Functional Interrogation of HOXA9 Regulome in Leukemia via Endogenous Reporter-based CRISPR/Cas9 screen
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PROVIDER: PRJNA590480 | ENA |
REPOSITORIES: ENA
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Project description:Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, including a vast majority of human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL). HOXA9 overexpression not only predicts poor patient diagnosis and outcome, but also plays critical roles in leukemia transformation and maintenance. Besides in few clinical cases as a translocation partner, HOXA9 gene generally shows lower frequencies of somatic mutations and is rarely amplified, indicating that transcriptional and epigenetic regulatory mechanisms might be predominantly utilized. However, our current understanding on the upstream regulation of HOXA9 in acute leukemia is still very limited, hindering development of effective methods to treat leukemia driven by aberrant HOXA9 expression. To mitigate these challenges, we have established the first HOXA9 endogenous reporter in an MLL-rearranged leukemia cell line to faithfully dictate HOXA9 expression by mCherry. By utilizing the reporter cell line and CRISPR/Cas9 genome editing tools, we have exploited comprehensive loss-of-function screenings to systematically discover novel transcription factors controlling HOXA9 expression. USF2 was discovered as a novel regulator of HOXA9 that directly binds to HOXA9’s promoter. USF2 depletion significantly down-regulated HOXA9 expression. Notably, in patients with MLL-rearranged B-ALL, transcriptional expression of USF2 and that of HOXA9 were positively correlated. In contrast, chromatin looping factor CTCF was dispensable for controlling HOXA9 expression in MLL-rearranged SEM cells. Collectively, these studies have functionally interrogated the regulome of HOXA9 and advance our understanding of the molecular regulation network in HOXA9-driven leukemia.
2020-10-30 | GSE140664 | GEO
Project description:Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.
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Project description:Mass Spectrometry analysis of the interactome of the oncoprotein NUP98-HOXA9
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Project description:Characterization of gene expression changes 72 hours after withdrawal of tamoxifen in murine hematopoietic progenitors transformed by Hoxa9-ER/Meis1 using RNAseq. In the presence of tamoxifen (4OHT), Hoxa9-ER localizes to the nucleus of cells allowing for transformation, while withdrawal of 4OHT (culture in EtOH) leads to loss of nuclear Hoxa9-ER. Loss of Hoxa9-ER leads to a decrease in cellular proliferation and differentiation along the myeloid lineage.
2014-06-11 | GSE58359 | GEO
Project description:Identification of the genome-wide binding sites of Hoxa9 and C/EBPα in a murine myeloblastic cell line transformed by Hoxa9/Meis1. Over 50% of Hoxa9 binding sites are co-bound by C/EBPα, providing mechanistic insight into the requirement of C/EBPα for Hoxa9-mediated leukemogenesis. Additionally, genome-wide occupancy of H3K4 monomethylation and H3K27 trimethylation provide additional information on the functionality of Hoxa9/C/EBPα cobound loci.
2014-06-11 | GSE58361 | GEO
Project description:Characterization of gene expression changes 72 hours after withdrawal of tamoxifen in murine hematopoietic progenitors transformed by Hoxa9-ER/Meis1 using RNAseq. In the presence of tamoxifen (4OHT), Hoxa9-ER localizes to the nucleus of cells allowing for transformation, while withdrawal of 4OHT (culture in EtOH) leads to loss of nuclear Hoxa9-ER. Loss of Hoxa9-ER leads to a decrease in cellular proliferation and differentiation along the myeloid lineage. Examination of gene expression by RNAseq in two conditions in biological replicates.
2014-06-11 | E-GEOD-58359 | biostudies-arrayexpress
Project description:Genetic studies, including genome-wide association studies, have identified many common variants that are associated with autoimmune diseases. Strikingly, in addition to being frequently observed in healthy individuals, a number of these variants are shared across diseases with diverse clinical presentations. This highlights the potential for improved autoimmune disease understanding which could be achieved by characterising the mechanism by which variants lead to increased risk of disease. Of particular interest is the potential for identifying novel drug targets or of repositioning drugs currently used in other diseases. The majority of autoimmune disease variants do not alter coding regions and it is often difficult to generate a plausible hypothetical mechanism by which variants affect disease-relevant genes and pathways. Given the interest in this area, considerable effort has been invested in developing and applying appropriate methodologies. Two of the most important technologies in this space include both low- and high-throughput genomic perturbation using the CRISPR/Cas9 system and massively parallel reporter assays. In this review, we introduce the field of autoimmune disease functional genomics and use numerous examples to demonstrate the recent and potential future impact of these technologies.
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Project description:Identification of the genome-wide binding sites of Hoxa9 and C/EBPα in a murine myeloblastic cell line transformed by Hoxa9/Meis1. Over 50% of Hoxa9 binding sites are co-bound by C/EBPα, providing mechanistic insight into the requirement of C/EBPα for Hoxa9-mediated leukemogenesis. Additionally, genome-wide occupancy of H3K4 monomethylation and H3K27 trimethylation provide additional information on the functionality of Hoxa9/C/EBPα cobound loci. Examination of two transcription factor binding sites and two histone modifications in a transformed cell line.
2014-06-11 | E-GEOD-58361 | biostudies-arrayexpress
Project description:Rearrangements involving the NUP98 gene resulting in fusions to several partner genes occur in acute myeloid leukemia and myelodysplastic syndromes. This study demonstrates that the second FG repeat domain of the NUP98 moiety of the NUP98-HOXA9 fusion protein is important for its cell immortalization and leukemogenesis activities. We demonstrate that NUP98-HOXA9 interacts with MLL via this FG repeat domain and that, in the absence of MLL, NUP98-HOXA9-induced cell immortalization and leukemogenesis are severely inhibited. Molecular analyses indicate that MLL is important for the recruitment of NUP98-HOXA9 to the HOXA locus and for NUP98-HOXA9-induced HOXA gene expression. Our data indicate that MLL is crucial for NUP98-HOXA9 leukemia initiation.
2017-02-28 | GSE93923 | GEO
Project description:Aberrant expression of homeobox transcription factor HOXA9 is a central component of the leukemogenic program driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid progenitor cells and pro-B cells leads to significant rearrangement of the epigenetic landscape with prominent emergence of cancer specific de novo enhancers. HOXA9 acts as a pioneer factor at the de novo enhancers and is required for recruitment of transcription factor CEBP/a and the histone H3K4 methyltransferase MLL3/MLL4 complex. HOXA9 function at the de novo enhancer is distinct from its physiological role at distal enhancers during normal hematopoietic development. Genetic deletion of MLL3/4 specifically affects the active enhancer signatures at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis. Our study reveals a previously uncharacterized role of HOXA9 and the MLL3/4 complex in leukemogenesis and provide mechanistic insights in epigenetic deregulation during malignant transformation.
2018-09-05 | GSE103447 | GEO