Biology subjects

DeKoter, R. P.

Publications and source records attributed to DeKoter, R. P..

9 recordsLinked to original sources

bioRxiv2025

GATA2 Induces a Stem Cell-Like Transcriptional Program in Macrophages that Promotes Atherogenesis.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden necrotic macrophages within blood vessels walls. GATA2 is a normally hematopoietic transcription factor which in the bone marrow helps maintain the proliferative, non-differentiated phenotype of hematopoietic progenitors. Unexpectedly, GATA2 is upregulated in macrophages within atherosclerotic plaque, where it plays an unknown role in disease progression. Although GATA2 can be expressed from two promoters, we determined that the atherogenic stimuli oxidized low-density lipoprotein and TNF induce GATA2 expression via the internal (IG) GATA2 promoter, with GATA2 transcription initiated by the transcription factors NF-{kappa}B, STAT1, and the aryl hydrocarbon receptor. GATA2 had a divergent effect on promoter activity, with GATA2 upregulating genes associated with stem cell maintenance, proliferation, reactive oxygen species production, and migration, while downregulating genes central to macrophage function including those for cholesterol efflux, pathogen phagocytosis, and for the efferocytosis of apoptotic cells. Consequentially, GATA2-expressing macrophages had a pro-atherogenic phenotype typified by an invasive phenotype, poor cholesterol efflux, and impaired phagocytosis and efferocytosis. These results indicate that GATA2 upregulation induces an immature, stem cell-like phenotype in atheroma macrophages, thereby promoting plaque cellularity while compromising atheroprotective mechanisms such as cholesterol clearance and apoptotic cell removal.

immunology↗

bioRxiv2024

T505A variant of ETV5 promotes proliferation of precursor B cells in a mouse model of acute lymphoblastic leukemia

Precursor B cell acute lymphoblastic leukemia (pre-B-ALL) arises as a result of precursor B cells acquiring driver mutations that lead to arrested differentiation and increased proliferation. Identification of driver mutations and understanding their biological function is critical to understanding pre-B-ALL development and advancing disease treatment. Using a mouse model of pre-B-ALL driven by deletion of genes encoding the related E26-transformation-specific (ETS) transcription factors PU.1 and Spi-B, we performed whole exome sequencing to identify secondary driver mutations. We identified recurrent variants in E26 transformation-specific transcription variant 5 (ETV5) resulting in R392P, V444I, and T505A amino acid changes. We found that the R392P and V444I variants altered the ability of ETV5 to bind to DNA using electrophoretic mobility shift assay. R392P and V444I variants did not activate a Dual-Specificity-Phosphatase 6 (DUSP6) reporter. In contrast, T505A ETV5 could interact with DNA and activate the DUSP6 promoter. To determine biological function, we forced expression of wild type, R392P, V444I, or T505A ETV5 in an interleukin-7-dependent pre-B cell line. Proliferation and apoptosis assays showed that T505A ETV5 conferred a proliferative advantage to pre-B cells. RNA sequencing showed that expression of ETV5 variants significantly altered gene expression in cultured cells. Through gene set enrichment analysis, T505A was suggested to downregulate the p53 pathway and the anti-proliferative protein, B cell translocation gene 2 (encoded by Btg2). In summary, these data suggest that ETV5 mutations play a role in pre-B-ALL by affecting proliferation and cell survival.

immunology↗

bioRxiv2024

Negative regulation of Activation-Induced Cytidine Deaminase gene transcription in developing B cells by a PU.1-interacting intronic region

Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a key role in somatic hypermutation and class switch recombination in germinal center B cells. However, off-target effects of AID are implicated in human leukemia and lymphoma. A mouse model of precursor B cell acute lymphoblastic leukemia driven by deletion of the related transcription factors PU.1 and Spi-B revealed C->T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Aicda mRNA transcript levels were increased in leukemia cells and preleukemic bone marrow pre-B cells lacking PU.1 and/or Spi-B, relative to wild type cells. Using chromatin immunoprecipitation, PU.1 was found to interact with a negative regulatory region (R2-1) within the first intron of Aicda. CRISPR-Cas9-induced mutagenesis of R2-1 in cultured pre-B cells resulted in upregulation of Aicda in response to lipopolysaccharide stimulation. Mutation of the PU.1 interaction site and neighboring sequences resulted in reduced repressive ability of R2-1 in transient transfection analysis followed by luciferase assays. These results show that a PU.1-interacting intronic region negatively regulates Aicda transcription in developing B cells.

immunology↗

bioRxiv2023

N-Acetylcysteine alters disease progression and increases Janus Kinase mutation frequency in a mouse model of precursor B cell acute lymphoblastic leukemia

B cell acute lymphoblastic leukemia (B-ALL) is the most prevalent type of cancer in young children and is associated with high levels of reactive oxygen species (ROS). The antioxidant N-acetylcysteine (NAC) was tested for its ability to alter disease progression in a mouse model of B-ALL. Mb1-Cre{Delta}PB mice have deletions in genes encoding PU.1 and Spi-B in B cells and develop B-ALL at 100% incidence. Treatment of Mb1-Cre{Delta}PB mice with NAC in drinking water significantly reduced the frequency of CD19+ pre-B ALL cells infiltrating the thymus at 11 weeks of age. However, treatment with NAC did not reduce leukemia progression or increase survival by median 16 weeks of age. NAC significantly altered gene expression in leukemias in treated mice. Mice treated with NAC had increased frequencies of activating mutations in genes encoding Janus Kinases 1 and 3. In particular, frequencies of Jak3 R653H mutations were increased in mice treated with NAC compared to control drinking water. NAC opposed oxidization of PTEN protein ROS in cultured leukemia cells. These results show that NAC alters leukemia progression in this mouse model, ultimately selecting for leukemias with high Jak3 R653H mutation frequencies.

cancer biology↗

bioRxiv2023

EZH2 inhibition stimulates viral mimicry in resting splenic B cells

In mammalian cells expression of repetitive genomic sequences is repressed by heterochromatin, underscoring the potential threat of repeat expression to cellular homeostasis. However, the specific consequences of ectopic repeat expression remains unclear. Here we demonstrate that EZH2 inhibitors stimulate repeat misexpression and cell death in resting splenic B cells. We show that B cells are uniquely sensitive to these agents because of high levels of H3K27me3 at repeats and correspondingly low DNA methylation. We generated a pattern recognition receptor loss-of-function mouse model called RIC with mutations in Rigi, Ifih1 (MDA5), and Cgas to specifically block the consequences of repeat misexpression. In both WT and RIC mutant B cells, EZH2 inhibition caused focused loss of H3K27me3 at repetitive elements and upregulated their expression. However, expression of inflammatory chemokines and cell death were interrupted by the RIC mutations. Furthermore, the chemokine expression patterns induced by EZH2 inhibitors resemble the B cell response to Epstein-Barr virus infection. This study demonstrates a viral mimicry effect induced by pharmacological activation of repeat expression that induces inflammation and B cell death.

pathology↗

bioRxiv2022

Spi-C and PU.1 counterregulate Rag1 and Igκ transcription to effect immunoglobulin kappa recombination in small pre-B cells

B cell development requires the ordered rearrangement of Ig genes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags. Ig{kappa} rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26-transformation-specific (ETS) transcription factor Spi-C is activated in response to double-stranded DNA breaks (DSBs) in small pre-B cells to negatively regulate pre-BCR signaling and Ig{kappa} rearrangement. However, it is not clear if Spi-C regulates Ig{kappa} rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation of Ig{kappa} light chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulated Ig{kappa} rearrangement, Ig{kappa} transcript levels, and Rag1 transcript levels. We found that Ig{kappa} and Rag1 transcript levels were increased in small pre-B cells from Spic-/- mice. In contrast, Ig{kappa} and Rag1 transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in the Rag1 promoter region. These results suggest that Spi-C and PU.1 counterregulate Ig{kappa} transcription and Rag1 transcription to effect Ig{kappa} recombination in small pre-B cells.

immunology↗

bioRxiv2021

The E26 Transformation-Specific-family transcription factor Spi-C is dynamically regulated by external signals in B cells

Spi-C is an E26 transformation-specific transcription factor closely related to PU.1 and Spi-B. Spi-C has lineage-instructive functions important in antibody-generating responses, B cell development, and red pulp macrophage generation. Spi-C is inducible by heme- and NF-{kappa}B-dependent pathways in macrophages. The present research aimed to examine the regulation of Spi-C expression in B cells. RT-qPCR analysis revealed that Spic expression was reduced in B cells following addition of lipopolysaccharide, anti-IgM antibodies, CD40L, or cytokines BAFF + IL-4 + IL-5. Cytochalasin treatment partially prevented downregulation of Spic. Unstimulated B cells upregulated Spic over time in culture. To determine the mechanism of Spic regulation, we examined the Spic promoter and upstream regulatory elements. The Spic promoter had unidirectional activity, which was reduced by mutation of an NF-{kappa}B binding site. Spic was repressed by an upstream regulatory region interacting with the heme-binding regulator Bach2. Taken together, these data indicate that Spi-C is dynamically regulated by external signals in B cells and provide insight into the mechanism of regulation.

immunology↗

bioRxiv2019

A role for ATP Citrate Lyase in cell cycle regulation during myeloid differentiation

Differentiation of myeloid progenitor cells into macrophages is accompanied by increased PU.1 concentration and increasing cell cycle length, culminating in cell cycle arrest. Induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration results in decreased levels of mRNA encoding ATP-Citrate Lyase (ACL) and cell cycle arrest. ACL is an essential enzyme for generating acetyl-CoA, a key metabolite for the first step in fatty acid synthesis as well as for histone acetylation. We hypothesized that ACL may play a role in cell cycle regulation in the myeloid lineage. In this study, we found that acetyl-CoA or acetate supplementation was sufficient to rescue cell cycle progression in cultured BN cells treated with an ACL inhibitor or induced for PU.1 expression. Acetyl-CoA supplementation was also sufficient to rescue cell cycle progression in BN cells treated with a fatty acid synthase (FASN) inhibitor. We demonstrated that acetyl-CoA was utilized in both fatty acid synthesis and histone acetylation pathways to promote proliferation. Finally, we found that Acly mRNA transcript levels decrease during normal macrophage differentiation from bone marrow precursors. Our results suggest that regulation of ACL activity is a potentially important point of control for cell cycle regulation in the myeloid lineage.

immunology↗

bioRxiv2018

Deletion of genes encoding PU.1 and Spi-B leads to B cell acute lymphoblastic leukemia associated with driver mutations in Janus Kinases

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with recurrent mutations that occur in cancer-initiating cells. There is a need to understand how spontaneous driver mutations influence clonal evolution in leukemia. The ETS-transcription factors PU.1 and Spi-B (encoded by Spi1 and Spib) execute a critical role in B cell development and serve as complementary tumour suppressors by opposing the proliferative events mediated by IL-7R signaling. Here, we used a mouse model to conditionally delete Spi1 and Spib genes in developing B cells. These mice developed B-ALL with a median time to euthanasia of 18 weeks. We performed RNA and whole-exome sequencing (WES) on leukemias isolated from Mb1-Cre{Delta}PB mice and identified single-nucleotide variants (SNVs) in Jak1, Jak3 and Ikzf3 genes, resulting in amino acid changes and in the gain of early stop-codons. JAK3 mutations resulted in amino acid substitutions located in the pseudo-kinase (R653H, V670A) and in the kinase (T844M) domains. Introduction of these mutations into wild-type pro-B cells conferred survival and proliferation advantages. We conclude that mutations in Janus kinases represent secondary drivers of leukemogenesis in the absence of Spi-B and PU.1 transcription factors. This mouse model represents an useful tool to study clonal evolution and tumour heterogeneity in B-ALL.

cancer biology↗