Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies.

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.

Transcription-factor-mediated supervision of global genome architecture maintains B cell identity.

Recent studies have elucidated cell-lineage-specific three-dimensional genome organization; however, how such specific architecture is established or maintained is unclear. We hypothesized that lineage-defining transcription factors maintain cell identity via global control of genome organization. These factors bind many genomic sites outside of the genes that they directly regulate and thus are potentially implicated in three-dimensional genome organization. Using chromosome-conformation-capture techniques, we show that the transcription factor Paired box 5 (Pax5) is critical for the establishment and maintenance of the global lineage-specific architecture of B cells. Pax5 was found to supervise genome architecture throughout B cell differentiation, until the plasmablast stage, in which Pax5 is naturally silenced and B cell-specific genome structure is lost. Crucially, Pax5 did not rely on ongoing transcription to organize the genome. These results implicate sequence-specific DNA-binding proteins in global genome organization to establish and maintain lineage fidelity.

Antagonism of B cell enhancer networks by STAT5 drives leukemia and poor patient survival.

The transcription factor STAT5 has a critical role in B cell acute lymphoblastic leukemia (B-ALL). How STAT5 mediates this effect is unclear. Here we found that activation of STAT5 worked together with defects in signaling components of the precursor to the B cell antigen receptor (pre-BCR), including defects in BLNK, BTK, PKCß, NF-κB1 and IKAROS, to initiate B-ALL. STAT5 antagonized the transcription factors NF-κB and IKAROS by opposing regulation of shared target genes. Super-enhancers showed enrichment for STAT5 binding and were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4 and IKAROS. Patients with a high ratio of active STAT5 to NF-κB or IKAROS had more-aggressive disease. Our studies indicate that an imbalance of two opposing transcriptional programs drives B-ALL and suggest that restoring the balance of these pathways might inhibit B-ALL.

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.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5.

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation.

While PAX5 is an important tumor suppressor gene in B-cell acute lymphoblastic leukemia (B-ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5-JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating exogenous gene mutation. The DNA-binding function and kinase activity of Pax5-Jak2 as well as IL-7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild-type Pax5 allele, allowing efficient DNA-binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B-ALL tumors, implying that nuclear Pax5-Jak2 phosphorylates STAT5. Together, these data reveal Pax5-Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Interactions between lineage-associated transcription factors govern haematopoietic progenitor states.

Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states and lineage choice remain ill-defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8-FL) to knock out 39 transcription factors (TFs) followed by RNA-Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B-lymphoid TF Ebf1 and T-lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3-mediated repression of Pax5 emerges as a mechanism to prevent precocious B-lymphoid differentiation, while Hox-mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.

Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop.

BACKGROUND: Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and mechanisms of circRNAs in human clear cell renal cell carcinoma (ccRCC) have yet to be thoroughly investigated. METHODS: Patient cohorts from online databases were used to screen candidate circRNAs, while another cohort from our hospital was obtained for validation. CircSOD2 was identified as a potential oncogenic target, and its relevant characteristics were investigated during ccRCC progression through various assays. A positive feedback loop containing downstream miRNA and its target gene were identified using bioinformatics and validated by luciferase reporter assays, RNA pull-down, and high-throughput sequencing. RESULTS: CircSOD2 expression was elevated in tumor samples and significantly correlated with overall survival (OS) and the tumor stage of ccRCC patients, which appeared in the enhanced proliferation, invasion, and migration of tumor cells. Through competitive binding to circSOD2, miR-532-3p can promote the expression of PAX5 and the progression of ccRCC, and such regulation can be salvaged by miR-532-3p inhibitor. CONCLUSION: A novel positive feedback loop, PAX5/circSOD2/miR-532-3p/PAX5 was identified in the study, indicating that the loop may play an important role in the diagnosis and prognostic prediction in ccRCC patients.

Diagnostic efficacy of SEPT9 and PAX5 gene methylation in gastrointestinal cancer and precancerous lesions.

To assess the diagnostic efficacy of SEPT9 along with PAX5 gene methylation detection in gastrointestinal cancer and precancerous lesions, the peripheral blood of 62 patients with gastric cancer (GC) and 60 patients with no evidence of disease (as the control group) were retrospectively collected. The methylation rates of PAX5 and SEPT9 gene promoters in blood samples of GC group were detected by PCR. At the same time, the differences in methylation rates of genes in the two groups were compared, and the predictive value of plasma methylation PAX5 and SEPT9 in GC was evaluated by receiver operating characteristic (ROC) curve. We found that there were 41 cases of methylated PAX5 gene promoter region and 39 cases of methylated SEPT9 gene promoter region in GC group. The control group contained 14 cases of PAX5 gene promoter methylation and 12 cases of RNF¹80 gene promoter methylation. The occurrence of PAX5 promoter methylation was correlated with age of GC patients. There were statistically significant differences in mSEPT9 gene in patients with different TNM stages. Kaplan-Meier survival curve analysis revealed that the three-year overall survival rate of GC patients with PAX5 methylation was lower than that of GC patients without PAX5 methylation. No significant difference was discovered in 3-year overall survival rate between GC patients with SEPT9 methylation and those without SEPT9 methylation. Combined detection could not improve the diagnostic value of GC, but could promote diagnosis sensitivity. In summary, the risk of PAX5 and SEPT9 gene methylation in GC patients presents higher when compared with healthy people. PAX5 gene methylation is closely related to age, while SEPT9 is closely related to tumor TNM stage, and PAX5 gene methylation can decrease the survival rate of GC patients. Detection of PAX5 gene methylation level can assist in evaluating the prognosis of GC patients.

Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program.

In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.

Mycovirus-Containing Aspergillus flavus Alters Transcription Factors in Normal and Acute Lymphoblastic Leukemia Cells.

Transcription factors control genes to maintain normal hemopoiesis, and dysregulation of some factors can lead to acute lymphoblastic leukemia (ALL). Mycoviruses are known to alter the genetics of their fungal host. The present study evaluates the effects of the products of a mycovirus-containing Aspergillus flavus (MCAF), isolated from the home of a patient with ALL, on certain transcription factors of normal and ALL cell lines. Our published studies have shown that ALL patients have antibodies to MCAF, and that exposure of the mononuclear leukocytes of patients in complete remission to its products, unlike controls, results in the re-development of genetic and cell surface phenotypes characteristic of ALL. For the present study, normal, pre-B, and B-cell leukemia cell lines were exposed to the culture of MCAF. Pre- and post-exposure levels of PAX5, Ikaros, and NF-κB were assessed. Exposure to MCAF resulted in apoptosis, cell cycle changes, and complete downregulation of all transcription factors in normal cell lines. In acute leukemia cell lines, cellular apoptosis and alterations in the cell cycle were also noted; however, while there was downregulation of all tested transcription factors, residual levels were retained. The noted alterations in the transcription factors caused by MCAF are novel findings. The possible role of MCAF in leukemogenesis needs to be further investigated. Mycovirus-containing Aspergillus flavus was initially isolated from a leukemia patient's home. Our prior published studies have illuminated intriguing associations of this organism with leukemia. Unlike controls, patients diagnosed with acute lymphoblastic leukemia (ALL) harbor antibodies to this organism. Furthermore, the exposure of mononuclear cells from patients with ALL in complete remission to the products of this organism reproduced genetic and cell phenotypes characteristic of ALL. These findings underscore the potential role of environmental factors in leukemogenesis and hint at novel avenues for therapeutic intervention and preventive strategies.

PAX5 and circ1857 affected DLBCL progression and B-cell proliferation through regulating GINS1.

PAX5, a member of the paired box gene family of transcription factors, is a B-cell-specific activator protein that plays important roles during B lymphopoiesis. Two putative PAX5 binding sites in the human GINS1 promoter region were identified. EMSA, ChIP and luciferase assay showed that PAX5 functions as a positive transcription factor for GINS1 expression. Furthermore, coordinated expression of PAX5 and GINS1 was observed in mice B cells under physiological conditions and LPS stimulation situations. A similar pattern was also observed in human DLBCL cell lines under differentiation-inducing conditions. In addition, both PAX5 and GINS1 were highly expressed and significantly correlated in DLBCL specimens and cell lines. These findings suggested that dysregulation of PAX5 played an extremely important role in controlling the universal phenomenon of tumor progression through increased expression of GINS1 in DLBCL. In addition, circ1857 that was generated using back splicing of PAX5 pre-mRNA could further stabilize GINS1 mRNA, modulate GINS1 expression and promote lymphoma progression. To the best of our knowledge, this report is the first to demonstrate the role of GINS1 in DLBCL progression, and the mechanism of GINS1 upregulation using both circ1857 and PAX5 in DLBCL was revealed. Our results suggested that GINS1 may be a possible therapeutic target for DLBCL.

PAX5-miR-142 feedback loop promotes breast cancer proliferation by regulating DNMT1 and ZEB1.

BACKGROUND: Breast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain. METHODS: We identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP. RESULTS: PAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3'UTR region, respectively. CONCLUSION: In summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy.

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.

B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

Continuous Culture of Mouse Primary B Lymphocytes by Forced Expression of Bach2.

Stable, long-term culture of primary B lymphocytes has many potential scientific and medical applications, but remains an elusive feat. A major obstacle to long-term culture is that in vitro mitogens quickly drive B cells to differentiate into short-lived plasma cells (PCs). PC differentiation is governed by opposing teams of transcription factors: Pax5, Bach2, and Bcl6 suppress PC commitment, whereas IFN regulatory factor 4 and Blimp1 promote it. To determine whether transcriptional programming could prolong B cell culture by blocking PC commitment, we generated mouse primary B cells harboring gain- or loss-of-function in the key transcription factors, continuously stimulated these cells with CD154 and IL-21, and determined growth potential and phenotypes in vitro. We found that transgenic expression of Bach2 prohibits PC commitment and endows B cells with extraordinary growth potential in response to external proliferation and survival cues. Long-term Bach2-transgenic B cell lines have genetically stable BCRs [i.e., do not acquire V(D)J mutations], express high levels of MHC class II and molecules for costimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established transgenic cell line causes the cells to secrete large quantities of Ig. This system has potential applications in mAb production, BCR signaling studies, Ag presentation to T cells, and ex vivo clonal expansion for adoptive cell transfer. Additionally, our results provide insight into molecular control over activated B cell fate and suggest that forced Bach2 expression in vivo may augment germinal center B cell or memory B cell differentiation at the expense of PC commitment.

Metabolic gatekeeper function of B-lymphoid transcription factors.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

A structural hierarchy mediated by multiple nuclear factors establishes IgH locus conformation.

Conformation of antigen receptor gene loci spatially juxtaposes rearranging gene segments in the appropriate cell lineage and developmental stage. We describe a three-step pathway that establishes the structure of the 2.8-Mb immunoglobulin heavy chain gene (IgH) locus in pro-B cells. Each step uses a different transcription factor and leads to increasing levels of structural organization. CTCF mediates one level of compaction that folds the locus into several 250- to 400-kb subdomains, and Pax5 further compacts the 2-Mb region that encodes variable (VH) gene segments. The 5' and 3' domains are brought together by the transcription factor YY1 to establish the configuration within which gene recombination initiates. Such stepwise mechanisms may apply more generally to establish regulatory fine structure within megabase-sized topologically associated domains.