Casirati et al. Epitope Editing of Hematopoietic Stem Cells
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ABSTRACT: Data for the manuscript Casirati et al. "Epitope Editing of Hematopoietic Stem Cells Enables Adoptive Immunotherapies for Acute Myeloid Leukemia"
Project description:A Scalable Epitope Tagging Approach for High Throughput ChIP-seq Analysis ChIP-seq comparison between CRISPR editing cells using epitope antibody and non-editing cells using endogeneous TF antibody
Project description:Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. We found that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification was associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cell (HSC) function and AML, such as HoxA cluster genes. In vivo, HMGN1 overexpression was linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperated with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieved the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.
Project description:Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly.
Project description:In this project, we studied how the nucleosome binding protein HMGN1 affects myeloid differentiation and hematopoietic and leukemia stem cell activity.
Project description:In this project, we studied how the nucleosome binding protein HMGN1 affects myeloid differentiation and hematopoietic and leukemia stem cell activity.
Project description:In this project, we studied how the nucleosome binding protein HMGN1 affects myeloid differentiation and hematopoietic and leukemia stem cell activity
Project description:Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly. 3 old mice and 5 young mice were assayed
Project description:Acute myeloid leukemia (AML) with rearrangement of the mixed-lineage leukemia (MLL) gene are the most aggressive hematopoietic malignancies. Previous studies demonstrated the distribution of several epigenetic modifications including H3K9me3, H3K79me2, H3K36me3, H3K4me3 and H3K27me3, in MLL-AF9 transformed murine cells. Here, we examined the H3K9me3 distribution in c-Kit+ cells (enriched with stem/progenitor cells) from both MLL-AF9 transformed murine cells in parallel with control wild-type cells, and found an overall lower distribution of H3K9me3 in leukemia stem cells than normal hematopoietic stem/progenitor cells.
Project description:To identify direct transcriptional targets of RFX6, we performed chromatin immunoprecipitation of HA epitope tagged RFX6 followed by massively parallel DNA sequencing (ChIP-seq). Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Pluripotent cells were then differentiated into PDX1+RFX6+ pancreatic progenitors and endogenous RFX6-HA was immunoprecipitated with an anti-HA antibody. To eliminate background signal caused by non-specific antibody binding, a control experiment using wild-type H9 hESC was performed in parallel.