Genomic studies controvert the existence of the CUX1 p75 isoform

CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.

Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection

CD8+ T-cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T-cells expresses CXCR5, the chemokine receptor required for cell migration into B cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T-cells (fCD8s) frequencies, trafficking pattern and CXCR5 regulation. We show that, although HIV infection results in a marginal increase of fCD8s in LN, the majority of HIV-specific CD8+ T-cells are CXCR5 negative (non-fCD8s) (p<0.003). Mechanistic investigations using ATAC-seq showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprints analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In-vitro stimulation of non-fCD8s with recombinant TGF-β resulted in significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-β signaling as a viable strategy for increasing fCD8s frequencies in follicular areas of the LN where they are needed to eliminate HIV infected cells, with implications for HIV cure strategies.

Pax5 mediates the transcriptional activation of the CD81 gene

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between − 130 and − 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between − 130 and − 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

EPCO-12. STING IS SILENCED IN GBM BY PROMOTER METHYLATION THAT IS ESTABLISHED BY TISSUE OF ORIGIN

STING (Stimulator of interferon genes) is an initiator of endogenous antitumor immunity. Loss of STING expression has been observed in various cancer types. Here we describe a mechanism of STING suppression that is common to brain tumors, occurring through regional promoter hypermethylation (RPH) near its transcriptional start site. The RPH-mediated suppression of STING expression is selective to normal brain and neuroectoderm-derived tumors, but not present in most other tumor types and cells in the brain tumor microenvironment. A pan-cancer comparison revealed that STING RPH levels in tumors correlate with the respective tissue of origin, including tumors metastasizing to the brain. We demonstrate that STING RPH suppression in glioma cells may be reversed by treatment with decitabine, a DNA methyltransferase inhibitor that is used in the treatment of hematologic malignancies. Collectively, our results suggest that the epigenetic silencing of STING occurs early in brain development and may provide an immunosuppressive context for the genesis of brain tumors. Furthermore, our findings that STING suppression in brain tumor cells can be reversed by epigenetic modulation have significant therapeutic implications for the treatment of glioblastoma and potentially other STING-silenced, immunologically-cold cancers.

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

Purpose: Targeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. Results: We validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA.

The chromatin remodeler Ino80 mediates RNAPII pausing site determination

Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

HPA axis dysregulation is associated with differential methylation of CpG-sites in related genes

DNA methylation shifts in Hypothalamic–pituitary–adrenal (HPA) axis related genes is reported in psychiatric disorders including hypersexual disorder. This study, comprising 20 dexamethasone suppression test (DST) non-suppressors and 73 controls, examined the association between the HPA axis dysregulation, shifts in DNA methylation of HPA axis related genes and importantly, gene expression. Individuals with cortisol level ≥ 138 nmol/l, after the low dose (0.5 mg) dexamethasone suppression test (DST) were classified as non-suppressors. Genome-wide methylation pattern, measured in whole blood using the EPIC BeadChip, investigated CpG sites located within 2000 bp of the transcriptional start site of key HPA axis genes, i.e.: CRH, CRHBP, CRHR-1, CRHR-2, FKBP5 and NR3C1. Regression models including DNA methylation M-values and the binary outcome (DST non-suppression status) were performed. Gene transcripts with an abundance of differentially methylated CpG sites were identified with binomial tests. Pearson correlations and robust linear regressions were performed between CpG methylation and gene expression in two independent cohorts. Six of 76 CpG sites were significantly hypermethylated in DST non-suppressors (nominal P < 0.05), associated with genes CRH, CRHR1, CRHR2, FKBP5 and NR3C1. NR3C1 transcript AJ877169 showed statistically significant abundance of probes differentially methylated by DST non-suppression status and correlated with DST cortisol levels. Further, methylation levels of cg07733851 and cg27122725 were positively correlated with gene expression levels of the NR3C1 gene. Methylation levels of cg08636224 (FKBP5) correlated with baseline cortisol and gene expression. Our findings revealed that DNA methylation shifts are involved in the altered mechanism of the HPA axis suggesting that new epigenetic targets should be considered behind psychiatric disorders.

Cutting-free application of CRISPR-mediated endogenous gene activation in human induced pluripotent stem cell derived cardiomyocytes and engineered human myocardium

Background Imbalanced transcriptional networks characterize cardiomyocyte stress and result in cardiac remodelling. We hypothesize that re-establishing homeostatic gene networks in cardiomyocytes will prevent further tissue damage. To tackle this challenge, we applied CRISPR-based endogenous gene activation (CRISPRa) in vivo and in vitro. Methods We employ precision transcriptome editing tools based on CRISPR/Cas9 with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) to induce target gene expression by directing dCas9VPR to promoter regions by guide RNAs (gRNA). Results Homozygous CRISPRa hiPSC cell lines were generated by targeted integration of a CAG promoter driven dCas9VPR-T2A-tdTomato expression cassette into the AAVS1 locus by CRISPR/Cas9 editing and homology directed repair. Expression of dCas9VPR was evaluated by immunoblotting and co-expressed reporter fluorescence in spontaneously beating hiPSC-CM. We previously identified a crosstalk between WNT signalling and Krueppel-like factor 15 (KLF15) necessary for controlling cardiac homeostasis. We designed and tested 8 non-overlapping gRNAs in the –400 bp region upstream of the KLF15 transcriptional start site (TSS) and tested individual gRNA effectiveness for gene activation in HEK293T cells. Five gRNAs were identified inducing KLF15 transcript levels between 2- and 5-fold compared to non-targeted (NT) gRNA transfected cells (n=3 experiments). The single most effective gRNA was transduced by lentiviral particles into CRISPRa hiPSC-CM increasing KLF15 transcript levels to 1.5-fold compared to NT-gRNA control. Synergistic effects of 3 instead of single gRNA increased KLF15 transcript levels by 3-fold compared to controls (n≥3 experiments). We hypothesized that dCas9VPR expression could be harnessed as an additional option for gene dose titration and we generated hiPSC lines with enhanced dCas9VPR expression (v2.0). We observed up to 5-fold KLF15 gene activation when triple gRNA and v2.0 were combined (n≥4 experiments). Engineered human myocardium (EHM) was generated consisting of CRISPRa cardiomyocytes, fibroblasts and collagen and we observed similar contractility in 4-week cultured EHM suggesting innocuous dCas9VPR and gRNA expression. CRISPRa component expression was maintained over the entire culture period as evaluated by dCas9VPR immunoblotting and KLF15 transcriptional activation (1.4 fold, v1.0 CRISPRa hiPSC-CM, n≥8 tissues) indicating sustained gene activition. Conclusions Targeted gene activation with CRISPR/Cas9 is a precise and effective tool for transcriptional activation in hiPSC-CM. We observed titratability of gene activation by 1.) dCas9VPR expression levels and 2.) single versus multiple gRNA use. We furthermore elucidated general rules for effective gRNA targeting within the 5' TSS of genes of interest which confirmed a dependency of baseline gene activity as a limiting factor for endogenous gene activation. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG) - Collaborative Research Center 1002German Center for Cardiovascular Research (DZHK)

Interaction of hypoxia and nicotine acetylcholine receptor signaling network reveals a novel mechanism for lung adenocarcinoma progression in never-smokers

High incident of lung cancer among never smokers and their disease pathogenesis is an unexplained phenomenon. We have analyzed 1727 lung cancer patient data to understand the impact of smoking on overall survival of lung cancer patients and have observed a difference of only 47 days between smokers and never smokers in adenocarcinoma patients suggesting that the disease is equally fatal in never-smokers irrespective of gender. In this study, we have investigated the possible collaboration between the nAChR and hypoxia signaling pathway to elucidate a mechanism of disease progression in never-smokers. We report a previously unidentified increase in both acetylcholine and nAChR-α7 levels in non small cell lung cancer cells in hypoxia. Similar increase in ubiquitously expressed nAChR-α7 transcripts was also observed in other cancer lines. A direct binding of HIF-1α with the hypoxia response element (HRE) present at -48 position preceding the transcriptional start site in nAChR-α7 promoter region was established. Significantly, the increased acetylcholine levels in hypoxia drove a feedback loop via modulation of PI3K/AKT pathway to stabilize HIF-1α in hypoxia. Further, Bungarotoxin, an antagonist of nAChR-α7 significantly reversed hypoxia mediated metastasis and induction of HIF-1α in these cells. Our study gives a plausible explanation for the equally worse prognosis of lung adenocarcinoma in never-smokers wherein the nAChR signaling is enhanced in hypoxia by acetylcholine, in the absence of nicotine.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.