gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the gene loci. We discovered that the CTCF binding site located between and genes (CBS7/9) is critical for establishing and maintaining aberrant gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior genes. Consistent with the role of the CBS7/9 boundary in locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.
The upstream open reading frame (uORF) is a post-transcriptional regulatory element in the 5' untranslated region (5'UTR), which modulates the translation levels of main open reading frame (mORF). Earlier studies showed that disturbed uORF-mediated translation control can result in drastic changes in translation levels of mORF, leading to genetic disorders. To date, there has been no systematic investigation into the relationship between variations in patients and uORF status. Here, taking the advantage of several datasets, including gene ontology (GO) annotations and sequence feature analysis, we have examined uORF impacts in human transcripts. GO annotations indicate that uORF-containing genes are enriched in certain features such as oncogenes and transcription factors. Sequence feature analysis reveals that uORF is a factor for determination of the translation initiation site (TIS) in human transcripts. We show that genes with uORFs have lower protein expression levels than genes without uORFs in multiple human tissues. Moreover, by examining three disease variation databases, we identified uORF-altering mutations from a total of 3,740,225 variations, which are highly suspected to be associated with changed levels of gene expression. For an experimental validation, we found four mutations with significant effects on protein expression but with only modest changes in transcription levels. These findings will provide researchers on related diseases with new insights into the importance of known mutations.
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