Abstract
Egr2 and Egr3 genes encode early growth response (EGR) transcription factors, act as immediate early genes following B cell receptor (BCR) signalling and are highly up-regulated in anergic ...mouse B cells. Anergy preserves microbial epitope responsiveness from a finite pool of pre-immune B cells, but is also reversible – creating a risk of autoimmune disease. Pathological proliferation of self-reactive B cells can also cause chronic lymphocytic leukemia (CLL), 3.8% of which harbour somatic missense EGR2 mutations resulting in loss- or change-of-function and correlated with poor prognosis. We analysed B cells in mice lacking one or both alleles of Egr2 and/or Egr3 and show that Egr2 and Egr3 deletion cause the cell-intrinsic accumulation in spleen, blood and bone marrow of populations enriched for self-reactive BCRs: B1a and CD21low CD23low age-associated B cells. Global single-cell RNA profiling of these expanded populations in vivo demonstrated their differential expression of genes involved in their survival and maintenance. We used chromatin immunoprecipitation sequencing (ChIP-Seq) to show that several of these genes are direct EGR2 transcriptional targets in human CLL. This is the first report on the roles of Egr2/3 in B cells and we demonstrate herein that Egr2 and Egr3 transcription factors are crucial in suppressing the accumulation of B1a and age-associated or atypical memory B cells.
•Tumor mutational load is a strong prognostic factor for progression to therapy among individuals with HCMBL, independent of CLL-IPI.•Accounting for both CLL-IPI and tumor mutational load, we ...identified individuals with HCMBL who have a worse prognosis than patients at low risk with CLL.
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High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.
Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, ...including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL.
Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85.
CD21
age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and ...immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21
and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21
B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.
Restriction digestion and real-time PCR (qAMP) Oakes, Christopher C; La Salle, Sophie; Trasler, Jacquetta M ...
Methods in molecular biology (Clifton, N.J.),
2009, Letnik:
507
Journal Article
DNA methylation in mammals has been shown to play many important roles in diverse biological phenomena. Here we describe a simple and straightforward method that quantitatively measures site-specific ...levels of DNA methylation in a quick and cost-effective manner. The quantitative analysis of DNA methylation using real-time PCR (qAMP) technique involves the digestion of genomic DNA using methylation-sensitive and methylation-dependent restriction enzymes followed by real-time PCR. This approach generates accurate and reproducible results without the requirement for prior treatment of the DNA with sodium bisulfite.
Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of ...tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."
We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.
The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation.
The osteoconductive and osteoinductive potential of two human allogeneic demineralized bone matrix putties were compared in a critical-sized athymic rat femoral defect model. Defects were treated ...with (1) a demineralized bone matrix in a hyaluronic acid carrier, (2) a demineralized bone matrix in a glycerol carrier, (3) a hyaluronic acid carrier alone, or (4) with no implant. Radiographic examinations and histologic analyses were done at 4, 8, and 16 weeks postoperatively. Eight of the 48 defects treated with a demineralized bone matrix and none of the 36 surgical controls showed complete radiographic healing by 16 weeks and no statistically significant difference between the radiographic scores for the two demineralized bone matrix preparations was found. On histologic review, both preparations of demineralized bone matrix had passive remineralization. The largest foci of endochondral ossification were seen in limbs treated with a demineralized bone matrix in a hyaluronic acid carrier. The 8-mm rat femoral defect allows for stringent assessment of the osteoinductive potential of bone graft substitutes. Hyaluronic acid and glycerol are viable carriers for demineralized bone matrices. As both de-mineralized bone matrices tested provided an adequate osteoconductive matrix and showed some, although limited, osteoinductive capacity, these materials should be used in clinical practice only as bone graft extenders or enhancers.
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