Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A genome-wide map of transcription start sites specific for acute promyelocytic leukemia reveals deregulation of full-length transcripts

ABSTRACT: Transcription start sites are the focal points of transcriptional regulation, where information from regulatory elements is integrated to stabilize initiation of transcription. In humans, most genes have more than one transcription start site, and these often exhibit different tissue specificity, serving as distinct regulatory frameworks for the same gene. Usage of such promoters can also result in differential gene function manifested on the protein level. Alternative promoter usage has been shown to be increased in several disease states, especially cancer. In this study, we have applied the nanoCAGE method to create a genome-wide map of TSS usage in sorted leukemic blasts from acute myeloid leukemia patients, and corresponding normal controls. We show that the nanoCAGE method can replace similar experiments made with microarrays in terms of expression, but also that it uniquely, allow for the identification of alternative promoter usage in cancer cells. We identify 2,162 putative promoters that are significantly differentially regulated between APL and controls. Interestingly, promoters whose usage is upregulated in APL have an increased propensity to be downstream alternative promoters, and conversely, the promoters producing the annotated longest gene variants are commonly downregulated in cancer. We show examples of genes with upregulated downstream promoters, and demonstrate protein domain loss that could contribute to leukemic induction and maintenance. In conclusion, we present the first genome wide promoterome study from rare purified human leukemic cells. nanoCAGE-seq of human acute myeloid leukemia samples vs. normal hematopoietic counterparts, both in replicates

ORGANISM(S): Homo sapiens

SUBMITTER: Johannes Waage

PROVIDER: E-GEOD-46561 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The origin of aberrant DNA methylation in cancer remains largely unknown. In this study, we elucidated the DNA methylome in primary Acute Promyelocytic Leukemia (APL) and the role of PML-RARa in establishing these patterns. APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34+ cells, promyelocytes and remission bone marrow. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score and Flt3-mutation status characterized methylation subtypes. Transcription factor binding sites, e.g. c-myc binding sites were associated with low methylation. SUZ12 and REST binding sites identified in embryonic stem cells were, however, preferentially DNA hypermethylated in APL. Unexpectedly, PML-RARa binding sites were also protected from aberrant DNA methylation in APL. In line, myeloid cells from pre-leukemic PML-RARa knock-in mice did not show altered DNA methylation and expression of PML-RARa in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. ATRA treatment of APL blasts did also not result in DNA methylation changes. These results suggest that aberrant DNA methylation is associated with leukemia phenotype but not required for PML-RARa-mediated initiation of leukemogenesis. We used Reduced Representation Bisulfite Sequencing (RRBS) to determine the genome-wide methylation signature of 18 primary APL patient samples. We then compared the APL methylation signature with methylation patterns found in CD34+ progenitor cells (n=4), promyelocytes (n=4) and remission bone marrow samples (n=8). Differentially methylated regions found in all three comparisons (APL vs. all three control specimens) were then further analyzed for genomic localization, variability and association with clinical parameters. Finally, the relationship between differentially methylated regions in APL and specific transcription factor binding sites was analyzed. For this purpose, ChiP-Sequencing of SUZ12 and REST was performed in primary APL patient blasts. To further determine the contribution of the leukemogenic transcription factor PML-RARa to methylation in APL, we also performed RRBS in pre-leukemic PML-RARa knock-in mice and hematopoetic progenitor cells retrovirally transduced with PML-RARa.

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A genome-wide map of transcription start sites specific for acute promyelocytic leukemia reveals deregulation of full-length transcripts

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