DNA Methylation of CDKN2A-B suppressor genes: mechanism and consequences

Tumor suppressor genes, p15INK4B and p16INK4A p14ARF, are the most frequently silenced genes in human cancers (Kim and Sharpless, 2006) because they represent a biological barrier to transformation (Skalska et al., 2013). Frequently, silencing is the result of DNA methylation of promoter regions, and to date, the precise mechanism is not known. Here, we present evidence that DNA methylation is associated with DNA damage caused by collisions between DNA and RNA polymerases near the transcription start sites. Inhibition of replication or transcription greatly reduces DNA damage and ultimately, local CpG methylation. This mechanism generates in vivo many variants (alleles) of p15INK4B p16INK4A and p14ARF genes, which differ only by the occurrence of methylated CpG within the sequence (epialleles). If some epialleles are positively selected, we expect to find them amplified in a complex population of proliferating cells. We have tested this notion by systematically investigating: 1. the configuration of methylated CpGs in each p16INK4A sequenced molecule (epiallele) and; 2. the frequency of families of epialleles during the progression of acute myeloid leukemia (AML) in cells from the bone marrow or the blood of three patients. The relevant results can be summarized as follows: 1. The epialleles, defined by unique methylation profiles, were stable and patient-specific. 2. The epialleles displayed gene specific methylation signatures and were somatically inherited; 3. Some epialleles were eliminated by the demethylating therapy (5-AzadC, Vidaza®), others were resistant or amplified by the demethylating drug and their frequency increased before and during the disease relapse; 4. The presence of high levels of families of epialleles after the therapy predicted the chemoresistance; 5. The epialleles of another suppressor, p21/WAF, in the same patients, did not evolve and remained stable throughout the progression of the disease; 6. Specific p16INK4A gene CpGs function as “seeds” for further methylation. Collectively, these data indicate: 1. An epigenetic mechanism driving the evolution of AML by dynamic methylation of alleles of p16INK4A suppressor gene; 2. The methylation changes during AML progression may be facilitated by DNA damage; 3. The distribution and the frequency of families of p16INK4A epialleles overtime accurately describe the evolution of myeloid acute leukemia

Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

Abstract The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30–90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT

Methylation of the Suppressor Gene p16INK4a: Mechanism and Consequences

Tumor suppressor genes in the CDKN2A/B locus (p15INK4b, p16INK4a, and p14ARF) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence

Binding of Carbonic Anhydrase IX to 45S rDNA Genes Is Prevented by Exportin-1 in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45S rDNA gene promoters

Pharmacological inhibition of LSD1 triggers myeloid differentiation by targeting GSE1 oncogenic functions in AML

The histone demethylase LSD1 is over-expressed in hematological tumors and has emerged as a promising target for anticancer treatment, so that several LSD1 inhibitors are under development and testing, in preclinical and clinical settings. However, the complete understanding of their complex mechanism of action is still unreached. Here, we unraveled a novel mode of action of the LSD1 inhibitors MC2580 and DDP-38003, showing that they can induce differentiation of AML cells through the downregulation of the chromatin protein GSE1. Analysis of the phenotypic effects of GSE1 depletion in NB4 cells showed a strong decrease of cell viability in vitro and of tumor growth in vivo. Mechanistically, we found that a set of genes associated with immune response and cytokine-signaling pathways are upregulated by LSD1 inhibitors through GSE1-protein reduction and that LSD1 and GSE1 colocalize at promoters of a subset of these genes at the basal state, enforcing their transcriptional silencing. Moreover, we show that LSD1 inhibitors lead to the reduced binding of GSE1 to these promoters, activating transcriptional programs that trigger myeloid differentiation. Our study offers new insights into GSE1 as a novel therapeutic target for AML.</p