Early B-cell Factor Research Articles

A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

CircEYA3 aggravates intervertebral disc degeneration through the miR-196a-5p/EBF1 axis and NF-κB signaling

Intervertebral disc degeneration (IDD) is a well-established cause of disability, and extensive evidence has identified the important role played by regulatory noncoding RNAs, specifically circular RNAs (circRNAs) and microRNAs (miRNAs), in the progression of IDD. To elucidate the molecular mechanism underlying IDD, we established a circRNA/miRNA/mRNA network in IDD through standardized analyses of all expression matrices. Our studies confirmed the differential expression of the transcription factors early B-cell factor 1 (EBF1), circEYA3, and miR-196a-5p in the nucleus pulposus (NP) tissues of controls and IDD patients. Cell proliferation, apoptosis, and extracellular mechanisms of degradation in NP cells (NPC) are mediated by circEYA3. MiR-196a-5p is a direct target of circEYA3 and EBF1. Functional analysis showed that miR-196a-5p reversed the effects of circEYA3 and EBF1 on ECM degradation, apoptosis, and proliferation in NPCs. EBF1 regulates the nuclear factor kappa beta (NF-кB) signalling pathway by activating the IKKβ promoter region. This study demonstrates that circEYA3 plays an important role in exacerbating the progression of IDD by modulating the NF-κB signalling pathway through regulation of the miR196a-5p/EBF1 axis. Consequently, a novel molecular mechanism underlying IDD development was elucidated, thereby identifying a potential therapeutic target for future exploration.

EBF1 Limits the Numbers of Cochlear Hair and Supporting Cells and Forms the Scala Tympani and Spiral Limbus during Inner Ear Development.

Early B-cell factor 1 (EBF1) is a basic helix-loop-helix transcription factor essential for the differentiation of various tissues. Our single-cell RNA sequencing data suggest that Ebf1 is expressed in the sensory epithelium of the mouse inner ear. Here, we found that the murine Ebf1 gene and its protein are expressed in the prosensory domain of the inner ear, medial region of the cochlear duct floor, otic mesenchyme, and cochleovestibular ganglion. Ebf1 deletion in mice results in incomplete formation of the spiral limbus and scala tympani, increased number of cells in the organ of Corti and Kölliker's organ, and aberrant course of the spiral ganglion axons. Ebf1 deletion in the mouse cochlear epithelia caused the proliferation of SOX2-positive cochlear cells at E13.5, indicating that EBF1 suppresses the proliferation of the prosensory domain and cells of Kölliker's organ to facilitate the development of appropriate numbers of hair and supporting cells. Furthermore, mice with deletion of cochlear epithelium-specific Ebf1 showed poor postnatal hearing function. Our results suggest that Ebf1 is essential for normal auditory function in mammals.

Early B-Cell Factor 1: An Archetype for a Lineage-Restricted Transcription Factor Linking Development to Disease.

The development of highly specialized blood cells from hematopoietic stem cells (HSCs) in the bone marrow (BM) is dependent upon a stringently orchestrated network of stage- and lineage-restricted transcription factors (TFs). Thus, the same stem cell can give rise to various types of differentiated blood cells. One of the key regulators of B-lymphocyte development is early B-cell factor 1 (EBF1). This TF belongs to a small, but evolutionary conserved, family of proteins that harbor a Zn-coordinating motif and an IPT/TIG (immunoglobulin-like, plexins, transcription factors/transcription factor immunoglobulin) domain, creating a unique DNA-binding domain (DBD). EBF proteins play critical roles in diverse developmental processes, including body segmentation in the Drosophila melanogaster embryo, and retina formation in mice. While several EBF family members are expressed in neuronal cells, adipocytes, and BM stroma cells, only B-lymphoid cells express EBF1. In the absence of EBF1, hematopoietic progenitor cells (HPCs) fail to activate the B-lineage program. This has been attributed to the ability of EBF1 to act as a pioneering factor with the ability to remodel chromatin, thereby creating a B-lymphoid-specific epigenetic landscape. Conditional inactivation of the Ebf1 gene in B-lineage cells has revealed additional functions of this protein in relation to the control of proliferation and apoptosis. This may explain why EBF1 is frequently targeted by mutations in human leukemia cases. This chapter provides an overview of the biochemical and functional properties of the EBF family proteins, with a focus on the roles of EBF1 in normal and malignant B-lymphocyte development.

EBF2 Links KMT2D-Mediated H3K4me1 to Suppress Pancreatic Cancer Progression via Upregulating KLLN.

Mono-methylation of histone H3 on Lys 4 (H3K4me1), which is catalyzed by histone-lysine N-methyltransferase 2D (KMT2D), serves as an important epigenetic regulator in transcriptional control. In this study, the authors identify early B-cell factor 2 (EBF2) as a binding protein of H3K4me1. Combining analyses of RNA-seq and ChIP-seq data, the authorsfurther identify killin (KLLN) as a transcriptional target of KMT2D and EBF2 in pancreatic ductal adenocarcinoma (PDAC) cells. KMT2D-dependent H3K4me1 and EBF2 are predominantly over-lapped proximal to the transcription start site (TSS) of KLLN gene. Comprehensive functional assays show that KMT2D and EBF2 cooperatively inhibit PDAC cells proliferation, migration, and invasion through upregulating KLLN. Such inhibition on PDAC progression is also achieved through increasing H3K4me1 level by GSK-LSD1, a selective inhibitor of lysine-specific demethylase 1 (LSD1). Taken together, these findings reveal a new mechanism underlying PDAC progression and provide potential therapeutic targets for PDAC treatment.

Urologic manifestations of hypotonia, ataxia, and delayed development syndrome (HADDS), a rare neurodevelopmental disorder

Early B-cell factor 3 (EBF3) is a transcription factor involved in neuronal differentiation and maturation. Pathogenic variants are associated with hypotonia, ataxia, and delayed development syndrome (HADDS) (MIM#617330). Urologic manifestations are common and may have implications regarding long term renal function. To review all known patients with pathogenic variants of the EBF3 gene resulting in HADDS with urologic manifestations. We hypothesize a high rate of bladder dysfunction secondary to the EBF3 variant's impact on relaxation of the urinary sphincter leading to detrusor sphincter dyssynergia (DSD). The PubMed database was queried for publications of the EBF3 mutation between January 2017 and January 2023. Search terms were "EBF3 mutation OR HADDS AND urology OR phenotype". Retrospective analysis of HADDS patients cared for in our institution was performed. Demographic and clinical information was collected. We identified 52 patients (33F:19M) through literature (28F:18M) and retrospective review (5F:1M). There was a high prevalence of genitourinary physical exam abnormalities, history of urinary tract infection, vesicoureteral reflux (VUR), and diagnosis of neurogenic bladder. Within the literature review cohort, 67% had a urologic diagnosis. Females were disproportionately affected with urologic manifestations. In our cohort, four of six children were diagnosed with VUR and severe voiding dysfunction consistent with neurogenic bladder (67%). These children were managed with a vesicostomy. Five children had bowel dysfunction requiring therapy. Urodynamics suggested a high prevalence of external sphincter dyssynergia. Less severe forms of DSD were felt to be implicated in the abnormal voiding parameters in children who presented later in life based on non-invasive flow studies. There is significant variability in the phenotypic presentation of patients with HADDS. While EBF3 plays a clear role in neurodevelopment, it also impacts muscle development and may impact muscle relaxation. The location of the genetic variant may impact the degree of DSD, with more severe forms leading to earlier presentations. Initial work-up should include a renal ultrasound (RUS) and post void residual (PVR). Consideration can be given to obtaining a VCUG, DMSA scan or urodynamic studies. Yearly screening should be pursued with an RUS and PVR in those with an initial unremarkable work-up given the variable timing and severity of presentation. Urologic manifestations of HADDS include high rates of bladder dysfunction secondary to DSD, vesicoureteral reflux, urinary tract infection, and cryptorchidism. These patients are at risk of renal deterioration if urinary abnormalities are not properly diagnosed and managed.

Elucidation of adipogenic differentiation regulatory mechanism in human maxillary/mandibular bone marrow-derived stem cells

ObjectiveThis study aims to investigate the underlying molecular mechanisms that regulate the adipogenic differentiation of maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs). DesignMBMSCs and iliac bone marrow-derived MSCs (IBMSCs) were compared for osteogenic, chondrogenic, and adipogenic differentiation. Cell surface antigen expression was examined using flow cytometry, and stem cell marker expression was assessed using real-time polymerase chain reaction (PCR). Various adipogenic regulatory factors’ expression was evaluated using real-time PCR and western blotting. ResultsNo significant differences in cell surface antigen profiles or stem cell marker expression in MBMSCs and IBMSCs were observed. MBMSCs and IBMSCs displayed similar osteogenic and chondrogenic potentials, whereas MBMSCs showed significantly lower adipogenic potentials than those shown by IBMSCs. Expression of CCAAT/enhancer binding protein β (C/EBPβ), C/EBPδ, early B-cell factor 1 (Ebf-1), and Krüppel-like factor 5 (KLF5), which are early adipogenic differentiation factors, was suppressed in MBMSCs compared to that in IBMSCs. Peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which play important roles in the terminal differentiation of adipocytes, was lower in MBMSCs than that in IBMSCs. Furthermore, the level of zinc finger protein 423 (Zfp423), which is involved in the commitment of undifferentiated MSCs to the adipocyte lineage, was significantly lower in MBMSCs than that in IBMSCs. ConclusionsMBMSCs are negatively regulated in the commitment of undifferentiated MSCs to the adipocyte lineage (preadipocytes) as well as in the terminal differentiation of preadipocytes into mature adipocytes. These results may elucidate the site-specific characteristics of MBMSCs.

Caveolin-1 rs1997623 Single Nucleotide Polymorphism Creates a New Binding Site for the Early B-Cell Factor 1 That Instigates Adipose Tissue CAV1 Protein Overexpression.

Caveolin-1 (CAV1) is implicated in the pathophysiology of diabetes and obesity. Previously, we demonstrated an association between the CAV1 rs1997623 C > A variant and metabolic syndrome (MetS). Here, we decipher the functional role of rs1997623 in CAV1 gene regulation. A cohort of 38 patients participated in this study. The quantitative MetS scores (siMS) of the participants were computed. CAV1 transcript and protein expression were tested in subcutaneous adipose tissue using RT-PCR and immunohistochemistry. Chromatin immunoprecipitation assays were performed using primary preadipocytes isolated from individuals with different CAV1 rs1997623 genotypes (AA, AC, and CC). The regulatory region flanking the variant was cloned into a luciferase reporter plasmid and expressed in human preadipocytes. Additional knockdown and overexpression assays were carried out. We show a significant correlation between siMS and CAV1 transcript levels and protein levels in human adipose tissue collected from an Arab cohort. We found that the CAV1 rs1997623 A allele generates a transcriptionally active locus and a new transcription factor binding site for early B-cell factor 1 (EBF1), which enhanced CAV1 expression. Our in vivo and in vitro combined study implicates, for the first time, EBF1 in regulating CAV1 expression in individuals harboring the rs1997623 C > A variant.

PRP8-Induced CircMaml2 Facilitates the Healing of the Intestinal Mucosa via Recruiting PTBP1 and Regulating Sec62.

Multiple organ dysfunction syndrome (MODS) occurs in the gastrointestinal tract and injured intestinal mucosa is the anatomical basis for various diseases. The expression of circular RNAs (circRNAs) is implicated in many diseases; however, the role of circRNAs in intestinal mucosal injury is yet to be discovered. Our preliminary gene microarray analysis revealed a novel circular RNA, circMaml2, with a significant intestinal mucosal protection effect. Its expression was found to decrease in severely burned intestinal mucosal tissue, whereas its overexpression might facilitate the reconstruction of the injured intestinal mucous membrane. The function of circMaml2 in cell proliferation and migration was studied in MC38 cells. The repair function of circMaml2 was tested on the intestinal mucosa of mice. RNA-binding protein polypyrimidine tract-binding protein 1(PTBP1) was selected by pull-down assay and mass spectrometry (MS). RNA immunoprecipitation (RIP) was performed to confirm the binding of circMaml2 and PTBP1 and to study PTBP1 and its downstream target, early B-cell factor 1(Ebf1). Bioinformatics software forecast analysis and dual-luciferase reporter assay were performed to ascertain miR-683 and Sec62 as the downstream targets of circMaml2 and miR-683, respectively. Furthermore, PRP8 was discovered to promote the biogenesis of circMaml2. CircMaml2 promotes cell proliferation and migration of MC38 cells and the repair of the intestinal mucosa of mice. This effect is brought about by combining with PTBP1 to improve Ebf1 and interacting with miR-683 to regulate Sec2. Furthermore, PRP8 was discovered to promote the biogenesis of circMaml2. This is the first reported study of the effect of circMaml2 on intestinal mucosal repair.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.

Factors affecting the induction of uncoupling protein 1 in C2C12 myogenic cells

Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)-3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes.

Duplication/triplication mosaicism of EBF3 and expansion of the EBF3 neurodevelopmental disorder phenotype.

Deleterious variants in the transcription factor early B-cell factor 3 (EBF3) are known to cause a neurodevelopmental disorder (EBF3-NDD). We report eleven individuals with EBF3 variants, including an individual with a duplication/triplication mosaicism of a region encompassing EBF3 and a phenotype consistent with EBF3-NDD, which may reflect the importance of EBF3 gene-dosage for neurodevelopment. The phenotype of individuals in this cohort was quite mild compared to the core phenotype of previously described individuals. Although ataxia tended to wane with age, we show that cognitive difficulties may increase, and we recommend that individuals with EBF3-NDD have systematic neuropsychological follow-up.

EBF3 reactivation by inhibiting the EGR1/EZH2/HDAC9 complex promotes metastasis via transcriptionally enhancing vimentin in nasopharyngeal carcinoma.

Metastasis is the major reason for treatment failure and accounts for cancer-related death in patients with nasopharyngeal carcinoma. However, the genetic alterations and molecular mechanisms that cause nasopharyngeal carcinoma metastasis are elusive. Herein, we performed RNA sequencing in patients with or without metastasis, and found that the early B-cell factor 3 (EBF3) was significantly elevated in the samples with metastasis. Mechanistically, EBF3 promoted metastasis by directly combining with the promoter of Vimentin and transcriptionally upregulating it. In addition, EBF3 was epigenetically silenced by EGR1/EZH2/HDAC9 complexes via sustaining the high level of H3K27-Me3 at its promoter. Clinically, there was a positive correlation between EBF3 and Vimentin in nasopharyngeal carcinoma tissues. Moreover, high expression of EBF3 or Vimentin was correlated with poor overall survival, while the combination of high EBF3 and Vimentin expression was associated with more significant poor prognosis. Therefore, specific agents targeting EBF3 or stabilizing the EGR1/EZH2/HDAC9 complex could be novel therapeutic strategies for cancer metastasis.

Expression and function of Ebf1 gene during chondrogenesis in chick embryo limb buds

The expression profile of early B-cell factor (Ebf) genes and loss of function experiments denote a crucial role for these genes during the late stage of skeletogenesis. However, little is known regarding the expression and function of these genes during the early stage of skeletogenesis. Therefore, this study aimed to detail the spatiotemporal expression pattern of cEbf1, in comparison to cEbf2 and cEbf3, in chick limb buds and investigate its function during chondrogenesis. cEbf1-3 were co-expressed in the distal mesenchyme from a very early stage and later in the outer perichondrium and the surrounding noncartilaginous mesenchymal cells. Ebf1 loss of function through injection of RCASBP virus-carrying Ebf1 dominant-negative form (ΔEbf1) into the wing buds resulted in shortened skeletal elements with a clear defect in the chondrocyte differentiation program. In RCASBP-ΔEbf1 injected wing, the chondrogenesis was initiated normally but hindered at the maturation stage. Subsequently, the chondrocytes failed to become mature or hypertrophic and the long bone diaphysis was not properly developed. The final phenotype included shorter, thicker, and fused long bones. These phenotypic changes were associated with downregulation of the early [Sox9 and collagen type II (Col2a1)] and the late [alkaline phosphatase (AP)] chondrocytes differentiation markers in the limb buds. These results conclude that cEbf1 could be involved in a molecular cascade that promotes the terminal stages of chondrogenesis in the long bone anlagen.

Long noncoding RNA IL6-AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR-149-5p and early B-cell factor1.

Chronic obstructive pulmonary disease is a complex condition with multiple etiologies, including inflammation. We identified a novel long noncoding RNA (lncRNA), interleukin 6 antisense RNA 1 (IL6‐AS1), which is upregulated in this disease and is associated with airway inflammation. We found that IL6‐AS1 promotes the expression of inflammatory factors, especially interleukin (IL) 6. Mechanistically, cytoplasmic IL6‐AS1 acts as an endogenous sponge by competitively binding to the microRNA miR‐149‐5p to stabilize IL‐6 mRNA. Nuclear IL6‐AS1 promotes IL‐6 transcription by recruiting early B‐cell factor 1 to the IL‐6 promoter, which increases the methylation of the H3K4 histone and acetylation of the H3K27 histone. We propose a model of lncRNA expression in both the nucleus and cytoplasm that exerts similar effects through differing mechanisms, and IL6‐AS1 probably increases inflammation via multiple pathways.

Early B-cell factors involve in the tumorigenesis and predict the overall survival of gastric cancer.

Gastric cancer (GC) is a heavy health burden around the world, which is the fifth most frequent tumor and leads to the third most common cancer-related deaths. It is urgent to identify prognostic markers as the guideline for personalized treatment and follow-up. We accessed the prognostic value of Early B-cell factors (EBFs) in GC. A total of 415 GC tissues and 34 normal tissues from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) cohort, 616 external patients from GSE15459, GSE22377, GSE51105, GSE62245 were enrolled for analysis. Univariate and multivariate Cox regression analyses were employed to evaluate the sole and integrative prognostic value of EBFs, respectively. Genetic alterations, DNA methylation of EBFs were also evaluated, as well as the involved signaling pathways. We revealed that increased EBFs associated with the poor prognosis of GC patients, the prognostic model was established in TCGA-STAD cohort, and validated in Gene Expression Omnibus (GEO) cohorts, with effectiveness in both HER2 positive and negative patients. DNA methylation was involved in the impact on prognosis. Cell cycle, immune-associated, and MAPK pathways were influenced by EBFs. Anti-CTLA4 immunotherapy is more suitable for EBFs determining high-risk groups, but not anti-PD-1/PD-L1 therapy. 5-Fluorouracil, methotrexate, vorinostat are suitable to inhibit the function of EBFs. Our new findings provide novel insight into the prediction of prognosis and clinical treatment of GC patients based on EBFs.

Identification of CXCL12‐abundant reticular cells in human adult bone marrow

SummaryA population of mesenchymal stem cells, termed CXC chemokine ligand (CXCL)12‐abundant reticular (CAR) cells or leptin receptor‐expressing cells, are the major cellular component of niches for haematopoietic stem cells (HSCs) in murine bone marrow. CAR cells are characterized by several salient features, including much higher expression of CXCL12, stem cell factor (SCF), forkhead box C1 (FOXC1) and early B‐cell factor 3 (EBF3), which are essential for HSC maintenance, than other cells. However, the human counterpart of CAR cells has not been fully described. Here, we show the presence of cells expressing much higher CXCL12 than other cells in human adult bone marrow using a flow cytometry‐based in situ technique that enables high‐throughput detection of mRNA at single‐cell resolution. Most CXCL12hi cells expressed high levels of SCF, FOXC1 and EBF3 and had the potential to differentiate into adipocytes and osteoblasts. Histologically, the nuclei of CXCL12hi cells were identified and quantified by EBF3 expression in fixed marrow sections. CXCL12hi cells sorted from residual bone marrow aspirates of chronic myeloid leukaemia patients expressed reduced levels of CXCL12, SCF, FOXC1 and EBF3 in correlation with increased leukaemic burden. Together, we identified the human counterpart of CAR cells, enabling the evaluation of their alterations in various haematological disorders by flow cytometric and histological analyses.

Genome-wide interference of ZNF423 with B-lineage transcriptional circuitries in acute lymphoblastic leukemia

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9–mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis.

EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

Genes encoding B lineage–restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation–sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9–mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation–sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro–B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.