the cohesion complex responsible for regulation of sister chromatid separation undergoing cell division [STAG2 alterations [STAG2 variant and loss of expression were noted in high proportion  [EWS-FLI1 transgenes, STAG2 inhibition has been associated with sarcoma formation and reduced survival [TP53 and CDK2NA have also been observed [CDK2NA and TP53 have both been postulated to mediate cell cycle regulation in conjunction with EWS-ETS [TP53 and CDK2NA and this association was characterized by a poorer prognosis [EWSR1, FLI1, and TP53 failed to result in significant colony growth. Alternatively, co-alteration of CDK2NA or STAG2 resulted in a significant increase in size furthering intrigue into how additional, recurrent variants may impact ES development [Additional GAs and associated pathway alterations have been evaluated for their potential to induce sarcoma oncogenesis ,17,18. Pdivision ,20. Recesurvival . Recurreobserved ,20,21,22 EWS-ETS . In prevelopment . Furtherelopment ,17,18.FGFR4) has been investigated and therapeutically targeted in a variety of solid tumors. Acting as a receptor tyrosine kinase (RTK) protein, FGFR4 is activated by a family of ligands, fibroblast growth factors (FGFs), at the extracellular domain resulting in intracellular transmission signals via transmembrane domain and intracellular tyrosine kinase [FGFR4Gly38Arg (G388R) single nucleotide variant (SNV) results in the substitution of arginine (Arg) for glycine (Gly) in the transmembrane domain of the receptor. The reported prevalence of the FGFR4G388R SNV is approximately 32% in the general population [Fgfr4G385R mice display a transformed phenotype with increased STAT3 signaling confirmed in vivo [FGFR4G388R SNV has been associated with FGFR4 protein damage and increased FGFR4 expression [FGFRG388R SNV in high-grade soft tissue sarcomas has previously been shown to have a deleterious effect on overall survival [FGFR4G338R SNV, other recurrent GAs, and their corresponding pathway alterations in ES with CGP.Fibroblast factor receptor 4 , gene amplifications, copy number alterations (CNAs), gene fusions, rearrangements (REs), and single nucleotide variations (SNVs) by next generation sequencing (NGS). 189 samples were assayed by hybrid-capture based CGP, including 406 DNA-sequenced genes in addition to 265 RNA-sequenced genes commonly reported to be rearranged in cancer, which was previously documented by He et al. [As part of routine clinical care, formalin-fixed and paraffin-embedded (FFPE) tissue from 189 Ewing sarcoma patients were sent to Foundation Medicine for CGP between 2012 and 2018 . The presence of the e et al. . At leasTissue from 189 ES clinical samples were analyzed with CGP. Demographic information is described in EWS-FL1 translocation.CGP identified several GAs in ES represented by the heat map in n = 89, affecting 24% of individual samples), HRR , Notch1  Histone/Chromatin remodeling , and PIK3  with additional pathways illustrated in TP53 , CDKN2A/B , and STAG2  as illustrated in PCLO, RAD21, and KDMSC as demonstrated in n = 5, 2.6%) and chromosome 8q . No CNVs were observed involving chromosome 16q.The highest incidence of pathway alterations affected MAPK  and coincided with GAs in high frequency , Notch1 , HRR , Histone/chromatin remodeling , and Cyclin pathways . Additional recurrent GAs noted in combination with the FGFR4G388R SNV are displayed in The requency . PathwayWNT represented the only pathway most frequently characterized by CN alterations (80%). Although recurrent, pathogenic variants were observed in potential tumor-agnostic targets , none of the observed variants were rearrangements/fusions for which the current FDA approvals exist.Observed GAs included single nucleotide variations (SNV), copy number (CN) alterations, and rearrangements (RE). Overall, SNVs were noted in the largest proportion accounting for 81% of observed GAs, with a smaller proportion resulting from CNs (18%) and REs (4%). GAs affecting the MAPK pathway were comprised of SNVs, CNs, and REs at 80%, 18%, and 2% respectively. Similarly, GAs impacting HRR included 78% SNVs and 22% CNs, without any observed REs. Both Notch1 and SWI/SNF pathway GAs were exclusively comprised of SNVs (100%). FGFRs are currently under investigation in multiple solid tumor types [FGFR4, an overall response rate (ORR) of 17% and median duration of response (DOR) of 5.3 months were achieved in patients with hepatocellular carcinoma [FGFR4G388R SNP has been evaluated in high-grade soft tissue sarcoma and associated with a poor prognosis [FGFR4G388R SNV. Recently, an evaluation of FGFR alteration targeting is underway advanced sarcomas harboring pre-specified alterations in FGFR1-4 . In our analysis, the FGFR4G388R SNV was detected in over half of the samples evaluated in our analysis. This is notably higher than would be expected in the general population based on previous large-scale analysis [n = 92, 37%). The FGFR4G388R SNV often co-occurred with recurrent GAs. How the FGFR4G388R SNV may affect GAs and pathways implicated in sarcoma formation is not clearly understood. Interestingly, while the FGFR4G388R SNV co-occurrence appeared random with most pathway alterations, the PI3KCA pathway was disproportionately altered in the absence of FGFR4G388R SNV co-alteration. This could suggest that downstream signaling alterations may modulate ES formation in patients without an existing FGFR4 alteration. Further investigation with clinical correlative data is necessary to better understand the potential pathogenicity of this particular SNV in ES.As reviewed by Chae et al., small molecule inhibitors and monoclonal antibodies targeting various or types . In a pharcinoma . The FGFrognosis . These fanalysis . Owing tTP53 , CDK2NA/B , and STAG2  represented the most frequently altered genes in our cohort after excluding VUS. Our data illustrated a higher proportion of TP53 mutations (19%) when compared to previous analyses of the genomic landscape in ES, which have demonstrated TP53 mutations in approximately 5.7 to 7% of tumor samples [TP53 is a tumor suppressor gene and its loss of function is frequently implicated in tumor development. TP53 mutational loss has previously been identified in ES and associated with higher TMB and shorter overall survival (OS) [In line with previous analyses,  n = 33, %, and ST samples ,22. Poteval (OS) .CDK2NA/B and STAG2. Interestingly, in previous genomic analyses of ES, a mutual exclusivity appears to exist between these two alterations [CDK2NA and STAG2 were mutated in 17% and 12% of cases, respectively. Co-occurrence of CDK2NA and STAG2 variants appeared in 5 (3%) of the cases. In a recent evaluation of STAG2 in ES, researchers concluded that STAG2 loss affects the gene-regulatory architecture resulting in promotion of disease progression [STAG2 is thought to function through its interaction with CTCF subsequently impacting gene expression regulated by EWS-FLI1 [CTCF has also been shown to interact with CDKN2A locus, regulating transcription [Additional genes frequently mutated in our cohort included erations . In our gression . FurtherEWS-FLI1 . Interescription .PI3K with additional pathways illustrated in both PI3KCA and MAPK pathway alterations represented two of the most common pathways altered in our evaluation. PI3K signaling pathway is frequently implicated in oncogenesis and typically mediated through loss of the inhibitory protein, PTEN [PI3K activity enhanced by PIK3R3 and loss of PTEN [MEK/MAPK or PI3K pathways via insulin growth factor-1 receptor (IGF-1R) neutralizing antibodies being associated with functional consequences including delayed time to primary tumor development and attenuated growth [PI3K and MAPK inhibitors resulting in suppression of tumor growth [PI3K inhibition, further investigation is warranted and currently underway . Perhaps, CGP would play a role in more appropriate patient selection for these agents.GAs affecting the following pathways were most prevalent in our analysis: MAPK, HRR, Notch1, Histone/Chromatin remodeling, and in, PTEN . Previou of PTEN . Furtherd growth . In vitrr growth . ConsideNotch signaling is highly conserved through evolution in multicellular organisms resulting in control of cellular proliferation, differentiation, and apoptosis. The Notch pathway can influence development of neighboring cells via juxtracrine signaling. Four receptors (Notch1-4) act in a canonical receptor-ligand interaction resulting in a series of cleavages to the Notch receptor leading to release of the Notch intracellular domain (NICD) [CBF-1/Su(H)/LAG1 (CSL) transcription factors that together recruit additional transcriptional co-activators (Co-A) and displacement of transcription co-repressors (Co-Rs) [FGFR4G388R SNP as demonstrated in n (NICD) . Thereaf (Co-Rs) . Further (Co-Rs) . Inhibit (Co-Rs) . In our HRR is pivotal in repair of double-strand breaks generated during crosslinking of DNA. Deficiency of HRR results from both germline alterations in BRCA1 and BRCA2, as well as with genetic or epigenetic inactivation with somatic variants contributing to a BRCA-like phenotypic expression [HRR have unrepaired DNA break due to inhibition, has been successfully exploited with the use of Poly (ADP-ribose) polymerase (PARP) inhibitors [EWS-FL1 or EWS-ERG lead to significant reduction in homologous recombination activity thought to be secondary to loss of EWSR1 function [BRCA1 deficiency was noted in ES cells suggesting a possible role for therapeutic strategies involving PARP-1 inhibition. ES mouse xenografts have been shown to be highly sensitive to PARP-1 inhibition with EWS-FL1 transcription mediated through PARP1 [HRR including CHEK2 , BRCA1 , BRCA2 , BARD1 , CHK1 , and RAD51D . Potentially, CGP may help to identify a subpopulation of patients with enhanced susceptibility to PARP inhibition. Multiple clinical trials are actively investigating the role of targeting homologous recombination deficiency (HRD) in ES via CHK1  and PARP inhibition .pression . Synthethibitors . ES cellhibitors . Expressfunction . Furthergh PARP1 . Furthergh PARP1 . In a mugh PARP1 . Our cohMSH3 and RUNX1T1 in a small proportion of samples represented an additional subgroup of the DDR pathway in our analysis. MSH3 functions as a heterodimer with MSH2 and is utilized in mismatch repair of detected insertion-deletion loops. Somatic alterations in MSH3 are associated with dysfunctional MMR and microsatellite instability [RUNX1T1 functions as a transcriptional co-repressor and interacting with histone deacetylases (HDACs) and is involved in multiple cellular processes including neuronal differentiation, microglial proliferation, endothelial angiogenesis, and adipocyte differentiation [DDR pathway as they relate to the pathogenesis of ES.GAs of tability . RUNX1T1ntiation ,54,55,56SWI/SNF), epigenetic modification, and the cyclin pathway. The SWI/SNF chromatin remodeling complex, a highly conserved ATP-dependent chromatin remodeling complex influencing transcriptional activity, is identifiable by next generation sequencing in genes including ARID1A, EZH2, INI1/SMARCB4, SMARCA4 among others [ARID1A-deficient cells lead to reduction of SWI/SNF regulation of enhancers associated with tumor generation [ARID1A variants were present in 4% of ES samples analyzed in this study. The role of SWI/SNF complex in oncogenesis is an active area of interest highlighted with the development of tazemetostat, a selective inhibitor of EZH2. In an open-label, phase I trial investigated tazemotostat in relapsed or advanced solid tumors, tazemotostat was found to be well-tolerated with promising activity, notably in epithelioid sarcoma patients [LSD1, an epigenetic modifying demethylase, has previously been shown to be upregulated in ES [LSD1 is thought to contribute to ES formation and overall survival. In a phase 1, non-randomized trial, SP-2577 (Seclidemstat), a reversible LSD1 inhibitor, is being evaluated in treatment of recurrent or refractory ES (NCT03600649). In previous evaluation of epigenetic modification, functional genomics revealed an activated cyclinD1/CDK4 pathway with potential sensitivity to chemical inhibition [cyclin D1 binding to cyclin dependent kinases, such as CDK4 and CDK6. Subsequent phosphorylation of RB, a cell cycle regulator, mediates cell cycle progression after dissociating from G1 to S phase-promoting transcription factors [Additional pathways frequently altered in our analysis included switch/sucrose-nonfermentable , nor does it exert high levels of PD-L1 expression. Interestingly, ES cells have been observed to have a high frequency of partial or complete absence of HLA class I expression, which has been associated with absence of CD8+ T cell infiltration . Tumor mFGFR4G388R SNV were demonstrated alone and in conjunction with additional GAs in a high proportion of a large cohort of ES tumors. TP53 was mutated in higher proportion than previously reported. Additional recurrent GAs included STAG2 and CDKN2A with demonstrated co-occurrence in a small proportion of the evaluable cases. The role and interplay between these genomic alterations are unclear and warrant further investigation. Unfortunately, patient clinical outcomes were not available for this cohort to further define the prognostic or predictive implications, which represents a major limitation of the analysis. Furthermore, age-matched controls were not available to analyze for a true enrichment of the FGFR4G388R SNV in this population. The lack of validation studies analyzing patient-derived cell lines represents another major limitation. Variant allele frequency (VAF) was not evaluable and may affect the pathogenicity in a continuous manner rather than purely in a binary manner warranting further exploration. Future investigation should be directed at the association of these GAs and their potential impact on clinical correlates including grade, time to progression, frequency of metastasis, and treatment response.In summary, alterations of the


Transposable elements (TEs) are dispersed repetitive DNA sequences that can move within a genome. Even though hundreds of years of evolution have led to the accumulation of mutations that render most TEs unable to transpose, they still exert multiple important functions. They play a role in hematopoiesis, especially during periods of high cellular plasticity, such as development, regeneration and aging. This is because TEs can populate functional elements, such as enhancers. Furthermore, TE RNA can be sensed by innate immune sensors that play a role in inflammation and inflammaging. TEs also play an important role in different aspects of leukemia and lymphoma, leading to either beneficial or detrimental outcomes. Further studies into the function of TEs in healthy or diseased hematopoietic systems are necessary to manipulate them for therapeutic benefit. Summary: Transposable elements exert a functional role on hematopoiesis and disease throughout life. Further characterization of these repetitive elements will open new diagnostic and therapeutic avenues. Drosophila (Transposable elements (TEs) – also known as transposons, interspersed repeats, selfish genetic elements, jumping genes or parasitic DNA – are dispersed repetitive sequences that can move within a genome by a mechanism called transposition. They were discovered in the 1940s in maize by Barbara McClintock . McClintosophila . Since tosophila , making osophila .Dictyostelium intermediate repeat sequence 1 (DIRS-1)-like elements : a type of cancer in which the bone marrow makes too many lymphocytes.Basic helix-loop-helix ARNT-like (BMAL1): a protein that, in humans, is encoded by the BMAL1 gene on chromosome 11. It plays a key role as a positive component of the mammalian autoregulatory transcription-translation feedback loop (TTFL), a  negative feedback loop that is responsible for generating molecular circadian rhythms. BMAL1 is the only essential clock gene for the circadian rhythm in humans.Breakage−fusion–bridge cycle: genetic instability in which the end-to-end fusion of broken sister chromatids yields a dicentric chromosome that forms a bridge in mitosis during anaphase.cGAS–STING (cyclic GMP–AMP synthase-stimulator of interferon genes) pathway: a key component of the innate immune system that functions to detect the presence of cytosolic DNA, triggering the expression of inflammatory genes that can lead to senescence or activation of cell-autonomous defense mechanisms.Deletion: genomic instability in which part of a chromosome or DNA sequence is omitted during DNA replication.Dictyostelium intermediate repeat sequence 1 (DIRS-1)-like elements: members of the non-LTR retrotransposon subclass. They use a tyrosine recombinase instead of an integrase to integrate into the genome.DNA methylation: a process predominantly involving the nucleotide cytosine during which a methyl group is transferred onto its C5 position, yielding 5-methylcytosine. This mostly acts to suppress gene transcription and repress TEs.DNA methyltransferase (DNMT) inhibitors: drugs used to study the role of DNA methylation in different tissues and model systems. They represent a possible therapeutic option for disorders that involve altered DNA methylation.DNA strands: polymers or chains of the monomer deoxyadenosine monophosphate (dAMP) that are linked by phosphodiester bonds.DNA transposons: DNA sequences that can move to and integrate at different locations within the genome.Duplication : genomic instability in which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene.Endogenous retroviruses (ERVs): endogenous viral elements of the genome that closely resemble and might be derived from retroviruses. Human ERVs (HERVs) are a type of LTR retrotransposon and account for 5–8% of the human genome.Endothelial-to-hematopoietic transition: a highly plastic process during which an endothelial cell becomes a hemogenic endothelial cell from which HSPCs will emerge.Evolutionarily young TEs: a subset of TEs that have more recently transposed into the human germline ’.Human silencing hub (HUSH) complex: a novel epigenetic complex responsible for the variability of the positional effect of integrated transgenes in human cells. It is associated with genomic regions dense in H3K9me3.Hutchinson–Gilford progeria syndrome (HGPS): a rare genetic disorder caused by a mutation in the LAMIN A gene LMNA that leads to accelerated aging.Leukemic stem cells (LSCs) in acute myeloid leukemia (AML): a low-frequency subpopulation of leukemic cells that possess stem cell properties distinct from those of the majority of leukemic cells, including the ability of self-renewal and drug resistance.Long interspersed nuclear elements (LINEs): a subclass of ∼7000 bp-long non-LTR retrotransposons that are widespread in the genomes of many eukaryotes (21.1% of human genome). The most abundant LINE in humans is LINE-1. Their integration into the genome is coupled to a process called target-primed reverse transcription : a pair of identical DNA sequences that are several hundred base pairs long and flank a retrotransposon. LTRs occur in eukaryotic genomes and flank gene or pseudogene series; they contain signals that promote and terminate transcription and are required for reverse transcription processes.LTR retrotransposon: class I transposable elements, i.e. retrotransposons, characterized by the presence of LTRs directly flanking an internal coding region. Their chromosomal integration relies on cleavage and strand-transfer reactions catalyzed by integrase : transposons that undergo replicative transposition without RNA intermediates : a protein component of the human silencing hub (HUSH) complex that specifically binds H3K9me3 histone H3. It is vital for the formation of heterochromatin and has specific roles in cancer metastasis.Mutagenesis: a process by which the DNA of an organism is permanently changed and a gene mutation occurs. It can be natural or artificial, and can cause cancer, hereditary diseases or evolutionary innovations. It is the main cause of species biodiversity.Non-LTR retrotransposon: class I transposable elements, i.e. retrotransposons, characterized by the lack of LTRs. cDNA synthesis does not take place in the cytoplasm of the cell but at the site of insertion of the new copy.Pancreatic ductal adenocarcinoma (PDAC): the most-prevalent type of pancreatic neoplasm, developed in the exocrine compartment and accounting for >90% of pancreatic cancer cases.Penelope-like elements (PLEs): a subclass of eukaryotic retroelements characterized by a reverse transcriptase domain with similarity to telomerases and group II introns.R/G ratio (TE repeat transcript to gene transcript ratio): ratio between the median of the normalized counts for all TE transcripts relative to that of the normalized counts for all gene transcripts.Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs): a type of intracellular pattern recognition receptor involved in viral recognition by the innate immune system. RIG-I (retinoic-acid inducible gene) is the best characterized receptor of the RLR family. Together with MDA5 (melanoma differentiation-associated 5) and LGP2 (laboratory of genetics and physiology 2), they are sentinels for intracellular viral RNA, the product of viral infection.Retrotransposon: mobile genetic element amplified by reverse transcription via an RNA intermediate, which can then move to and integrate at – i.e. transpose to – different locations within the genome. Copy numbers of a retrotransposon are, therefore, increased quicker than DNA transposons.SET domain bifurcated histone lysine methyltransferase 1 (SETDB1): a prominent member of the Suppressor of Variegation 3–9 (SUV39)-related protein lysine methyltransferases (PKMTs). It is widely expressed in human tissues, methylating Histone 3 lysine 9 (H3K9) residues, promoting chromatin compaction and exerting negative regulation on gene expression (pression .Short dispersed nuclear elements (SINEs): a subclass of retrotransposons that are ∼100-700 bp. They make up about 13% of the mammalian genome. Their integration is also coupled to target-primed reverse transcription (cription .Sirtuin 6 (SIRT6): a histone deacetylase of the sirtuin family expressed in the nucleus. It is involved in DNA repair, telomere maintenance, carbohydrate and lipid metabolism, and plays a protective role against senescence.Sirtuin 7 (SIRT7): a NAD-dependent deacetylase that interacts with RNA polymerase I and has a key role in chromatin remodeling.Target-primed reverse transcription: a process in which an element-encoded endonuclease (an endonuclease that is encoded directly by the transposable element) cuts the target DNA, generating an exposed 3′ hydroxyl group that serves as a primer for reverse transcription of the RNA of that element.Translocation: genomic instability characterized by the reciprocal exchange of chromosomal material between non-homologous chromosomes, i.e. those that do not belong to the same pair.Werner syndrome (WS): a rare autosomal recessive disorder with mutations in the WRN gene. It is characterized by the appearance of premature aging and is also known as adult progeria.TEs are grouped into two main classes according to their mode of transposition . Class 1elements , (3) Pents PLEs; , (4) lonts PLEs; . Class 2ts PLEs;   and detrimental  – during hematopoietic development, regeneration, aging and disease.Synthetic TEs, like pression . TE-deripression , can be pression .Among thOwing to the many diverse functions of TEs, it is important to understand their function, especially since they have been understudied for many years. Among the different systems they affect, TEs have been shown to exert roles – both beneficial  and detrimental  – during hematopoietic development, regeneration, aging and disease. These roles are the focus of this Perspective.RIG-I  and melanoma differentiation-associated gene 5 , which activated inflammatory signaling to enhance the formation of HSPCs. This suggests that, during a plastic process, TE expression increases and – until their repression – that these TEs are used as ligands by RLRs to ‘sense’ plasticity. In this scenario, moderate and tolerable plastic changes result in beneficial inflammatory signaling that drives HSPC formation; however, extreme changes may lead to inflammation that kills the cells.Hematopoietic stem and progenitor cells (HSPCs) are formed in vertebrates by an endothelial-to-hematopoietic transition , which iMda5−/− HSCs were more quiescent compared with WT HSCs, therefore performing better in the long term. However, during rapid acute stress, such as serial injections of chemotherapeutic fluorouracil, Mda5−/− mice were unable to reconstitute their blood system fast enough (Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are present in a quiescent state, and, under homeostatic conditions, human HSCs exhibit very low transcription and transposition levels of LINE-1 subclass retrotransposons  see Glo. Howevert enough . These vNatural stress, such as aging, is also linked to alterations in the transcription of TEs . Aging lCollectively, TEs exert diverse roles in highly plastic biological processes, such as developmental transitions during normal physiological processes or in response to stress and aging.de novo retrotransposition can occur  family is expressed in acute megakaryocytic and erythroid leukemia . HERVs a HERV-K3 . Additio HERV-K3 .[…] the different regulatory pathways of TEs need to be carefully studied and dissected to enable better management of cancer.One type of leukemia that has been more extensively studied for its association with TE expression is acute myeloid leukemia (AML), a heterogeneous cancer of the myeloid lineage. Leukemic stem cells LSCs;  propagatAML is often associated with epigenetic alterations in DNA methylation and modification of histones. In a 2021 preprint, Nadorp et al. revealed that LSCs and mature leukemic cells display different chromatin accessibility profiles for diverse TE subfamilies  preprintIn the context of cancer, including AML, TEs play a dual role . On the […] we must aim to better understand how HSC-responsive genes might be regulated by elements populated by TEs.The accumulation of genomic data over the past two decades has led to substantial progress in the field of TE research. Emerging genomics methods, including long-read and single-cell RNA sequencing, have the potential to revolutionize our understanding of TE biology and regulation. As the quality and quantity of genomics resources increase, many more instances of TE function in development and disease are likely to be discovered .[…] we mCurrent evidence shows that TEs are not only junk DNA but that they have multiple functions in all the roadblocks of life – from development to regeneration, to aging and disease. Indeed, as mentioned above, all these are phases in life during which cells exhibit high plasticity. Upon changes that lead to increased cellular plasticity TEs are expressed and have a role in innate immune signaling or as functional genomics elements. For the latter, evidence is emerging mostly from studies of development , but notFurther studies that increase our understanding of the role of TEs in different systems will substantially contribute to our knowledge of the biology behind normal physiology and pathogenesis and may also reveal novel therapeuticsRegarding aging, it has been shown in other systems – including adipose tissue, liver, kidney and muscle – that manipulating the biology of TEs, e.g. with reverse L1 transcriptase inhibitors, can reverse phenotypes of aging in mice . TherefoKMT2A . An additional clinical trial is investigating the use of azacytidine in combination with other drugs for AML .Advancing our knowledge of TEs in cancer should allow a more personalized treatment approach. In the case of AML, treatment could start with analyzing deregulated TEs in LSCs obtained through biopsies, followed by assessing of whether TE inhibition or TE re-expression is needed for therapy. Immuno- or chemotherapy could then be combined with treatment targeting regulators of TE expression to force LSC differentiation into mature leukemic cells and to reactivate immune signaling pathways leading to cell death . Such meTEs have emerged as new players in multiple physiological and pathological phenomena within and beyond the hematopoietic system. Further studies that increase our understanding of the role of TEs in different systems will substantially contribute to our knowledge of the biology behind normal physiology and pathogenesis and may also reveal novel therapeutics.


Integration of retroviruses into the host genome can impair the genomic and epigenomic integrity of the cell. As a defense mechanism, epigenetic modifications on the proviral DNA repress retroviral sequences in mouse embryonic stem cells (ESC). Here, we focus on the histone 3 variant H3.3, which is abundant in active transcription zones, as well as centromeres and heterochromatinized repeat elements, e.g., endogenous retroviruses (ERV).H3f3a or H3f3b, are knocked out. Depletion of H3f3a resulted in a transient upregulation of incoming retroviral expression and ERVs, while the depletion of H3f3b did not have the same effect and repression was maintained. However, the depletion of both genes resulted in a stable activation of the retroviral promoter. These findings suggest that H3.3 is important for regulating retroviral gene expression in mouse ESC and provide evidence for a distinct function of the two H3.3 genes in this regulation. Furthermore, we show that Trim28 is needed for depositing H3.3 in retroviral sequences, suggesting a functional interaction between Trim28 recruitment and H3.3 loading.To understand the involvement of H3.3 in the epigenetic silencing of retroviral sequences in ESC, we depleted the H3.3 genes in ESC and transduced the cells with GFP-labeled MLV pseudovirus. This led to altered retroviral repression and reduced Trim28 recruitment, which consequently led to a loss of heterochromatinization in proviral sequences. Interestingly, we show that H3.3 depletion has a differential effect depending on which of the two genes coding for H3.3, Identifying the molecular mechanisms by which H3.3 and Trim28 interact and regulate retroviral gene expression could provide a deeper understanding of the fundamental processes involved in retroviral silencing and the general regulation of gene expression, thus providing new answers to a central question of stem cell biology.The online version contains supplementary material available at 10.1186/s13148-023-01499-5. The replication of murine leukemia virus (MLV) is efficient in most dividing cells; however, it is restricted to pluripotent cells , 2. AfteH3f3a and H3f3b), is a replacement histone and can be incorporated into nucleosomes in a replication-independent manner. H3.3 deposition has long been associated with gene activation, but in ESC, H3.3 is also deposited in lineage-specific bivalent genes ). Cells were incubated for 10 min on ice. A similar volume of high salt buffer  was added, and cells were incubated 15 min on ice. Cells were then centrifuged  that carries a single copy of tagged histone H3.3A located downstream of the Type I Collagen Col1A1) locus, under the control of Doxycycline . A poolA1 locus,H3f3a and H3f3b) that translate into identical protein products (H3.3A and H3.3B) [H3f3b gene and obtained two CRISPR KO lines (g1- and g2-H3.3B-). KO was verified using Sanger sequencing . Depletion was verified using RT-qPCR  . First, g1- and g-H3.3B-. g1- and g-H3.3B-. g1- and g-H3.3B-. g1- and g-H3.3B-. g1- and g-H3.3B-. g1- and g-H3.3B-. g1- and g-H3.3B-. However, the GFP levels proved similar in both depletion methods  of selected genomic regions and retroviral sequences in control and H3.3-depleted ESCs. Trim28 recruitment to the PBSpro sequence by ZFP809  is cruciTo pinpoint the factors that recruit H3.3 to retroviral sequences, we eliminated one of the specific chaperones of H3.3, DAXX. The protein was depleted using gRNA targeting the third exon of the gene. KO was confirmed by RT-qPCR Fig. A and immThe effort to decipher the molecular mechanism of retroviral silencing in pluripotent cells has been ongoing for decades; however, many key questions remain . Here, wThe different mechanisms underlying histone deposition and eviction  relate to the dynamics of H3.3 at different regions of the chromatin . In ESCsTherefore, a model can be suggested in which the efficient recruitment of Trim28, mediated by the PBSpro sequence of the MLV genome, is responsible for the hyperdynamic H3.3 turnover on these sequences. Additionally, it should be noted that our experimental system is highly attuned to the new H3.3 variants, while the repositioning of old H3.3 is invisible to us. This is because the incoming proviral sequences do not have any old H3.3 to recycle; therefore, only new H3.3 can be acquired on their chromatin. Furthermore, the one-hour DOX induction labels only newly transcribed H3.3, while the old ones are only observed after ~ 8 h. Together, these might explain the hyperdynamic turnover rate in integrated proviruses and its link to heterochromatinization, since the old H3.3 are more enriched for active PTMs , 35.Despite the high correlation between H3.3 dynamic deposition and Trim28 recruitment to retroviral sequences, its cause remains unknown. Therefore, we deleted or depleted the two H3.3 genes and examined the effect on MLV repression onset and maintenance. The results show that H3.3A depletion leads to short-term up-regulation of retroviral-derived transcription, while H3.3B deletion does not have such an effect. However, the effect of H3.3A is limited, as silencing is restored after two weeks due to H3.3B expression. This, with the consistent expression upregulation observed when both genes were eliminated, highlights the importance of H3.3 incorporation into the silenced retroviral chromatin.H3f3a and H3f3b appear regulated by different transcriptional and post-transcriptional factors, such as microRNAs [H3f3b also have distinct expression patterns in various tumors [H3f3b is evolutionarily adapted for broad expression patterns in diverse cellular programs, including cell differentiation, while H3f3a is more fine-tuned for a specific transcriptional program [Although H3.3A and B are identical on the protein level, icroRNAs  and H3f3s tumors , 36), ans tumors , 37. Thi program . ConsequOur data demonstrated that depletion of Trim28 results in reduced H3.3 occupancy on retroviral sequences, and that depletion of H3.3 leads to less Trim28 enrichment. Hence, the mutual dependence between Trim28 and H3.3 controls the epigenetic silencing in these regions. Trim28 was previously shown to recruit ESET and DAXX-ATRX, which modify the H3K9me3 mark and maintain H3.3 on the chromatin, respectively. Therefore, the effect of Trim28 KD on the occupancy of the retroviral sequences with H3.3 (Fig. Previous studies have shown that the chaperone complex composed of ATRX and DAXX is responsible for the deposition of H3.3 on heterochromatin throughout the genome of mouse ESCs , 38, 39.H3.3 Histone variant deposition plays a key role in endogenous retroviral regulation in ESCs , 42, 43.We identified a role for H3.3 in establishing and maintaining the repression of newly integrated retroviral sequences in ESCs. This silencing is epigenetic and the loss of H3.3, mainly H3.3A, up-regulates retroviral expression via the erasure of H3K9me3. This can serve as a paradigm for understanding the unique properties of the “retroheterochromatin” in mESC.Additional file 1: Figure S1. Controlling H3.3 deposition and dynamics.anti-HA ChIP on full length or integration deficient MLV-infected, Dox-induced KH2 cells followed by RT-qPCR on proviral site. Values are % Input normalized to the b-Globin gene. Data are the mean ± s.e.m..Accumulation of H3 into PBSpro, PBSgln, and genomic sequences at 1, 4 and 8 h after DOX induction. Presented are bound values normalized to the 8 h Bound fraction. ChIP-qPCR data are the mean ± s.e.m..Comparable levels of GFP positive cells in H3.3B KOand KDinfected with PBSpro or PBSgln MLVImmunoprecipitationof nuclear extracts from ESCs carrying the H3.3-HA-tag, followed by immunoblotting with the indicated Antibodies.ChIP-qPCR for H3 in cells depleted with WT and Trim28 infected with PBSpro or PBSgln virus. Data are the mean ± s.e.m..Additional file 2: Table S1. Primers list.


The structure of σ32-RPo reveals key interactions essential for the assembly of E. coli σ32-RNAP holoenzyme and for promoter recognition and unwinding by σ32. Specifically, a weak interaction between σ32 and −35/−10 spacer is mediated by T128 and K130 in σ32. A histidine in σ32, rather than a tryptophan in σ70, acts as a wedge to separate the base pair at the upstream junction of the transcription bubble, highlighting the differential promoter-melting capability of different residue combinations. Structure superimposition revealed relatively different orientations between βFTH and σ4 from other σ-engaged RNAPs and biochemical data suggest that a biased σ4–βFTH configuration may be adopted to modulate binding affinity to promoter so as to orchestrate the recognition and regulation of different promoters. Collectively, these unique structural features advance our understanding of the mechanism of transcription initiation mediated by different σ factors.Bacterial RNA polymerases (RNAP) form distinct holoenzymes with different σ factors to initiate diverse gene expression programs. In this study, we report a cryo-EM structure at 2.49 Å of RNA polymerase transcription complex containing a temperature-sensitive bacterial σ factor, σ The data were plotted in SigmaPlot  and fitted to eqn: F = F0 + a1 × ) + a2× ). The amplitudes (a1 and a2) and observed rates  were estimated as previously reported [The promoter for the stop flow assay was prepared as previously reported . The Cy3reported . To moni filter 5/9.3 nm areported .Grids (Quantifoil 1.2/1.3 Au 300 mesh) were glow-discharged for 25 s at 15 mA prior to application of 4 μL complex at a concentration of 1.2 mg/mL, then plunge-frozen in liquid ethane using a Vitrobot  with 100% chamber humidity at 4 °C.2. A total of 8555 movies were imported and processed using Relion. All 40 frames were aligned using MotionCor2 [32 interaction area to 3.40 Å.Grids were transferred to a 300 kV Titan Krios electron microscope equipped with a Gatan K3 summit direct electron detector and a GIF energy filter with a slit width of 20 eV. All the images were auto-collected via EPU  with a mtionCor2  before ctionCor2 . From thtionCor2 . 2D averE. coli core RNAP (PDB: 7MKP) [32 were manually built in COOT [The coordinates of core RNAP and promoter DNA from the structure of B: 7MKP)  were fitB: 7MKP)  and manuB: 7MKP) . The coo in COOT . The coo in COOT . Due to E. coli σ32-RPo, we reconstituted the complex with the E. coli RNAP core enzyme, σ32, and a nucleic acid scaffold  and σ4 (residues 231–279)  and σ32-RNAP results in an RMSD of 1.633 Å across 3268 residues [32-RPo remains in a closed conformation (We obtained a cryo-EM map at 3.0 Å for the d 2.49 Å . The map231–279) B,C. The 231–279) C,D. Simiresidues . Similarormation C. The om32-RPo, the σ2 and σ4 domains bind to the surface of RNAP . In the structure, σ4 adopts a helix-turn-helix fold -RNAP holoenzyme is significantly slower than that of wild-type σ32-RNAP. Mutations in the protein pockets of A−11  also exhibited slowed RPo equilibration. It is worth noting that the curves could well fit to a double exponential where the amplitudes and observed rates for the fast  and slow  phases were fixed [a1, a2) describe that phase’s contribution to the total fluorescent enhancement signal. As show in E. coli RNAP complex. Specifically, our results show that substituting W108, H107, and F104 with alanine greatly destabilizes the RPo, while substituting V97 and N94 with alanine only slightly destabilizes the RPo  and QR (σ28), and tested the corresponding transcription activity using a Mango-dependent in vitro transcription assay. As shown in WW > σHW (WT) > σQR. Similarly, the RPo formation activity was ranked as σWW > σHW (WT) > σQR  > σQR , indicatobs,k1~0.1s−1, obs,k2~0.01s−1) in E. coli σ24-RNAP [M. tuberculosis CarD-RNAP [obs,k1~1s−1) report on protein–DNA interaction is not related to opening  and the observed slow rate  is related to RPo equilibration. These results indicated the importance of RPo formation for transcription, and also highlighted the multiplicity of RPo equilibration in different bacteria.Notably, the curves and parameters  σ28 and σ38 belong to the same group 3 category; and (3) the tail of σ24 did not observably interact with βFTH, we chose σ28 and σ38 as our research targets. The chimera of σ32 and σ28, designated as σ32–28, was designed by replacing the βFTH-interacting σ4 segment of σ32 with the βFTH-interacting σ4 segment of σ28. The chimera of σ32 and σ38, designated as σ32–38, was designed by replacing the βFTH-interacting σ4 region of σ32 with the βFTH-interacting σ4 region of σ38  [32 I54  [2.2 and the clamp helix of the β’ subunit, we speculate that cytoplasmic σ32 faces two mutually exclusive fates: either bind to RNAP to exert their active function or be inactivated through membrane localization.Our structure may also help solve or explain some previous questions. Based on our structure, we speculate that phosphorylation of Y260 may inhibit σactivity  by attenl region ,58,59, aaperones . Sandwicegion C)  and resiegion C)  are main70. While the presence of W-dyad is common in group 1 σ factors [32 replaces W433 in the W-dyad in σ70 to wedge the upstream transcription bubble, while W108 in σ32 resembles W434 in the W-dyad of σ70. Our data also show that different σ factors employ distinct wedge residues in initiating transcription with the following activity ranking: WW > HW > QR. This result is consistent with the result of the stopped-flow assay. Our result is in congruence with previous hypothesis that the W-dyad is the optimal choice for wedging the upstream ds/ss junction of the transcription bubble [From first engaging with promoter DNA to finally producing RNA, RNAP holoenzymes harboring promoters undergo several sequential conformational states, including promoter recognition (RPc), promoter melting (RPi), and bubble formation (RPo) . Promote factors ,67, resin bubble , giving n bubble . Variatin bubble .4 contacts the βFTH and mediates recognition of the promoter −35 element [4–βFTH module, which plays a significant role in promoter recognition [4–βFTH modules of different σs can allosterically affect promoter recognition has not been explored. Here, two chimeric σ factors (σ32–28 and σ32–38) with partial σ4 domain replaced were designed to test the impact on DNA binding affinity and transcription activity, and it was surprising to find variation in DNA binding affinity and enhanced transcription activity with the chimeric proteins  a biased σ4–βFTH configuration to modulate the affinity of binding to the promoter so as to orchestrate the recognition and regulation of different promoters. Our work will deepen our understanding of transcription initiation mechanisms of different σ32 factors as well as the biological function of σ32.In summary, our structure and supporting biochemical data identified a different promoter recognition and promoter melting mode for transcription initiation, including (1) a relatively weak interaction between σ


NRF2 is a transcription factor that plays a pivotal role in carcinogenesis, also through the interaction with several pro-survival pathways. NRF2 controls the transcription of detoxification enzymes and a variety of other molecules impinging in several key biological processes. This perspective will focus on the complex interplay of NRF2 with STAT3, another transcription factor often aberrantly activated in cancer and driving tumorigenesis as well as immune suppression. Both NRF2 and STAT3 can be regulated by ER stress/UPR activation and their cross-talk influences and is influenced by autophagy and cytokines, contributing to shape the microenvironment, and both control the execution of DDR, also by regulating the expression of HSPs. Given the importance of these transcription factors, more investigations aimed at better elucidating the outcome of their networking could help to discover new and more efficacious strategies to fight cancer. Nuclear factor E2-related factor 2 (NRF2) is a transcription factor that plays a pivotal role in cytoprotection, mainly because it induces the transcription of phase I and II phase I detoxification enzymes, limiting oxidative stress . NRF2 acSignal transducer and activator of transcription 3 (STAT3) is a multifunctional transcription factor, classically activated by the Interleuchin-6 (IL-6)-type family cytokines, through Janus kinase (JAK) . STAT3 eBesides cytokines, STAT3 can be activated also by tyrosine kinases, such as v-Fps, v-Ros, Etk/BMX and v-Abl or by proteins encoded by oncoviruses such as Epstein-Barr virus (EBV) .The interplay between NRF2 and STAT3 is quite controversial and interestingly, it may also involve autophagy. Therefore, in this perspective, we will discuss the literature reports and attempt to shed some light on this interplay, particularly in the context of cancer cells.Autophagy is a process controlled by several molecular pathways, including phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) (PI3K/AKT/mTOR)  and STATAmong other consequences, the impairment of autophagy leads to SQSTM1/p62 accumulation . This reAmong the numerous processes controlled by NRF2, there is the inflammatory process , that, wInterestingly, a recent study has reported that Brusatol can also lead to the inhibition of STAT3 and of that of the upstream kinases responsible for its activation, in Head and Neck Squamous Cell Carcinoma , underscPost-translational modifications, such as phosphorylation, can strongly influence the activity of NRF2. Phosphorylation of NRF2 may occur at different residues and may be mediated by several kinases, resulting either in the activation or inhibition of NRF2 . Among tAmong other post-translational modifications, methylation has been reported to influence NRF2 and STAT3 activity, as indeed methylation has been found to modify several nonhistone proteins including NRF2 and STAT3. Regarding NRF2, it has been shown that methylation can occur at arginine 437 residue and that this effect can moderately affect NRF2 activity , 62. RegOf note, methylation of NRF2 and STAT3 promoters can also influence the expression and activity of these transcription factors. For example, in the context of Alzheimer Disease, NRF2 activity is reduced by methylation and the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) may enhance NRF2 expression and transcriptional activity . RegardiAn interesting point that deserves to be discussed is how the interplay between NRF2 and STAT3 affects the DNA damage response (DDR). In the physiological execution of DDR, Heat Shock Proteins (HSPs) plays a pivotal role, as many molecules, involved in both single- and double-stranded DNA breaks, are HSP clients . For exaHSPs could contribute to STAT3 activation mediated by NRF2, reported in previous studies  as, amonIn conclusion, with this perspective, we attempted to have shed some light into the complex interplay between NRF2 and STAT3, pathways playing a key role in cancers, either of hematologic and solid origin. As these transcription factors may either activate or inhibit each other Figs.  and 2 an


In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is  , between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects. In contrast to carcinomas, which are solid tumors that develop from epithelial cells, sarcomas originate in mesenchymal cells. These sarcomas can be classified into three main groups .1) Soft Twenty-one percent of pediatric cancers are defined as sarcomas , of whicES was first described by James Ewing in 1921 as a new bone tumor called “diffuse bone endothelioma” . InitialThese tumors were distinguished from “Ewing sarcoma-like”, having morphological similarities with ES, but being characterized by other fusion genes and different clinical and pathological features . This clThe majority of ES are osseous, arising mainly in the pelvis and ribs, but also in the diaphysis of long bones  . Twenty EGR2, Early Growth Response 2; ADO, 2-Aminoethanethiol Dioxygenase; TARDBP, TAR DNA Binding Protein; RREB1, Ras Responsive Element Binding Protein 1; KIZ, Kizuna Centrosomal Protein and NKX2-2, NK2 Homeobox 2) facilitating the binding of the EWS-FLI1 fusion protein  and FLI1 genes, generating the EWS-FLI1 fusion protein , BCOR (BCL6 Corepressor), ZMYM3 (Zinc finger MYM-type containing 3) or CDKN2A (Cyclin Dependent Kinase Inhibitor 2a)  .ES is a fast-growing tumor, forming osteolytic lesions, which can lead to bone pain and sometimes to pathological fractures. The clinical picture of ES is not very distinctive, which often leads to a delay in diagnosis, ranging from several weeks to several months. The first signs that may lead to consultation are the appearance of swelling in the affected bone, associated with a slight pain that may become more pronounced at night or following physical activity. In more advanced cases, pain and the presence of a mass are accompanied by less specific symptoms, such as fever, fatigue or weight loss, which may be a sign that the tumor has become metastatic .Diagnosis includes imaging studies before confirmation with surgical biopsy and histological and molecular analyses. Imaging consists of conventional radiography, through which it is possible to see the osteolytic damage created by the tumor mass in the diaphyseal-metaphyseal bone . Magnetiin situ hybridization (FISH) or even quantitative Polymerase Chain Reaction (qPCR) techniques.Histological features of the sample include the observation of small, round, undifferentiated cells. These cells have a prominent nucleus and sparse cytoplasm with glycogen deposits. Classically, the marker CD99 (Cluster of Differentiation 99), a transmembrane glycoprotein, is used for the diagnosis of ES . Other mvia the bloodstream , ENO2 (Neuron-specific Enolase), S-100 or genes of the Notch signaling pathway . NeuroseMSCs are multipotent, self-renewing stem cells derived in particular from bone marrow, able to differentiate into osteoblasts, adipocytes, chondrocytes or myocytes.in vivo, with characteristics (CD99 expression) and morphology similar to those of ES , EWSR1  and TAF15  genes belong to the FET family of RNA-binding proteins. Structurally, the FUS, EWS and TAF15 genes are very similar, with several common domains  repeats. Its composition suggests its involvement in protein-protein interactions,- A central Recognition RNA Motif (RRM),- Arginine-glycine-glycine (RGG)-rich domains, also involved in protein self-assembly and RNA binding,- A Zinc-Finger Domain, with the same role as the RGG domains in protein-RNA binding.Located on chromosomes 16, 22 and 17 respectively, the  domains .- An N-tDue to their structure, the ubiquitously expressed genes that compose the FET protein family are involved in a wide variety of processes, such as transcription, post-transcriptional regulation and DNA damage repair . These pFLI1) is one of 26 genes in the E-twenty-six Transformation-Specific (ETS) protein family. The ETS proteins are divided into 12 subfamilies, including the ERG subfamily, which comprises 3 proteins: ERG (ETS-Related Gene), FLI1 and FEV (Fifth Ewing Variant). All proteins in this ETS family have a DNA-binding domain (ETS domain), binding a purine-rich motif (GGA [A/T]) (Friend Leukemia Integration 1 (A [A/T]) . More spA [A/T]) .These proteins are known to be involved in various biological processes, such as embryonic development, vasculogenesis, angiogenesis and hematopoiesis . In addiEWS-FLI1 fusion gene (under the control of the EWS promoter), composed of the N-terminal domain of the EWS gene and the C-terminal domain of FLI1 (EWS-FLI1 transcripts have been described in the literature (EWS gene and exon 6 of the FLI1 gene (EWS gene and exon 5 of the FLI1 gene (FLI1-EWS) also exists, but until recently was considered to be little or not expressed in ES cells   generates the  of FLI1 . Dependiterature , the majLI1 gene , or betwLI1 gene . It shouES cells . Indeed,ve in ES . Howeverferation .EWS-FLI1 fusion gene leads to the formation of the EWS-FLI1 fusion protein, whose activator domains are more potent than those of FLI1 alone as a result of N-terminal domain substitution during chromosomal translocation , a chromatin remodeling complex   and by tcription .EWS-FLI1 translation is modulated by compounds such as Lovastatin or Tunicamycin, which reduce the protein level of EWS-FLI1, thus reducing primary tumor growth , but incvia a lysosome-dependent pathway  to increase its transcriptional activity , or with pathway .etc.). In particular, EWS-FLI1 has been shown to regulate the expression of numerous transcription factors (c-Myc (Gli1 (Glioma-associated Oncogene Homolog 1) (NR0B1 (Nuclear Receptor subfamily 0 group B member 1) (FOXO1 (Forkhead Box O1) (IER3 (Immediate Early Response 3)   or NR0B1ember 1) , genes i Box O1) , or IER3ponse 3) , tumor sSOX2 enhancer (SRY-box transcription factor 2), thereby reducing its expression and altering tumor growth in vivo  and involved in the regulation of gene expression by limiting enhancer-promoter interactions within the same TAD , or by same TAD .BRCA1 (Breast cancer type 1 susceptibility) gene, preventing its expression, and its crucial role in the response to DNA damage  combining Olaparib or Niraparib, with Temozolomide, Irinotecan and Ceralasertib. With regard to cell-cycle targeting, it has been shown, for example, that ES cells require CDK4 and Cyclin D1 to survive and grow independently of anchoring  to study the tolerated dose of Abemaciclib, an inhibitor of these kinases involved in cell-cycle regulation, and in particular in G1 phase regulation. A clinical trial was also initiated last year  to determine whether LY2880070, a Checkpoint kinase 1 (Chk1) inhibitor capable of regulating the G2/M transition of the cell cycle in combination with the chemotherapeutic agent gemcitabine could be an effective treatment for ES  Polymerase 1 (PARP1), or the cell cycle, with inhibitors of Cyclin-Dependent Kinase (CDK) have been or are currently being studied . For exanchoring . Thus, at for ES . Anothert for ES . Differetrial.gov No NCT05046314), in combination with vincristine in patients with relapse or treatment-refractory ES. This clinical trial started after a first phase I clinical trial having demonstrated the non-toxicity of TK216 . YK-4–279 inhibits the interaction between EWS-FLI1 and RNA helicase A, which is known to enhance EWS-FLI1 activity . Indeed, pre-clinical studies have shown the interest of silencing EWS-FLI1 via RNA interference approaches (Because EWS-FLI1 i) is characteristic of 85% of ES cases, ii) regulates numerous oncogenes, and iii) that development of ES depends on this fusion protein, targeting EWS-FLI1 appears as an interesting approach in new therapies for ES. This section therefore focuses on approaches to target EWS-FLI1 that have led to clinical trials, by targeting its interactome or using RNA interference (see ). There activity , and is activity . Howeveractivity , rather proaches . The curA better understanding of the cellular and molecular mechanisms governing ES development has led to the emergence of targeted therapies, such as those targeting EWS-FLI1. In this context, new pre-clinical approaches are currently being studied, involving the targeting of EWS-FLI1 expression, epigenetic changes induced by this fusion protein, or the regulation of the EWS-FLI1 target expression. It has been shown that Proteolysis Targeting Chimeric Molecules (PROTACs) could be an interesting method for targeting EWS-FLI1 degradation. Indeed, one study demonstrated that polyubiquitination of EWS-FLI1 to Lysine 380 led to its degradation . PROTACs


The methods used in this study are the following: Luciferase reporter gene assays, immunoblot analysis, CRISPR-Cas9-mediated genome editing, and real-time quantitative PCR. In the beta-cell line HIT, overexpressed DLK increased c-FOS promoter activity twofold. Using 5′-,3′-promoter deletions, the promoter regions from − 348 to − 339 base pairs (bp) and from a − 284 to − 53 bp conferred basal activity, whereas the promoter region from − 711 to − 348 bp and from − 53 to + 48 bp mediated DLK responsiveness. Mutation of the cAMP response element within the promoter prevented the stimulatory effect of DLK. Treatment of HIT cells with KCl and the adenylate cyclase activator forskolin increased c-FOS promoter transcriptional activity ninefold. Since the transcriptional activity of those promoter fragments activated by KCl and forskolin was decreased by DLK, DLK might interfere with KCl/forskolin-induced signaling. In a newly generated, genome-edited HIT cell line lacking catalytically active DLK, c-Fos mRNA levels were reduced by 80% compared to the wild-type cell line. DLK increased c-FOS promoter activity but decreased stimulated transcriptional activity, suggesting that DLK fine-tunes c-FOS promoter-dependent gene transcription. Moreover, at least in HIT cells, DLK is required for FOS mRNA expression.The dual leucine zipper kinase (DLK) and the ubiquitously expressed transcription factor c-FOS have important roles in beta-cell proliferation and function. Some studies in neuronal cells suggest that DLK can influence c-FOS expression. Given that c-FOS is mainly regulated at the transcriptional level, the effect of DLK on The online version contains supplementary material available at 10.1007/s00210-023-02401-z. Both are highly conserved orthologues of DLK/Wallenda (App) promoter and increased amyloid-beta levels . The 5′- and 3′-deletions of − 711 c-fosLuc were generated by PCR using the primers listed in Tab. 1 of Supplementary Information (SAll expression vectors for DLK wild-type (WT) and its diverse mutants  have been described before  were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5% horse serum, 100 units/ml penicillin, and 100 µ/ml streptomycin /Cas9 method using the plasmid-based approach : Fos (Mm00487425_m1), Actb (Mm02619580_g1), and Hprt (Mm03024075_m1). The similarity between mouse and hamster Fos is 91.68%. For quantification, QuantStudio 7 Flex  was used. Expression of Dlk and Fos mRNA was normalized to the geometrical mean of murine Actb and Hprt using the formula 2−ΔΔCT.Total RNA from HIT WT and HIT-K185A cells was isolated by RNAzol RT  followed by a cleaning step  according to the manufacturer’s protocol. RNA concentration was determined by NanoDrop™ 2000c  and was reverse transcribed using the High Capacity cDNA reverse transcription kit . HIT WT cells and HIT-K185A cells were harvested in lysis buffer . Statistical analysis using Prism 8.0  was as stated in the legends.c-FOS promoter, a luciferase reporter gene under the control of the human c-FOS promoter (from –711 to + 48 bp) was transiently co-transfected into HIT cells with expression vectors for DLK wildtype (WT), the calcineurin binding-deficient mutant (V364A) and the ATP binding-deficient mutant (K185A). Both, DLK WT and DLK V364A enhanced c-FOS promoter transcriptional activity approximately twofold while DLK K185A had no effect , indicating that this promoter region represses c-FOS dependent gene transcription. Further 5′-deletion of the binding-site for the signal transducer and activator of transcription (STAT) transcription factors severely decreased c-FOS promoter activity and was not re-established by the deletion of additional bp  increased the basal promoter activity. Further 3′-deletions up to − 298 bp reduced the transcriptional activity and deletions beyond − 312 bp decreased the transcriptional activity by 95%  and calcium among others   Fig. . In the ion Fig. . The traion Fig. . To studity Fig. , suggestity Fig. . The traity Fig.  and D. Tc-Fos mRNA level, HIT cells were transiently transfected with the expression vectors for DLK WT and K185A. DLK WT but not K185A slightly increased c-Fos mRNA level . Thus, DLK enhances c-FOS activity through phosphorylation of the protein .To investigate whether DLK increases vel Fig. . Since tels Fig. . Preventels Fig. . These fels Fig. . In thision Fig. . In lineion Fig.  decrease60% Fig. , whereasThe transcription factor c-FOS is part of the activator protein (AP)-1 formed by heterodimers of members of the FOS and the JUN families. Both groups of proteins are basic region leucine zipper (bZip) proteins, heterodimerizing via their leucine zippers and binding to DNA-sites with the consensus motif 5′-TGA G/C TCA-3′ via their basic region   Kovács , c-FOS a) Kovács . For insc-FOS promoter activity: the kinase inhibited KCl/forskolin-induced transcription and increased basal promoter activity. These findings indicate that DLK interferes with KCl and/or forskolin-provoked signaling pathways. In HIT cells, KCl treatment resulting in membrane depolarization increases the intracellular calcium concentration. Forskolin might by increasing cAMP levels, activation of PKA, and phosphorylation of the voltage-dependent L-type calcium channel enhance the calcium concentration as well. Calcium then activates calcium-dependent enzymes among them calcineurin. DLK interacts with calcineurin via the phosphatase’s substrate recognition site thereby preventing the access of other calcineurin substrates to the phosphatase  and the calcium responsive transactivator CREST regulates calcium-dependent c-fos promoter activity Below is the link to the electronic supplementary material.