Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation Gene 2: About 2 Cases

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of inherited immunologic disorders with profound defects in cellular and humoral immunity. SCID is the most severe PID and constitutes a pediatric emergency. Affected children are highly susceptible to bacterial, viral, fungal, and opportunistic infections with life-threatening in the absence of hematopoietic stem cell transplantation. We report here two cases of SCID. The first case is a girl diagnosed with SCID at birth based on her family history and lymphocyte subpopulation typing. The second case is a 4-month-old boy with a history of recurrent opportunistic infections, BCGitis, and failure to thrive, and the immunology workup confirms a SCID phenotype. The genetic study in the two cases revealed a novel mutation in the RAG2 gene, c.826G > A (p.Gly276Ser), in a homozygous state. The novel mutation in the RAG2 gene identified in our study may help the early diagnosis of SCID.

Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.

KERAGAMAN GENETIK IKAN TIGER FISH (Datnioides sp.) ASAL KALIMANTAN DAN SUMATERA

Ikan tiger fish (Datnioides sp.) merupakan ikan hias air tawar yang memiliki nilai ekonomis penting. Distribusi populasi ikan ini meliputi Papua, Kalimantan, dan Sumatera, dengan tingkat eksploitasi yang cukup tinggi di dua lokasi terakhir. Penelitian ini dilakukan untuk mendapatkan informasi keragaman genetik ikan tiger fish yang mendiami perairan Kalimantan dan Sumatera. Sebanyak 24 sampel ikan uji dikoleksi dari Sungai Kapuas, Kalimantan Barat dan Sungai Musi, Sumatera Selatan. Penelitian dilakukan dalam dua tahap, tahap pertama yaitu identifikasi molekuler dengan menggunakan DNA barcoding gen cytochrome oxidase 1 (COI), tahap kedua adalah analisis keragaman genetik dengan menggunakan marka DNA mitokondria gen cytochrome b (Cyt b), dan DNA inti gen recombination activating gene (RAG2). Hasil identifikasi secara molekuler menunjukkan bahwa ikan hasil koleksi memiliki kesamaan genetik sebesar 100% dengan spesies D. undecimradiatus. Keragaman genetik ikan tiger fish antar populasi berkisar pada nilai 0,023 (standar deviasi 0,001) sedangkan keragaman intra populasi adalah sebesar 0,002 dan 0,003 masing-masing untuk populasi Kalimantan dan Sumatera. Jarak genetik sampel baik yang berasal dari Sumatera maupun Kalimantan dengan spesies D. undeciumradiatus masing-masing 0,003 dan 0,006; sedangkan dengan spesies D. microlepis yaitu 0,142. Analisis menggunakan gen RAG2 menunjukkan sampel yang diuji memiliki struktur populasi yang terpisah ditandai dengan terjadinya mutasi pada enam nukleotida dan tiga asam amino.The Tiger fish (Datnioides sp.) is a freshwater ornamental fish that has important economic value. The distribution of this fish included Papua, Kalimantan, and Sumatra, but intensive exploitation occurs in the last two population. This research was conducted to obtain the genetic diversity of tiger fish that inhabited in Kalimantan and Sumatra. A total of 24 fish were collected from Kapuas River, West Kalimantan and Musi River, at Sumatra. The study was conducted in two stages, the first stage is molecular identification of sample by using DNA barcoding cytochrome oxidase 1 (COI) gene, the second stage is analyses of genetic diversity of tiger fish within and between population by using the mitochondrial DNA cytochrome b (Cyt b) gene, and nucleus DNA recombination (RAG2) gene. The molecular identification has shown that the collected fish has a genetic similarity of 100% with D. undecimradiatus. The genetic diversity of tiger fish between populations is 0.023 (standard deviation of 0.001) whereas intra-population is 0.002 and 0.003 for Kalimantan and Sumatra, respectively. The genetic distance of samples with species D. undeciumradiatus were 0.003 and 0.006 for Kalimantan and Sumatera, respectively, whereas the genetic distance with D. microlepis was 0.142. The analysis of mutation on RAG2 gene shows there are six nucleotides and three amino acids have mutation.

Corrected and Republished from: BCL11A Is a Critical Component of a Transcriptional Network That Activates RAG Expression and V(D)J Recombination

ABSTRACT Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.