scholarly journals Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages

2021 ◽  
Vol 5 (13) ◽  
pp. 2673-2686
Author(s):  
Serine Avagyan ◽  
Margaret C. Weber ◽  
Sai Ma ◽  
Meera Prasad ◽  
William P. Mannherz ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Single Cell ◽  
Cell Lineage ◽  
Hematologic Malignancies ◽  
Deficiency Syndrome ◽  
Increased Risk ◽  
Lymphoid Progenitors ◽  
B Lymphoid ◽  
Normal Differentiation

Abstract Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.

scholarly journals Single-cell analysis of developing B cells reveals dynamic gene expression networks that govern B cell development and transformation

2020 ◽  
Author(s):  
Robin D. Lee ◽  
Sarah A. Munro ◽  
Todd P. Knutson ◽  
Rebecca S. LaRue ◽  
Lynn M. Heltemes-Harris ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Rna Binding ◽  
Single Cell Analysis ◽  
Cell Transformation ◽  
Cell Lineage ◽  
Cell Development ◽  
B Cell Development

SummaryIntegration of external signals and B-lymphoid transcription factor activities orchestrate B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We used single-cell transcriptomics and proteomics to characterize B cell development. Our analysis revealed unique transcriptional signatures that refine the pre-B cell expansion stages into novel pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with unexpected dynamic and reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings reveal the underlying heterogeneity of developing B cells and point to key developmental nodes linked to B cell transformation.

scholarly journals Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1.

1996 ◽  
Vol 183 (2) ◽  
pp. 393-401 ◽  
Author(s):  
A M Reimold ◽  
P D Ponath ◽  
Y S Li ◽  
R R Hardy ◽  
C S David ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Cell Lines ◽  
Binding Protein ◽  
Cell Lineage ◽  
Regulatory Elements ◽  
Target Sequence ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Activator Protein

The transcription factor human X-box binding protein 1 (hXBP-1) is a basic region-leucine zipper protein implicated in the regulation of major histocompatibility complex class II gene expression as well as in exocrine gland and skeletal development. Multiple regulatory elements in the hXBP-1 promoter lie 3' to the transcription start site, including the hX2 site, whose core sequence is an AP-1-like element identical to the hXBP-1 target sequence in the HLA-DRA promoter. One complex identified by electrophoretic mobility shift assay (EMSA), complex 3, was previously shown to protect the hX2 site and more 3' bases. Sequence analysis now shows that this region contains a consensus binding site for transcription factor BSAP (B cell lineage-specific activator protein). Complex 3 and BSAP have identical cell-type specificities, as they are found only in pre-B and mature B cell lines. In EMSAs, BSAP antibody specifically recognized complex 3, and in vitro translated BSAP could bind to an hXBP promoter fragment. Cotransfections using an hXBP-1 reporter construct indicated that BSAP downregulates the hXBP-1 promoter. The highest levels of hXBP-1 mRNA were found when BSAP was not expressed, in pre-Pro-B cells and in plasma cell lines. In addition, hXBP-1 and BSAP levels were inversely correlated along the early stages of B cell development. In the regulation of the hXBP-1 promoter, a strong positive transcriptional influence at the hX2 site is opposed by the downregulatory actions of BSAP.

Targeted Overexpression of the Transcription Factor XBP-1 in B Cells Promotes Plasma Cell and Lymphoplasmacytic Neoplasms in Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 359-359 ◽  
Author(s):  
Ruben D. Carrasco ◽  
Kumar Sukhdeo ◽  
Marina Protopopova ◽  
Masha German ◽  
Joel Henderson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
Growth Factor ◽  
B Cell ◽  
Signaling Pathways ◽  
Plasma Cell ◽  
Rna Processing ◽  
Cell Lineage ◽  
Growth Factor Signaling ◽  
Factor X

Abstract The transcription factor X-box binding protein-1 (XBP-1) plays critical roles in the unfolded protein response (UPR), the differentiation of plasma cells, and the regulation of growth factor signaling pathways. XBP-1 is subject to regulation by alternative RNA processing producing XBP-1-spliced (S) and -unspliced (U) mRNAs encoding proteins with identical DNA-binding and bZIP domains yet distinct C-terminal transactivation domains. The weaker transactivation potential of XBP-1(U) has prompted speculation that it may influence XBP-1(S) activity as a transdominant mutant. While elevated XBP-1 expression has been reported in transformed cells, the relative ratios of these XBP-1 isoforms and associated physiological relevance in cancer are uncertain. Here, we assessed the differential impact of enforced XBP-1(S) versus XBP-1(U) transgene expression in the B cell lineage. Both transgenes elicited early onset antibody-dependent autoimmune disease characterized by elevated levels of serum immunoglobulin (Ig) and IL-6 production, increased numbers of marginal zone and mature follicular B cells in the spleen, and expanded mature B cell populations in the bone marrow. Notably, aged XBP-1(S) mice developed clonal plasma cell expansions, culminating in the human-equivalent of Monoclonal Gammopathy of Undetermined Significance (MGUS) or Multiple Myeloma (MM). Conversely, XBP-1(U) mice develop multi-organ lymphoplasmacytic infiltrates and, with advancing age, succumb to neoplasms resembling human Lymphoplasmacytic Lymphoma/Waldenstrom’s Macroglobulinemia (LPL/WM). These unanticipated genetic observations in the mouse were translated to human disease with documentation of elevated levels of XBP-1(S) in MM and XBP-1(U) in LPL/WM. Together, these results indicate that imbalances in XBP-1(S) and XBP-1(U) alters B cell lineage homeostasis and can drive two distinct types of lymphoplasmacytic neoplasms in vivo. The findings of this study, together with the known capacity of XBP-1 to regulate various cancer-relevant growth factor signaling pathways, predicts that epigenetic dysregulation of alternate XBP-1 RNA processing can promote age-associated B cell malignancies in humans.

Mef2C Is a Lineage-Restricted Target Gene of Scl/Tal1 and Regulates Megakaryopoiesis and B-Cell Homeostasis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 278-278
Author(s):  
Katrin E Rhodes ◽  
Christos Gekas ◽  
Laurraine Gereige ◽  
Hildur Helgadottir ◽  
Roberto Ferrari ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Muscle Development ◽  
Target Genes ◽  
Fetal Liver ◽  
Premature Aging ◽  
Bhlh Transcription Factor ◽  
Functional Requirements ◽  
B Lymphoid ◽  
Deficient Mice

Abstract The bHLH transcription factor stem cell leukemia/T-cell acute leukemia gene (Scl/Tal1) is a master regulator for hematopoiesis, essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. To identify Scl target genes in hematopoietic cells, we performed gene expression analysis on HOX11-immortalized Sclfl/fl fetal liver cell lines. Analysis of the top 50 downregulated genes revealed several genes related to hematopoiesis including erythroid and megakaryocyte development, vasculogenesis, as well as genes/unknown ESTs that have not been previously linked to blood development. One of the top downregulated genes was transcription factor myocyte enhancer factor 2C (Mef2C). Mef2C−/− embryos die at E9.5, the same time as Scl−/− embryos, and exhibit severe defects in cardiac and muscle development. Analysis of Mef2C−/− embryos showed that, Mef2C, in contrast to Scl, is not required for specification into primitive or definitive hematopoietic lineages. To bypass the embryonic lethality, we utilized a conditionally targeted Mef2Cfl/fl strain and crossed it with a hematopoietic cell-specific VavCre strain that deactivates Mef2C shortly after the emergence of HSCs. Interestingly, adult VavCre+Mef2Cfl/fl mice exhibited severe platelet defects highly reminiscent to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reductions of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.

Lymphoid Blast Crisis Transformation and Development of Drug- Resistance in Chronic Myeloid Leukemia Are Driven by Aberrant Somatic Hypermutation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 571-571
Author(s):  
Lars Klemm ◽  
Cihangir Duy ◽  
Niklas Feldhahn ◽  
John Groffen ◽  
Yong-mi Kim ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Blast Crisis ◽  
Cell Lineage ◽  
Chronic Phase ◽  
Imatinib Resistance ◽  
B Lymphoid ◽  
Germinal Center B Cells

Abstract Chronic myeloid leukemia (CML) in chronic phase has a disease-free survival of 87% (DFS; 5 years) and long-term treatment with Imatinib is effective. In a large subgroup of patients with CML, however, the disease ultimately progresses into B lymphoid blast crisis (LBC) with only 6% DFS and resistance to Imatinib develops in virtually all cases. In most cases, acquired resistance to Imatinib can be attributed to somatic mutations within the BCR-ABL1 kinase domain. Whereas BCR-ABL1 kinase mutations are rare in chronic phase CML, such mutations are found in >80% of patients with B cell lineage LBC. Likewise, deletions of the ARF and INK4B genes are rare in chronic phase CML but found in ~50% of B cell lineage LBC. In a search for a B cell lineage-specific mutation mechanism responsible for BCR-ABL1 kinase mutations, we tested the hypothesis that aberrant activation of somatic hypermutation may give rise to drug-resistance and progression of chronic phase CML into LBC. Somatic hypermutation drives affinity maturation of immunoglobulins expressed by germinal center B cells and requires the cytidine deaminase AID. Expression of AID depends on PAX5, a transcription factor that determines B cell lineage commitment of hematopoietic progenitor cells. The dependence of AID expression on PAX5 limits somatic hypermutation to the B cell lineage. Consistent with aberrant activation of somatic hypermutation in B cell lineage LBC, we found both PAX5 and AID expression at the mRNA and protein level in B lymphoid but not myeloid subclones from patient-derived blast crisis CML. However, AID protein levels in LBC clones were 5–10-fold lower than in germinal center B cells. To confirm lineage-specific activation of AID-expression in BCR-ABL1 driven leukemia, we isolated bone marrow from Aid-GFP reporter transgenic mice and transformed the bone marrow cells with BCR-ABL1 under either myeloid (IL3, IL6, SCF) or B lymphoid (IL7) culture conditions. The Aid-GFP reporter drives GFP expression under control of upstream and downstream regulatory elements of the Aid locus (Crouch et al., 2007). BCR-ABL1-induced Aid-expression was only observed under B lymphoid culture conditions and was very heterogeneous among the leukemia cell population: Only about 5–10% of CD19+ B lymphoid leukemia clones express Aid-GFP. In these cells, however, Aid mRNA levels are 240-fold higher than in Aid-GFP-negative cells and even 1.5-fold higher than in normal germinal center B cells. Consistent with these findings, we found aberrant somatic hypermutation of the IGHM, BCL6 and MYC loci as well as evidence of ongoing DNA single-strand breaks at the ARF and INK4B loci in B cell lineage LBC but not myeloid CML clones. Ectopic expression of AID in seven otherwise AID-negative CML cell lines cells leads to the acquisition of Imatinib-resistance and sequence analysis of the Imatinib-resistant clones revealed accumulation of mutations within the BCR-ABL1 kinase domain that cause Imatinib-resistance in patients (e.g. L248V, E225K, T315I). Aberrant expression of AID also caused Imatinib-resistance of CML cells in vivo: NOD/SCID mice were injected with CML cells that were either transduced with AID/GFP or GFP alone. Whereas more than the half of the mice injected with GFP+ CML cells were still alive after 170 days, all mice in the AID/GFP+ CML group died within 54 days after injection despite Imatinib-treatment. Forced expression of the B cell-specific transcription factor PAX5 in otherwise PAX5-negative CML cells resulted in a partial B lymphoid lineage conversion similar to LBC. Of note, ectopic expression of PAX5 also resulted in aberrant AID expression, subsequent acquisition of BCR-ABL1 kinase mutations and development of drug-resistance. We conclude that B cell-specific activation of PAX5/AID-induced aberrant somatic hypermutation provides a genetic basis for the strikingly different outcome of myeloid lineage CML as compared to LBC.

Lineage-Specific Functions of LKB1 in CML and B Lymphoid Blast Crisis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 34-34
Author(s):  
Lai N Chan ◽  
Huimin Geng ◽  
Markus Muschen
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Cancer Cells ◽  
Aerobic Glycolysis ◽  
Blast Crisis ◽  
Cell Lineage ◽  
Imatinib Treatment ◽  
Hematopoietic Stem ◽  
B Lineage ◽  
B Lymphoid

Abstract Abstract 34 Background: The serine-threonine liver kinase B1 (LKB1, also called STK11) acts as negative regulator of aerobic glycoloysis, a metabolic pathway that is typically used in cancer cells (commonly referred to as ‘Warburg effect’). LKB1, together with AMPKs, shifts the metabolic balance from aerobic glycolysis to oxidative phosphorylation and thereby reverses the metabolic program of cancer cells and functions as tumor suppressor. Recently, it has been shown that LKB1 plays a critical role in the maintenance of quiescence and metabolic homeostasis of hematopoietic stem cells (HSCs). Hypothesis: In the present study, we focused on the roles of LKB1 in BCR-ABL1-driven leukemias including CML and B lymphoid blast crisis/Ph+ ALL (LBC). While LKB1 is widely seen as tumor suppressor in solid tumors, we found that high expression levels of LKB1 at diagnosis predict poor clinical outcome in patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=0.0204). In addition, high levels of LKB1 expression correlate with positive minimal residual disease (MRD+, p=0.0323) status in patients. These findings were unexpected and seem to contradict the common notion of LKB1 as a tumor suppressor. Results: To study the function of LKB1 in CML and B lymphoid blast crisis/Ph+ ALL (LBC), we developed a mouse model for inducible ablation of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (LBC). To this end, Lkb1-fl/fl bone marrow hematopoietic stem and progenitor cells and B cell precursor cells were transformed with BCR-ABL1 and transduced with tamoxifen-inducible Cre. Unexpectedly, Cre-mediated deletion of Lkb1 had opposite effects in CML and LBC. While Lkb1-deletion in CML results in an initial proliferative burst of the leukemia cells, the vast majority of B cell lineage LBC cells undergo rapid cell cycle arrest. These findings are consistent with changes of cell cycle checkpoint proteins in response to Lkb1 deletion in CML and B cell lineage LBC. While Lkb1 deletion in CML cells results in downregulation of Arf, p53 and p27 levels, Lkb1 deletion in B lineage LBC cells resulted in upregulation of Arf and p27. In addition, Lkb1 deletion in CML resulted in inactivation of AMPK, a known substrate of LKB1, as well as enhanced activation of mTORC1. By contrast, while deletion of Lkb1 in B cell lineage LBC cells resulted in inactivation of AMPK as shown by reduced phosphorylation of AMPKα T172, there was reduction in mTORC1 activity based on diminished levels of phospho-p70 S6 kinase and S6 following LKB1 deletion. The effects of LKB1 on sensitivity of BCR-ABL1 CML and B lineage LBC cells to Imatinib were also examined. Lkb1-deficient ALL cells became more sensitive to Imatinib treatment. On the other hand, initial Lkb1 deletion rendered CML cells more resistant to Imatinib treatment. When primary patient-derived Ph+ ALL cells (n = 3) were treated with Imatinib, upregulation of phospho-LKB1 (S428) was observed. Finally, LKB1 was also shown to regulate energy homeostasis in CML and B cell lineage LBC in different manners, as measured by monitoring ATP and lactate production. Conclusions: Here we show that Lkb1 plays critical roles in mediating proliferation and cell growth in BCR-ABL1-driven leukemias. While LKB1 is widely seen as a tumor suppressor that limits aerobic glycolysis in cancer cells according to the Warburg effect, our findings demonstrate that LKB1 has lineage-specific functions in BCR-ABL1 driven leukemias. While LKB1 function in CML resembles its tumor suppressor function in solid tumors, LKB1 is critical for survival and proliferation on B cell lineage CML blast crisis and Ph+ ALL. The finding of a divergent role of Lkb1 in CML and B cell lineage LBC/Ph+ ALL is relevant because small molecule inhibitors of AMPK and mTORC1 are currently under development for the treatment of BCR-ABL1-driven leukemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IRF-4 functions as a tumor suppressor in early B-cell development

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3798-3806 ◽  
Author(s):  
Jaime Acquaviva ◽  
Xiaoren Chen ◽  
Ruibao Ren
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Progenitors ◽  
B Lymphoid

Interferon regulatory factor-4 (IRF-4) is a hematopoietic cell–restricted transcription factor important for hematopoietic development and immune response regulation. It was also originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. In contrast to its oncogenic function in late stages of B lymphopoiesis, expression of IRF-4 is down-regulated in certain myeloid and early B-lymphoid malignancies. In this study, we found that the IRF-4 protein levels are increased in lymphoblastic cells transformed by the BCR/ABL oncogene in response to BCR/ABL tyrosine kinase inhibitor imatinib. We further found that IRF-4 deficiency enhances BCR/ABL transformation of B-lymphoid progenitors in vitro and accelerates disease progression of BCR/ABL-induced acute B-lymphoblastic leukemia (B-ALL) in mice, whereas forced expression of IRF-4 potently suppresses BCR/ABL transformation of B-lymphoid progenitors in vitro and BCR/ABL-induced B-ALL in vivo. Further analysis showed that IRF-4 inhibits growth of BCR/ABL+ B lymphoblasts primarily through negative regulation of cell-cycle progression. These results demonstrate that IRF-4 functions as tumor suppressor in early B-cell development and may allow elucidation of new molecular pathways significant to the lymphoid leukemogenesis by BCR/ABL. The context dependent roles of IRF-4 in oncogenesis should be an important consideration in developing cancer therapies targeting IRF-4.

scholarly journals Deltex1 Redirects Lymphoid Progenitors to the B Cell Lineage by Antagonizing Notch1

Immunity ◽  
2002 ◽  
Vol 16 (2) ◽  
pp. 231-243 ◽  
Author(s):  
David J Izon ◽  
Jon C Aster ◽  
Yiping He ◽  
Andrew Weng ◽  
Fredrick G Karnell ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lineage ◽  
Lymphoid Progenitors

scholarly journals Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3461-3471 ◽  
Author(s):  
Christos Gekas ◽  
Katrin E. Rhodes ◽  
Laurraine M. Gereige ◽  
Hildur Helgadottir ◽  
Roberto Ferrari ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Fetal Liver ◽  
Premature Aging ◽  
Functional Requirements ◽  
Helix Loop Helix ◽  
B Cell Homeostasis ◽  
B Lymphoid ◽  
Deficient Mice

Abstract The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Sclfl/fl fetal liver progenitor cell lines. Analysis of Mef2C−/− embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre+Mef2Cfl/fl mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced, whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore, megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.

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