Transcription Start Sites Of Genes Research Articles

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

Genome-wide R-loop analysis defines unique roles for DDX5, XRN2, and PRMT5 in DNA/RNA hybrid resolution

DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation.

The Dynamics of Transcriptional Activation by Hepatic Reprogramming Factors

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.

Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays.

In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.

Characterization of microRNA genes from Pigeonpea (Cajanus cajan L.) and understanding their involvement in drought stress

MicroRNAs (miRNA) are non-coding 20-24 nucleotide long RNAs regulating gene expression. In this study, we have characterized and analysed expression of miRNAs in Pigeonpea by using bioinformatics and experimental tools. We identified 116 miRNAs belonging to 32 phylogenetic families. Further, transcription start sites of miRNA genes revealed abundance and unique arrangement of adenine at +1 and thymine at -1 position. Promoter analysis exhibited presence of 19 most prevalent motifs which comprises majorly of TATA box and MYC domains. In total, 252 miRNA-targets were identified and found to be involved in various developmental processes and stress responses. Moreover, genome-wide localization studies demonstrated clustering of cca-miRNA 395 and 169 genes. The tandem and segmental duplication events were observed suggesting miRNA genes have been originated parallelly with protein coding genes. The expression analysis revealed induction of cca-miR169a, 398a and 408 miRNAs under drought stress highlighting their involvement. Conversely, down-regulation of their putative targets (NFYA, SOD, and UCLA, respectively) confirmed regulatory role of miRNAs in their expression emphasising the negative relationship between these miRNAs and targets in Pigeonpea. This study reports vast repertoire of miRNA genes which further can be experimentally characterized to elucidate their functions in various biological processes and can be recommended for Pigeonpea improvement programs.

Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways.

Modification of the cancer-associated chromatin landscape in response to therapeutic DNA damage influences gene expression and contributes to cell fate. The central histone mark H2Bub1 results from addition of a single ubiquitin on lysine 120 of histone H2B and is an important regulator of gene expression. Following treatment with a platinum-based chemotherapeutic, there is a reduction in global levels of H2Bub1 accompanied by an increase in levels of the tumor suppressor p53. Although total H2Bub1 decreases following DNA damage, H2Bub1 is enriched downstream of transcription start sites of specific genes. Gene-specific H2Bub1 enrichment was observed at a defined group of genes that clustered into cancer-related pathways and correlated with increased gene expression. H2Bub1-enriched genes encompassed fifteen p53 target genes including PPM1D, BTG2, PLK2, MDM2, CDKN1A and BBC3, genes related to ERK/MAPK signalling, those participating in nucleotide excision repair including XPC, and genes involved in the immune response and platinum drug resistance including POLH. Enrichment of H2Bub1 at key cancer-related genes may function to regulate gene expression and influence the cellular response to therapeutic DNA damage.

SAT-446 Transcriptome Comparison of a Natural T3 Regulated Process and a Treatment with T3

Thyroid hormones (TH) act mainly on the expression of the genome. It is well accepted that differential gene expression following treatment with TH recapitulates the expression profiles taking place during natural processes induced by these hormones. Although both processes seem to correlate at the scale of a few target genes, this hasn’t been addressed in a systematic manner. Our objectives were first to compare transcriptome variations after TH treatment with transcriptome variations during a TH controlled natural process and second to evaluate the proportion of the direct TH-response. The measurement of gene expression at genome scale (transcriptome) is obtained by sequencing all the messenger RNAs (RNA-seq). Direct response was sorted out by linking the transcription start sites of target genes using RNA-PET analysis with TR binding sites mapping using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Indeed, ChIA-PET not only allow to map TR binding sites but also the physical interactions between them and transcription start sites of regulated genes. Our model is one of the striking developmental processes orchestrated by TH: amphibian metamorphosis. Tadpole transformation is marked by dramatic changes including de novo morphogenesis (limb), tissue remodelling (brain, intestine...) and organ resorption through apoptosis (tail). These changes involve cascades of gene regulation initiated by TH and their receptors. Because metamorphosis has close and interesting parallels with the perinatal period in mammals (including human), metamorphosis is thus an attractive model to analyze in a physiological context, the functions and mechanisms of action of TH. Here, we have focused on one Xenopus tropicalis organs, the tail fin skin which will disappear through cell death. We have compared natural metamorphosis with 24h of 10nM triiodothyronine (T3) treatment. We were able to observe several differences between T3 treatment and natural development. First, the genes regulated by T3 only correspond to a proportion of genes differentially expressed during metamorphosis. Second, T3-response genes start to be regulated well before tail regression (several days). Finally, T3-direct target genes represent a few percent of all the genes differentially expressed during tail regression. In conclusion, the comparison of transcriptomes of natural and induced metamorphosis allow us to reach a more precise understanding of TH action

Transcription Factors and DNA Play Hide and Seek.

Transcription factors (TFs) bind to specific DNA motifs to regulate the expression of target genes. To reach their binding sites, TFs diffuse in 3D and perform local motions such as 1D sliding, hopping, or intersegmental transfer. TF-DNA interactions depend on multiple parameters, such as the chromatin environment, TF partitioning into distinct subcellular regions, and cooperativity with other DNA-binding proteins. In this review, how current understanding of the search process has initially been shaped by prokaryotic studies is discussed, as well as what is known about the parameters regulating TF search efficiency in the context of the complex eukaryotic chromatin landscape.

Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies.

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

Alveolar Macrophage Chromatin Is Modified to Orchestrate Host Response to Mycobacterium bovis Infection.

Bovine tuberculosis is caused by infection with Mycobacterium bovis, which can also cause disease in a range of other mammals, including humans. Alveolar macrophages are the key immune effector cells that first encounter M. bovis and how the macrophage epigenome responds to mycobacterial pathogens is currently not well understood. Here, we have used chromatin immunoprecipitation sequencing (ChIP-seq), RNA-seq and miRNA-seq to examine the effect of M. bovis infection on the bovine alveolar macrophage (bAM) epigenome. We show that H3K4me3 is more prevalent, at a genome-wide level, in chromatin from M. bovis-infected bAM compared to control non-infected bAM; this was particularly evident at the transcriptional start sites of genes that determine programmed macrophage responses to mycobacterial infection (e.g. M1/M2 macrophage polarisation). This pattern was also supported by the distribution of RNA Polymerase II (Pol II) ChIP-seq results, which highlighted significantly increased transcriptional activity at genes demarcated by permissive chromatin. Identification of these genes enabled integration of high-density genome-wide association study (GWAS) data, which revealed genomic regions associated with resilience to infection with M. bovis in cattle. Through integration of these data, we show that bAM transcriptional reprogramming occurs through differential distribution of H3K4me3 and Pol II at key immune genes. Furthermore, this subset of genes can be used to prioritise genomic variants from a relevant GWAS data set.

Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains

BackgroundCTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive.ResultsWe carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation.ConclusionsDynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.

A dual role of dLsd1 in oogenesis: regulating developmental genes and repressing transposons.

The histone demethylase LSD1 is a key chromatin regulator that is often deregulated in cancer. Its ortholog, dLsd1 plays a crucial role in Drosophila oogenesis; however, our knowledge of dLsd1 function is insufficient to explain its role in the ovary. Here, we have performed genome-wide analysis of dLsd1 binding in the ovary, and we document that dLsd1 is preferentially associated to the transcription start site of developmental genes. We uncovered an unanticipated interplay between dLsd1 and the GATA transcription factor Serpent and we report an unexpected role for Serpent in oogenesis. Besides, our transcriptomic data show that reducing dLsd1 levels results in ectopic transposable elements (TE) expression correlated with changes in H3K4me2 and H3K9me2 at TE loci. In addition, our results suggest that dLsd1 is required for Piwi dependent TE silencing. Hence, we propose that dLsd1 plays crucial roles in establishing specific gene expression programs and in repressing transposons during oogenesis.

2 The landscape of accessible chromatin in bovine oocytes and early embryos

Chromatin reorganization governs gene expression regulation during pre-implantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We analysed pools of 20 germinal vesicles or MII oocytes or 2-, 4-, 8-, 16-cell, morula, and blastocyst stage invitro-produced embryos. We conducted ATAC-seq on six pools for each stage and an additional four pools of invivo-derived morula and blastocysts and six replicates using individual Day 14 elongating embryos. We obtained ~110 million paired end reads uniquely mapped to the bovine reference genome for each stage. Hierarchical clustering, t-distributed stochastic neighbour embedding, and principal component analysis showed four distinct patterns for open chromatin status: (1) low accessibility in germinal vesicles and MII oocytes and in 2- and 4-cell embryos; (2) significantly elevated accessibility in 8-cell, 16-cell, and morula embryos; (3) less accessibility in blastocysts; and (4) extremely high accessibility in elongating embryos. This dynamic and sequential chromatin remodelling is consistent with transcription activation during the bovine minor embryonic genome activation from fertilization to 4-cell, major embryonic genome activation at 8-cell, first differentiation at blastocyst and drastic transcription initiation for embryo elongation. Genome-wide characteristics of accessible chromatin showed (1) accessible chromatin near the transcription start sites of active genes and CpG-rich promoters; (2) widespread accessible chromatin regions extensively overlapped with transposable elements; (3) distal peaks preferentially enriched for repeats including LINE, SINE, and LTR from 8-cell to morula embryos, especially for LTR, whereas enrichment in simple repeats were found from oocytes to 4-cell and in elongating embryos; and (4) highly stage-specific transcription factor motifs in distal peaks were unveiled. By integrating the maps of chromatin accessibility with bovine embryo transcriptomes and DNA methylomes, we found promoter accessibility and DNA methylation in bovine embryos correlated with both gene activities and CpG densities. Most importantly, we constructed the regulatory networks of stage-specific expressed genes and stage-specific activated genes with three omics datasets in bovine early embryos and revealed conserved and distinctive transcriptional regulatory networks between invivo- and invitro-derived embryos. This comprehensive analysis revealed critical features of the chromatin landscape and epigenetic reprogramming during bovine early embryo development.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

SummaryHow repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Natural human genetic variation determines basal and inducible expression of PM20D1, an obesity-associated gene

PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene's transcription start site (TSS) in human but not mouse adipocytes. The -4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.

Co-Localization of DNA i-Motif-Forming Sequences and 5-Hydroxymethyl-cytosines in Human Embryonic Stem Cells.

G-quadruplexes (G4s) and i-motifs (iMs) are tetraplex DNA structures. Sequences capable of forming G4/iMs are abundant near the transcription start sites (TSS) of several genes. G4/iMs affect gene expression in vitro. Depending on the gene, the presence of G4/iMs can enhance or suppress expression, making it challenging to discern the underlying mechanism by which they operate. Factors affecting G4/iM structures can provide additional insight into their mechanism of regulation. One such factor is epigenetic modification. The 5-hydroxymethylated cytosines (5hmCs) are epigenetic modifications that occur abundantly in human embryonic stem cells (hESC). The 5hmCs, like G4/iMs, are known to participate in gene regulation and are also enriched near the TSS. We investigated genomic co-localization to assess the possibility that these two elements may play an interdependent role in regulating genes in hESC. Our results indicate that amongst 15,760 G4/iM-forming locations, only 15% have 5hmCs associated with them. A detailed analysis of G4/iM-forming locations enriched in 5hmC indicates that most of these locations are in genes that are associated with cell differentiation, proliferation, apoptosis and embryogenesis. The library generated from our analysis is an important resource for investigators exploring the interdependence of these DNA features in regulating expression of selected genes in hESC.

SREBP1-dependent de novo fatty acid synthesis gene expression is elevated in malignant melanoma and represents a cellular survival trait

de novo fatty acid biosynthesis (DNFA) is a hallmark adaptation of many cancers that supports survival, proliferation, and metastasis. Here we elucidate previously unexplored aspects of transcription regulation and clinical relevance of DNFA in cancers. We show that elevated expression of DNFA genes is characteristic of many tumor types and correlates with poor prognosis, especially in melanomas. Elevated DNFA gene expression depends on the SREBP1 transcription factor in multiple melanoma cell lines. SREBP1 predominantly binds to the transcription start sites of DNFA genes, regulating their expression by recruiting RNA polymerase II to promoters for productive transcription elongation. We find that SREBP1-regulated DNFA represents a survival trait in melanoma cells, regardless of proliferative state and oncogenic mutation status. Indeed, malignant melanoma cells exhibit elevated DNFA gene expression after the BRAF/MEK signaling pathway is blocked (e.g. by BRAF inhibitors), and DNFA expression remains higher in melanoma cells resistant to vemurafenib treatment than in untreated cells. Accordingly, DNFA pathway inhibition, whether by direct targeting of SREBP1 with antisense oligonucleotides, or through combinatorial effects of multiple DNFA enzyme inhibitors, exerts potent cytotoxic effects on both BRAFi-sensitive and -resistant melanoma cells. Altogether, these results implicate SREBP1 and DNFA enzymes as enticing therapeutic targets in melanomas.

Abstract 4331: Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling

Abstract To develop novel and improved cancer therapies, we aim to target the epigenome, which is often deregulated in cancer. We identified the splicing factor SF3B1 as a potential epigenetic regulator in a whole genome siRNA screen for reactivation of aberrantly silenced genes alone or in synergy with the DNMT inhibitor Decitabine (DAC). The screen was done in YB5 colon cancer cells which contain a methylated and silenced CMV promoter driving GFP expression, and confirmed in a similar model in HCT116, another colon cancer cell line. SF3B1 has been studied in splicing, but little is known about its effects on gene regulation outside of this function, prompting us to further investigate this. We validated the screen findings by siRNA (siSF3B1) and inhibition of SF3B1 with Pladienolide B (PB). We then performed RNA-Seq and Reduced Representation Bisulfite Sequencing (RRBS) in cells treated with siSF3B1 and DAC, and we performed ChIP-Seq to assess SF3B1 binding. By flow cytometry, we measured a ~7% and ~5% induction of GFP in YB5 cells after siSF3B1 and DAC, respectively. The combination treatment caused a synergistic increase to ~15% GFP positive cells (p < 0.001). siSF3B1 led to 423 up and 338 downregulated genes, while DAC induced 430 up and 135 downregulated genes. With the combination, there was activation of 1190 genes and downregulation of 904 genes (fold change > 2, FDR < 0.1). There were 695, 119, 1584 genes alternatively spliced following siSF3B1, DAC, and the combination treatment, respectively, but there was no significant overlap with the regulated genes (p < 0.05), suggesting a distinct mechanism for gene expression regulation. There were two major subsets of siSF3B1 upregulated genes. A set of genes had low promoter methylation (0-20% methylation) and were p53 activation targets such as CDKN1A and GADD45A. The other set of genes had high promoter methylation (80-100% methylation) and promoters containing TATA-box motifs. RRBS showed global DNA hypomethylation after DAC treatment as expected, with an average methylation of 76.2% compared to 84.7% methylation in siControl cells. Further demethylation occurred in the combination treated cells, which had an average methylation of 72.7% (p<0.001). ChIP-Seq showed differential binding of SF3B1 at the transcription start sites (TSSs) of genes based on their expression. SF3B1 was depleted at TSSs of expressed genes and enriched at nonexpressed genes, resembling the binding pattern of histone H3. Finally, SF3B1 inhibition with PB alone and with DAC also induced reactivation of gene expression and altered DNA methylation. Together, these findings suggest that SF3B1 may be playing a role in gene regulation outside of its role in splicing. In cancer, SF3B1 transcriptional target genes are potentially tumor suppressor genes and upon knockdown or inhibition of SF3B1, their expression is reactivated leading to antitumor effects. Citation Format: Sandra Deliard, Yasuyuki Okamoto, Jozef Madzo, Somnath Pandey, Jaroslav Jelinek, Jean-Pierre Issa. Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4331.

Abstract 953: JARID1 family inhibitor reduces generation of drug-resistant EGFR mutation-positive lung cancer cells

Abstract Epigenetic alteration contributes to tumor initiation, progression and invasion. Epigenetic machinery has become a major focus for the new development of molecular targeted cancer therapeutics. H3K4 trimethylation (H3K4me3) is epigenetic marks which exist surrounding transcription start site of active genes. JARID1 family is demethylase of H3K4me2/me3. JARID1a and JARID1b have been suggested, especially to have oncogenic properties and associations with anti-cancer drug resistance. We previously showed that JARID1 family inhibitor, 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), recovers sensitivity of drug-resistant lung cancer cells. Here we examined whether PBIT affect the generation of drug resistant cells. A recent report revealed that “secretomes” induced by targeted therapies paradoxically stimulate the outgrowth of drug-resistant subpopulation, which are driven by downregulation of transcription factor FRA1. We confirmed that EGFR mutation-positive PC9 and H1975 cells treated with EGFR-TKIs produced the secretomes that promoted EGFR-TKIs-resistant cell growth. Interestingly, PC9 and H1975 cells pre-treated with 1 μM PBIT did not produce such tumor-promoting secretomes. IGF-1 was upregulated in the secretomes by the EGFR-TKI treatment, but the upregulation was disrupted by the 1 μM PBIT pre-treatment. Immunoblot assay showed that PC9 and H1975 decreased expression of FRA1 after the treatment with EGFR-TKIs, while PC9 and H1975 pre-treated with PBIT mitigated such decrease in FRA1 expression after treatment with EGFR-TKIs. Consistently, chromatin immunoprecipitation assay showed that H3K4me3 at FRA1 promoter region was decreased after treatment with gefitinib in PC9, while the decrease did not occur in PC9 pre-treated with PBIT. To investigate whether PBIT inhibits generation of drug resistant cells, we treated 100 small groups (100 cells each) of cells with EGFR-TKIs with or without 1 μM PBIT for 4 weeks. EGFR-TKI-resistant cells were significantly reduced in combination with PBIT. Of note, 1 μM PBIT alone did not affect clonogenic cell growth in soft agar colony formation assay. Immunoblot assay showed that expression of IGF-1-AKT-S6 pathway molecules in TKI-resistant cells was mitigated by the PBIT combination therapy. These results indicate that PBIT reduces EGFR-TKI-induced secretomes containing IGF-1 as a relevant mediator by preventing downregulation of FRA1 expression via prevention of decrease of active marker H3K4me3 at FRA1 promoter regions. JARID1 inhibitor may have potential to overcome EGFR-TKI-resistance in lung cancer, especially via off-target mechanisms. Citation Format: Shin Ariga, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Hirotoshi Dosaka-Akita. JARID1 family inhibitor reduces generation of drug-resistant EGFR mutation-positive lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 953.

Abstract 4032: The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma

Abstract The refractory and relapsed disease is currently the challenge in Hodgkin Lymphoma (HL) treatment. There is no specific therapy but rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the HL molecular biology and the screening for therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by Rel-B and NIK, to survive. Its constitutive activation seems to be involved in the refractory and relapsed disease. To determine the specific Rel-B target genes we performed ChIP-Seq in UH01 and L1236 human HL cell lines for Rel-A, Rel-B, cRel, p50 and p52. We found 29,414 Rel-B peaks genome-wide distributed. To further identify genes with higher probabilities of being directly regulated by Rel-A, Rel-B, and/or cRel we concentrated on the peaks that localized within a +/- 2 kb window, relative to the gene transcription start site. The +/- 2 kb Rel-B peaks were distributed on 4,509 genes; meanwhile the +/- 2 kb cRel peaks were on 1,994 genes and the Rel-A peaks were on 830 genes. Out of the 4,509 genes with the Rel-B peaks, only 6% overlapped with the Rel-A peaks and 11% with the cRel peaks. The Rel factors distribution showed a Rel-B DNA-binding hierarchy in HL. The ChIP-Seq data were merged with gene expression arrays results. The gene expression assays were analyzed by comparing the signal from cells infected with noninduced shRNAs for the Rel factors to that from cells infected with induced shRNAs. Differentially expressed genes were identified via ANOVA analysis. Genes that were downregulated more than 2-fold with a p<0.001 were considered significant. We found that the Rel-B downstream controlled gene set was enriched for cell cycle and cell death regulation and DNA damage and repair signatures, among others. One of the exclusively Rel-B target genes was BCL2. We showed that exogenous BCL2 was able to partially rescue HL cells from dying in response to Re-B depletion. We also found that BCL2 was useful as a prognosis marker in terms of overall survival in a cohort of 96 HL patients [Log Rank Test (p=0.002)]. In this retrospective study [follow up period 47,4 (6-136) months], BCL2 was able to identify at diagnosis patients that were refractory to conventional first line treatment and patients that relapsed. The HL cell lines U-H01, L1236, SUPDHL1, KM-H2 and L540 were sensitive to the BCL2 inhibitor venetoclax. The HDM-2 cell line, which does not express BCL2, was a good control of target specificity since it did not die in response to venetoclax. In summary, we found that the alternative NFkB pathway plays an important role in the refractory and relapsed Hodgkin disease, being BCL2 a key downstream target. BCL2 performed well as a prognosis marker identifying refractory patients and those that would relapse. We propose BCL2 expression assessment in the lymph node biopsy at diagnosis and BCL2 as a directed therapy. Citation Format: Angélica María Gamboa-Cedeño, Mariángeles castillo, Victoria Otero, Natalia Schutz, Dorotea Fantl, Federico Jauk Vitali, Hernán García Rivello, Myriam Nuñez, Stella Maris Ranuncolo. The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4032.