EBF1 Research Articles

Specific Mutation Predict Relapse/Refractory Diffuse Large B-Cell Lymphoma.

The application of rituximab has significantly enhanced the overall survival rates in patients with diffuse large B-cell lymphoma (DLBCL). Regrettably, a significant number of patients still progress to relapse/refractory DLBCL (rrDLBCL). Herein, we employed targeted sequencing of 55 genes to investigate if gene mutations could predict the progression to rrDLBCL. Additionally, we compared the mutation profiles at the time of DLBCL diagnosis with those found in rrDLBCL cases. Our findings highlighted significantly elevated mutation frequencies of TP53, MEF2B and CD58 in diagnostic biopsies from patients who progressed to relapse or refractory disease, with CD58 mutations exclusively observed in the rrDLBCL group. In assessing the predictive power of mutation profiles for treatment responses in primary DLBCL patients, we found that the frequency of CARD11 mutations was substantially higher in non-response group as compared with those who responded to immunochemotherapy. In addition, we revealed mutations in HIST2H2AB, BCL2, NRXN3, FOXO1, HIST1H1C, LYN and TBL1XR1 genes were only detected in initial diagnostic biopsies, mutations in the EBF1 gene were solely detected in the rrDLBCL patients. Collectively, this study elucidates some of the genetic mechanisms contributing to the progression of rrDLBCL and suggests that the presence of CD58 mutations might serve as a powerful predictive marker for relapse/refractory outcomes in primary DLBCL patients.

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.

A human-specific insertion promotes cell proliferation and migration by enhancing TBC1D8B expression.

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution.

Aberrant Heterochromatin Silences Immune Response Genes in Chronic Lymphocytic Leukemia

Introduction: Recent studies have shown that clonal B cells derived from patients with chronic lymphocytic leukemia (CLL) have altered histone modification landscapes compared to B cells from healthy controls. We have also shown that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells, implicating the interaction between oncogenic signaling and epigenetic machinery in CLL (Wang et al., Blood Cancer J. 2022; Wang et al., Cancer Res (2023) 83 (7_Supplement): 4725). However, the majority of the chromatin studies in CLL concentrate on the active enhancers associated with oncogene expression; there is a very limited understanding of lost enhancers in CLL: 1) What is the function of lost enhancers; 2) How are these lost enhancers maintained in CLL; 3) Can these lost enhancers be detected at the precursor state of CLL, monoclonal B-cell lymphocytosis (MBL); 4) if this aberrant chromatin signature affected by CLL treatment. Methods: We systematically mapped the genome-wide histone modification profile (histone marks: H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, and H3K36me3) in 22 previously untreated CLL samples, 6 high count MBL samples, and peripheral blood B cells from 6 healthy donors by Cleavage Under Targets and Tagmentation (CUT&Tag). We also analyzed B cells from 8 patients before and after one year of continuous Bruton tyrosine kinase inhibitor (BTKi) ibrutinib treatment. The genomic regions with a decrease in both H3K4me1 and H3K27ac enrichment (analyzed by Deseq2, fold change > 1, p < 0.01) were named lost enhancers. We used CRISPR-mediated epigenetic editing to study the role of gained H3K9me3 on EBF1 gene expression. Results: Compared to normal B cells, the lost enhancers (genomic regions defined by decreased enrichment of H3K4me1 and H3K27ac) in CLL B cells showed increased heterochromatin marks (H3K9me3 and H3K27me3) enrichment. Importantly, these enhancers are associated with genes downregulated in CLL and involved in the immune response, indicating the source of epigenetic impact on impaired immunity in CLL. We further found that this aberrant chromatin structure also exits in clonal B cells from the precursor stage of MBL, implicating the importance of epigenetic silencing in CLL evolution. In particular, we found the lost enhancer signature across the gene locus EBF1, an important transcription factor that defines B cell lineage and regulates normal B cell activation. Using CRISPR-mediated epigenetic editing, we demonstrated that the gain of H3K9me3 is sufficient to silence the EBF1 enhancer and its gene expression. Finally, we found that while BTKi ibrutinib treatment partially inhibits the CLL-gained enhancers in some patients, it failed to restore the lost enhancers in CLL B cells. Conclusion: In summary, we have elucidated an epigenetic silencing mechanism that may contribute to the loss of physiological function of normal B cells, which places the clonal B cells at a selection advantage during CLL evolution and increases the risk of serious infections in CLL and MBL ( Fig 1).

Uncovering the Cis-Regulatory Program of Early Human B-Cell Commitment and Its Implications in the Pathogenesis of B-Cell Acute Lymphoblastic Leukemia

The early stages of the B-cell system are key for cellular immunity development. The development of functional B cells requires appropriate regulation of dynamic transcription-factor networks. These regulatory networks are responsible for lineage-specific gene expression activation and lineage commitment. Thus, understanding the gene regulatory network (GRN) of this system is essential for studying healthy development and malignant transformations. To this end, in the present study, we aim to uncover the complete chart of the cis-regulatory elements and their dynamic activity over early B-cell commitment in healthy humans. We generated matched human data for chromatin accessibility (ATAC-Seq) and transcriptome (RNA-Seq) in eight sorted B-cell subpopulations (HSCs, CLPs, pro-B cells, pre-B cells, Immature B cells, and Transitional B cells as well as CD5+ and CD5- Naive B cells) isolated from bone marrow and peripheral blood from the same healthy donors (n=13). These comprehensive profiles of the stepwise progression of B-cell precursors and the combined analysis of both omics allowed us to infer the regulatory elements of B-cell differentiation and the resulting GRNs. Our data identified 169 TFs, 7,310 genes, and 16,074 OCRs governing the early B-cell differentiation, resulting in a GRN with 377,268 TF-OCR-gene interactions. The inferred GRN included genes and OCRs able to draw the entire regulatory dynamics over differentiation behaving in accordance with established knowledge. As example, we identified genes and TFs related to homeostasis and stemness, such as MEIS1, CD34, and CDH2, and antiapoptotic genes such as BCL2, highly expressed in HSC, CLP, and pro-B cells. We identified surface marker genes and TFs involved in the commitment and differentiation of B cell lineage, including CD79A/B, PAX5, TCF3, EBF1, and FOXO1, to become upregulated in pro-B cells, as well as down-regulation in pre-B cells of genes linked to T cell development such as NOTCH1. To identify the key regulatory elements along B cell differentiation, we generated a snapshot of the GRN for each cell subpopulation. As a result, the networks' node hub architectures revealed those TFs with relevant transcriptional activity per subpopulation (Figure 1). In the analysis we recovered the primary TFs involved in B cell commitment, differentiation, maintenance, and class-switch recombination: TCF3, BCL11A, IRF8, PAX5, REL, IRF4, EBF1, POU2F2, FOXO1, and LEF1; but also identified ELK3 as a novel TF critical in pro-B cells. Interestingly, this ETS-factor, recently defined as a novel oncogene, has been reported to increase in ETV6-RUNX1+ acute lymphoblastic leukemia cases and other carcinogenic processes. Moreover, it has been associated with pro-B cell expansion. In a deeper approach, our GRNs allowed the exploration of upstream regulators of specific genes. In the case of ELK3, the TFs ERG, STAT5A/B, E2F3, ETS2, ETV6, SPIB, and POU2F2 were uncovered as putative regulators. As a positive control, we explored the upstream regulation of EBF1 gene, a well-known and key TF of B cell fate. Besides the already described regulators (PAX5, TCF3, FOXO1, RUNX1, etc.), we identified new candidates such as MLXIP. Finally, we translated our findings into a disease context using public data. Our resource facilitated the identification of triggers for specific B-cell acute lymphoblastic leukemia (B-ALL) subtypes, pinpointing the inflection point of malignant transformation. We found ETV6-RUNX1 fusions significantly enriched in pro-B cells, MEF2D fusions and TCF3-PBX1 mutations in pre-B cells, DUX4 fusions in HSCs, and KMT2A fusions in CLPs. Moreover, our data shed light on the functional implications of B-ALL susceptibility loci. The susceptibility locus rs17481869, associated with ETV6-RUNX1 patients, was identified within a small OCR specific to pro-B cells. The ALL-associated rs4762284 locus was observed in a closed chromatin region, but we could identify two proxies (rs2268508 and rs17736737) in open regions. Furthermore, from the list of SNPs associated with BCP ALL-related traits from the GWAS catalog (NHGRI-EBI), we identified 11 SNPs overlapping some of our OCRs. In summary, we are presenting the most comprehensive publicly available atlas of early B cell regulation (B-rex; https://brex.shinyapps.io/brex/), and therefore a powerful resource for understanding early B cell differentiation in health and disease.

EBF1 Promotes the Sensitivity of Cervical Cancer Cells to Cisplatin via Activating FBN1 Transcription

Cisplatin (DDP) is widely used in the chemotherapy of cervical cancer (CC), the fourth most common female malignancy worldwide. However, some patients progress to chemotherapy resistance, which leads to chemotherapy failure, tumor recurrence, and poor prognosis. Therefore, strategies to identify the regulatory mechanisms underlying CC development and increase tumor sensitivity to DDP will help improve patient survival. This research was designed to ascertain the mechanism of EBF1-dependent regulation of FBN1 which promotes chemosensitivity of CC cells. The expression of EBF1 and FBN1 was measured in CC tissues resistant or sensitive to chemotherapy and in DDP-sensitive or -resistant cells (SiHa and SiHa-DDP cells). SiHa-DDP cells were transduced with lentiviruses encoding EBF1 or FBN1 to evaluate the influence of these two proteins on cell viability, expression of MDR1 and MRP1, and cell aggressiveness. Moreover, the interaction between EBF1 and FBN1 was predicted and demonstrated. Finally, to further verify the EBF1/FB1-dependent mechanism of DDP sensitivity regulation in CC cells a xenograft mouse model of CC was established using SiHa-DDP cells transduced with lentiviruses carrying EBF1 gene and shRNA directed to FBN1.EBF1 and FBN1 showed decreased expression in CC tissues and cells, particularly in those resistant to chemotherapy. Transduction of SiHa-DDP cells with lentiviruses encoding EBF1 or FBN1 lead to decreased viability, IC50, proliferation capacity, colony formation ability, aggressiveness, and increased cell apoptosis. We have shown that EBF1 activates FBN1 transcription by binding to FBN1 promoter region. Additionally, it was revealed that FBN1 silencing reversed the promoting effect of EBF1 overexpression on chemosensitivity of CC cells in vivo. EBF1 facilitated chemosensitivity in CC cells by activating FBN1 transcription.

WITHDRAWN: Effect of shRNA‑mediated knockdown EBF1 gene expression on the proliferation of lung cancer cell line A549 invitro and invivo.

The paper entitled "Effect of shRNA‑mediated knockdown EBF1 gene expression on the proliferation of lung cancer cell line A549 invitro and invivo" by Lin Wang et al, which was published online on 16 March 2023, has been withdrawn at the authors' request.

EBF1 Promotes the Sensitivity of Cervical Cancer Cells to Cisplatin via Activating FBN1 Transcription

Cisplatin (DDP) is widely used in the chemotherapy of cervical cancer (CC), the fourth most common female malignancy worldwide. However, some patients progress to chemotherapy resistance, which leads to chemotherapy failure, tumor recurrence, and poor prognosis. Therefore, strategies to identify the regulatory mechanisms underlying CC development and increase tumor sensitivity to DDP will help improve patient survival. This research was designed to ascertain the mechanism of EBF1-dependent regulation of FBN1 which promotes chemosensitivity of CC cells. The expression of EBF1 and FBN1 was measured in CC tissues resistant or sensitive to chemotherapy and in DDP-sensitive or -resistant cells (SiHa and SiHa-DDP cells). SiHa-DDP cells were transduced with lentiviruses encoding EBF1 or FBN1 to evaluate the influence of these two proteins on cell viability, expression of MDR1 and MRP1, and cell aggressiveness. Moreover, the interaction between EBF1 and FBN1 was predicted and demonstrated. Finally, to further verify the EBF1/FB1-dependent mechanism of DDP sensitivity regulation in CC cells a xenograft mouse model of CC was established using SiHa-DDP cells transduced with lentiviruses carrying EBF1 gene and shRNA directed to FBN1 EBF1 and FBN1 showed decreased expression in CC tissues and cells, particularly in those resistant to chemotherapy. Transduction of SiHa-DDP cells with lentiviruses encoding EBF1 or FBN1 lead to decreased viability, IC50, proliferation capacity, colony formation ability, aggressiveness, and increased cell apoptosis. We have shown that EBF1 activates FBN1 transcription by binding to FBN1 promoter region. Additionally, it was revealed that FBN1 silencing reversed the promoting effect of EBF1 overexpression on chemosensitivity of CC cells in vivo. EBF1 facilitated chemosensitivity in CC cells by activating FBN1 transcription.

EBF1 Negatively Regulates Brassinosteroid-Induced Apical Hook Development and Cell Elongation through Promoting BZR1 Degradation

Brassinosteroids (BRs) are a group of plant steroid hormones that play important roles in a wide range of developmental and physiological processes in plants. Transcription factors BRASSINOZALE-RESISTANT1 (BZR1) and its homologs are key components of BR signaling and integrate a wide range of internal and environmental signals to coordinate plant growth and development. Although several E3 ligases have been reported to regulate the stability of BZR1, the molecular mechanism of BZR1 degradation remains unclear. Here, we reveal how a newly identified molecular mechanism underlying EBF1 directly regulates BZR1 protein stability via the 26S proteasome pathway, repressing BR function on regulating Arabidopsis apical hook development and hypocotyl elongation. BZR1 directly binds to the EBF1 gene promotor to reduce EBF1 expression. Furthermore, the genetic analysis shows that BZR1, EIN3 and PIF4 interdependently regulate plant apical hook development. Taken together, our data demonstrates that EBF1 is a negative regulator of the BR signaling pathway.

EBF1 Promotes Cell Proliferation, Migration and Inhibits Cell Apoptosis in AML

Background: Multiple studies reported that EBF1 played important roles in types of cancers. However, the potential molecular regulatory mechanism of EBF1 in acute myeloid leukemia (AML) remains to be further defined. In this study, we aimed to clarify the role and potential mechanism of EBF1 in AML. Method: The expression levels of EBF1 were assessed in AML patients and cell lines by RT-qPCR and western blots. The expression of EBF1 was up-regulated or knocked down in AML cell lines by lentivirus mediated overexpression and RNA interference. CCK-8, flow cytometry and trans well assays were used to analyze the effect of EBF1 on cell proliferation, cycle, apoptosis and migration. Results: The level of EBF1 expression was up-regulated in newly diagnosed AML patients and cell lines. The EBF1 gene expression significantly reduced in CR patients and significantly increased in relapsed patients as compared to newly diagnosed AML patients (both P <0.001). EBF1 knockdown repressed cell proliferation, migration, and induced cell apoptosis and cell cycle G0/G1 inhibition in AML cells. EBF1 overexpression promoted the malignancy of AML cells. Conclusion: Our findings manifested that EBF1 was remarkably upregulated in AML and downregulation of EBF1 repressed the malignant behaviors of AML cells, which would provide a new insight for the treatment of AML.

Gene copy number alterations in Indian children with B-acute Lymphoblastic Leukemia: Correlation with survival outcome

BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common lymphoid malignancy occurring in children. Copy number alterations like CDKN2A/B, IKZF1, ETV6, RB1 and BTG1 gene deletions have been shown to be associated with differential survival outcome. The literature on survival outcome of children with each gene deletion is very limited. The targeted therapy for some of these gene deletions are under trial in adult ALL. Our study will provide insight in the role of such genes in disease biology. MethodsA total of 91 children with newly diagnosed ALL were enrolled and monitored for response to treatment and were followed up for 4.0 years and analyzed for copy number alterations by Multiplex ligation dependent probe amplification assay (MLPA) using SALSA MLPA probemix P335–B2 ALL-IKZF1 kit. Survival analysis was performed using event free survival (EFS) as the end point.Survival curves between two groups were calculated using Kaplan-Meier analysis and compared using log-rank tests. Other comparisons were performed using the Chi-squared or Fisher's exact test. All analyses were performed using Stata 14.0. ResultsWe observed gene copy number alterations in 51.6% of children with B-ALL subgroup. CDKN2A/B was the most commonly observed gene deletion followed by PAX5, ETV6, IKZF1, RB1, BTG1 and EBF1. Children with CDKN2A/B, IKZF1, RB1 and BTG1 gene deletion had significantly lesser event free survival as compared to those without gene deletions in short term follow up while ETV6 and EBF1 gene deletions and PAR1 gene alterations did not have any impact on survival outcome. DiscussionEvent free survival in children with CDKN2A/B, IKZF1, RB1 and BTG1 gene deletions was significantly lower in short term possibly due to aggressive disease phenotype with faster progression. We found PAR1 gene alterations to be associated with better event free survival in long term follow up though it did not show significant association. ConclusionsCDKN2A/B, IKZF1 and RB1 gene deletions were independently associated with poor event free survival. We opine that individual gene deletions, and not integrated copy number alterations profile, may be used for risk stratification and the CDKN2A/B, IKZF1 and RB1 may serve as poor prognostic markers.

F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder.

Cold stress adversely affects plant production, both qualitatively and quantitatively. Banana (Musa acuminata) is sensitive to cold stress and suffers chilling injury (CI) when stored under 11°C, causing abnormal fruit softening. However, the mechanism underlying the abnormal fruit softening due to CI remains obscure. This study uncovered the coordinated transcriptional mechanism of ethylene F-box (EBF1) protein and abscisic acid-insensitive 5 (ABI5)-like protein in regulating chilling-induced softening disorders of Fenjiao banana. Cold stress severely inhibited the transcript and protein levels of EBF1, ABI5-like, and fruit softening-related genes. The ABI5-like protein bound to the promoters of key starch and cell wall degradation-related genes such as β-amylase 8 (BAM8), pectate lyase 8 (PL8), and β-D-xylosidase23-like (XYL23-like) and activated their activities. EBF1 physically interacted with ABI5-like and enhanced the transcriptional activity of the key starch and cell wall degradation-related genes but did not ubiquitinate or degrade ABI5-like protein. This promoted fruit ripening and ameliorated fruit CI in a manner similar to the effect of exogenous abscisic acid treatment. The ectopic and transient overexpression of EBF1 and ABI5-like genes in tomato (Solanum lycopersicum) and Fenjiao banana accelerated fruit ripening and softening by promoting ethylene production, starch and cell wall degradation, and decreasing fruit firmness. EBF1 interacted with EIL4 but did not ubiquitinate or degrade EIL4, which is inconsistent with the typical role of EBF1/2 in Arabidopsis (Arabidopsis thaliana). These results collectively highlight that the interaction of EBF1 and ABI5-like controls starch and cell wall metabolism in banana, which is strongly inhibited by chilling stress, leading to fruit softening and ripening disorder.

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.

Retraction.

Reraction: "EBF1 gene promotes the proliferation and inhibits the apoptosis of bone marrow CD34+cells in patients with myelodysplastic syndrome through negative regulation of mitogen-activated protein kinase axis," by Shuang Hou, Jie Hao, Yan-Yu Wang, Bing-Bing Zhao, Gong-Wei Xiao, Yan-Qing Li, Xi Liu, Zhi-Lan Zou, Ye Yao, Hong Xiong, J Cell Biochem. 2019; 1407-1419: The above article, published online on 18 October 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27177) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context. Thus, the editors consider the conclusions of this article to be invalid.

Thyroid Hormone Receptor α1 Mutants Impair B Lymphocyte Development in a Mouse Model.

Background: Mutations of the thyroid hormone receptor α (THRA) gene cause resistance to thyroid hormone (RTHα). RTHα patients exhibit very mild abnormal thyroid function test results (serum triiodothyronine can be high-normal to high; thyroxine normal to low; thyrotropin is normal or mildly raised) but manifest hypothyroid symptoms with growth retardation, delayed bone development, and anemia. Much has been learned about the in vivo molecular actions in TRα1 mutants affecting abnormal growth, bone development, and anemia by using a mouse model of RTHα (Thra1PV/+ mice). However, it is not clear whether TRα1 mutants affect lymphopoiesis in RTHα patients. The present study addressed the question of whether TRα1 mutants could cause defective lymphopoiesis. Methods: We assessed lymphocyte abundance in the peripheral circulation and in the lymphoid organs of Thra1PV/+ mice. We evaluated the effect of thyroid hormone on B cell development in the bone and spleen of these mice. We identified key transcription factors that are directly regulated by TRα1 in the regulation of B cell development. Results: Compared with wild-type mice, a significant reduction in B cells, but not in T cells, was detected in the peripheral circulation, bone marrow, and spleen of Thra1PV/+ mice. The expression of key transcription regulators of B cell development, such as Ebf1, Tcf3, and Pax5, was significantly decreased in the bone marrow and spleen of Thra1PV/+ mice. We further elucidated that the Ebf1 gene, essential for lineage specification in the early B cell development, was directly regulated by TRα1. Thus, mutations of TRα1 could impair B cell development in the bone marrow via suppression of key regulators of B lymphopoiesis. Conclusions: Analysis of lymphopoiesis in a mouse model of RTHα showed that B cell lymphopoiesis was suppressed by TRα1 mutations. The suppressed development of B cells was, at least in part, via inhibition of the expression of key regulators, Ebf1, Tcf3, and Pax5, by TRα1 mutations. These findings suggest that the mutations of the THRA gene in patients could lead to B cell deficiency.

Abnormal thymic B cell activation and impaired T cell differentiation in pristane-induced lupus mice

Changes in the thymus and potential mechanisms underlying the pathogenesis in pristane-induced lupus (PIL) mice are poorly understood. This study aimed to systematically and specifically examine changes in the thymus and the potential mechanisms responsible for immunological abnormalities in PIL mice. The results showed that PIL mice exhibit serious thymic hyperplasia, an elevated thymus index, a damaged histopathological structure and increased thymocyte apoptosis. We found that thymic T cell differentiation was impaired as the CD4+ CD8+ double-positive (DP) thymocyte frequency significantly decreased, becoming almost absent at 28 weeks after induction, while CD4 CD8- double-negative (DN) thymocytes and CD4+ CD8- single-positive (CD4+ SP) and CD4 CD8+ single-positive (CD8+ SP) cells were increased. This phenomenon might be explained by an inhibition of the DN-to-DP-cell transition and stimulation of DP cell conversion into CD4+ /CD8+ SP thymocytes. Moreover, we discovered a dramatic and abnormal increase in thymic B cells, that was associated with CD19, Irf8, Ebf1, Pax5, Irf4, Blk, CXCL13, CXCR5, CD79a, CD79b, Lyn, Syk, Btk, and BLNK gene accumulation, which exhibited positive interactions. We further verified that the mRNA expression of these genes was significantly upregulated and consistent with the RNA-seq results. These results suggest a role of these genes in the increase of B cells in the thymus of PIL mice. In summary, our results showed the changes in the thymus in PIL and elucidated the immunologic abnormalities of increased B cells, potentially providing insight into the associated molecular mechanisms and facilitating further research.

C/EBPα induces Ebf1 gene expression in common lymphoid progenitors.

C/EBPα is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7Rα-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D+ and 65% of upstream Ly6D- CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBPα during B lineage development, with reduced enhancer deletion in Ly6D- CLP mediated by IL7Rα-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced ~40-fold in Flt3+Sca-1intc-kitintIL7Rα+ CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4+ versus hCD4- CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBPα using the electrophoretic mobility shift assay. Mutation of this C/EBPα-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBPα-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBPα in a subset of CLP contributes to initiation of B lymphopoiesis.

SIAH2 is Expressed in Adipocyte Precursor Cells and Interacts with EBF1 and ZFP521 to Promote Adipogenesis.

Expression of zinc finger protein 423 (ZFP423), a key proadipogenic transcription factor in adipocyte precursor cells, is regulated by interaction of the proadipogenic early B-cell factor 1 (EBF1) and antiadipogenic ZFP521. The ubiquitin ligase seven-in-absentia homolog 2 (SIAH2) targets ZFP521 for degradation. This study asked whether SIAH2 is expressed in adipocyte precursor cells and whether SIAH2 interacts with ZFP521 and EBF1 to regulate ZFP521 protein levels during adipogenesis. SIAH2 expression in precursor cells was assessed in primary cells and tissues from wild-type and SIAH2 null mice fed a control or high-fat diet. Primary cells, 3T3-L1 preadipocytes, and HEK293T cells were used to analyze Siah2, Ebf1, and Zfp521 expression and SIAH2-mediated changes in ZFP521 and EBF1 protein levels. Siah2 is expressed in platelet-derived growth factor receptor α (PDGFRα)+ and stem cell antigen-1 (SCA1)+ adipocyte precursor cells. SIAH2 depletion reduces Ebf1 gene expression and increases EBF1 protein levels in early but not late adipogenesis. In early adipogenesis, SIAH2 forms a protein complex with EBF1 and ZFP521 to enhance SIAH2-mediated ubiquitylation and degradation of ZFP521 while increasing EBF1 protein levels. Siah2 is expressed in PDGFRα+ adipocyte precursor cells and is linked to precursor cell commitment to adipogenesis by interacting with EBF1 and ZFP521 proteins to target the antiadipogenic ZFP521 for degradation.

EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression, and folate metabolism.

EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.