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

Naik, Abani Kanta; Byrd, Aaron; Lucander, Aaron; Krangel, Michael S.

doi:10.1084/jem.20181402

Cited by 26 publications

(31 citation statements)

References 55 publications

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“…At the core of the recombination machinery are the RAG proteins 15 . The RAG gene locus, comprising RAG1 and RAG2, has been reported to be coordinately regulated by RUNX proteins and we confirm here Runx1 binding to the Rag1 core promoter 32,33 . The transcription factor RUNX1 is also involved in the regulation of T-cell-specific genes such as CD4 and CD8 23,34 .…”

Section: Discussionsupporting

confidence: 83%

Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

Seitz

Kleo

Dröge³

et al. 2020

Sci Rep

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T-cell receptor gene beta (TCRβ) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knockout mouse model and demonstrate by deep TCRβ sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRβ rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRβ gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRβ gene rearrangements in TALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1 +/− mouse model, heterozygous Runx1 mutations in TALL patients displayed no detectable impact on TCRβ rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions. RUNX1 belongs to the evolutionary conserved Runt transcription factor family and is indispensable for the establishment of definitive hematopoiesis in vertebrates and is an important regulator of cells of the immune system 1-3. RUNX1 is among the most frequently mutated genes in various hematological malignancies and RUNX1 alterations can lead to a loss of RUNX1 function or to a dominant-negative effect 4,5. Mono-allelic RUNX1 mutations occur in approximately 15% of T-Cell Acute Lymphoblastic Leukemia (T-ALL), predominantly in cases with an immature phenotype and a poor prognosis 6-8. In de novo Acute Myeloid Leukemia (AML) patients, somatic mutations in RUNX1 are detectable in approximately 3% of children and 15% of adults 4. In an AML subgroup with an immature phenotype (AML-M0), 30% of the cases are associated with bi-allelic inactivating RUNX1 point mutations and deletions 9. Patients with Myelodysplastic Syndrome (MDS) carrying RUNX1 mutations have a higher risk and shorter latency for progression to AML 10. Furthermore, there are over 50 different types of chromosomal translocations affecting RUNX1 4. We focus in this study on the most common translocation, t(12;21), occurring in approximately 20% of childhood B-cell

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Section: Discussionsupporting

confidence: 83%

Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

Seitz

Kleo

Dröge³

et al. 2020

Sci Rep

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“…Previous studies have identified several cis-regulatory elements in the Rag1/2 gene locus, including the anti-silencing element (ASE) and enhancer of Rag (Erag) (12,13). Deletion of these elements resulted in the partial loss of RAG1 and RAG2 activity, suggesting the presence of additional regulatory elements for Rag1/2 gene expression (5,(13)(14)(15). Besides, what TFs regulate these regulatory elements for Rag gene expression during T and B cell development remains to be determined.…”

Section: Introductionmentioning

confidence: 99%

The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells

et al. 2020

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Cell type–specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage–specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage–specific enhancer (R-TEn) and the two B cell–specific elements, R1B and R2B. By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.

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“…In addition, by using the VL3-3M2DP thymocyte cell line, this group demonstrated that the ASE requires Gata3 and E2A, while the Rag1 promoter relies on Runx1 and E2A as critical regulators. Together, the ASE and Rag1 promoter framework functions as a chromatin hub ( 65 ). Recently, we have identified cell type-specific CREs for Rag1/2 expression in B and T cells using E2A ChIP-seq and ATAC-seq data, because E2A specifies adaptive lymphocyte and is the driver of differences in enhancer repertoires in T cells and ILCs ( 24 , 52 ).…”

Section: The Regulation Of Rag1/2 Expression By CImentioning

confidence: 99%

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression

Miyazaki

2021

Front. Immunol.

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Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 (Rag1/2) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation.

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

Cited by 26 publications

References 55 publications

Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression

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