ABSTRACT
Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis.
Subject(s)
Cell Cycle Proteins , Chromatin , Chromosomal Proteins, Non-Histone , Cohesins , Leukemia, Myeloid, Acute , Mutation , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Mutation/genetics , Hematopoietic Stem Cells/metabolism , Cell Differentiation/genetics , Gene Expression Regulation, Leukemic , Antigens, Nuclear/metabolism , Antigens, Nuclear/genetics , Nuclear ProteinsABSTRACT
Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.
Subject(s)
Apoptosis , Cell Differentiation , Chromatin/metabolism , Histones/metabolism , Lysine/metabolism , Myeloid Cells/metabolism , Acetylation , Animals , Cells, Cultured , Chromatin/genetics , Epigenesis, Genetic , Humans , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/cytology , Protein Processing, Post-Translational , Transcription, GeneticABSTRACT
Chinese Hamster Ovary (CHO) cells are the preferred cell line for production of biopharmaceuticals. These cells are capable to grow without serum supplementation, but drastic changes in their phenotype occur during adaptation to protein-free growth, which typically include the change to a suspension phenotype with reduced growth rate. A possible approach to understand this transformation, with the intention to counteract the reduction in growth by targeted supplementation of protein-free media, is gene expression profiling. The increasing availability of genome-scale data for CHO now facilitates quests for a better understanding of metabolic pathways and gene networks. So far, systematic large-scale expression profiling in CHO cells by microarray was limited due to lack of publicly available array designs and limitations of alternative approaches. Based on the recent release of CHO and Chinese Hamster genome sequences, including an annotated RefSeq genome, we have constructed a publicly available microarray design for effective genome-scale expression profiling. The design employed microarray probes optimized for uniformity, sensitivity, and specificity, with probe properties computed using the latest thermodynamic models. We validated the platform in an analysis of gene expression changes in response to serum-free adaptation. The observed effects on the lipid metabolism as well as on nucleotide synthesis were used to successfully select media supplements that were able to increase growth rate.