Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells

Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery

Integrative Genomic Analysis Reveals Aberrant Epigenetic Marks in MDS That Can Be Seen in Peripheral Blood Leucocytes

Abstract Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though global epigenetic alterations in MDS have not been well described. The transmission of these epigenetic marks during hematopoietic differentiation and their role in disease pathophysiology is also unknown. We first compared global methylation profiles of 8 bone marrow samples with peripheral leucocytes by using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. We observed a high correlation (r=0.89–0.96) of epigenetic marks between bone marrow and peripheral blood samples suggesting that a majority of epigenetic marks can be also be seen in differentiated cells. We subsequently compared peripheral blood leucocytes from 20 patients with MDS with 10 age-matched normal and anemic controls. Parallel gene expression analysis was performed using 37K oligo maskless arrays on cDNA from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Epigenetic profiling demonstrated two clusters of MDS based on similarity of aberrant epigenetic changes. Overall, there was a trend towards hypermethylation in MDS, albeit not statistically significant given the large number of relatively unchanged genes. Detailed analysis revealed several novel differentially methylated genes that had corresponding changes in gene expression, when MDS samples were compared to the controls with a low false discovery rate of analysis. Interesting genes getting hypomethylated and overexpressed included TNF superfamily member 9, granulocyte pep A, microsomal glutathione S-transferase, homeo box B4, mitochondrial RPL11, and others. Similarly, the set of genes that were getting hypermethylated with associated decrease in gene expression included Evi-1, DAPK, HOXB3, Protein Phosphatase 1, CEBPB, mutated in colorectal cancer (MCC), myeloid-lymphoid or mixed-lineage leukemia 5 (MLL5), plasminogen-related protein B, ovarian cancer related protein 1 (ORP1), and others. In addition, we did array-based comparative genome hybridization (aCGH) to look at exact genome copy number changes in these samples. We found changes that were not detectable by conventional karyotyping in all samples. Commonly seen alterations were del(14q11), del(20q11), del(5q13), del(8p23), amp(1q42), amp(5q11), amp(17q12), amp(19q13) and amp(7q22). Integrative analysis revealed sets of genes that were either silenced by methylation or deletion in different patients. Thus, our data demonstrates that promoter DNA methylation changes are an important phenomenon in MDS evolution, and are associated with changes in expression of genes playing important roles in cancer development and/or progression. We also show that previously unrecognizable changes in copy number exist in most patients with MDS. In addition, our work shows that whole genome methylation assays, even when done on peripheral blood leukocytes, can be used for potential biomarker studies in the diagnosis of MDS

Trisomy 21 enhances human fetal erythro-megakaryocytic development

Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes

MicroRNA expression in maturing murine megakaryocytes

MicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously