Ikaros and RAG-2-mediated antisense transcription are responsible for lymphocyte-specific inactivation of NWC promoter.

Agnieszka Łaszkiewicz,Paweł Kisielow,Monika Kasztura,Łukasz Bzdzion,Małgorzata Cebrat,Sylwia Janik,Łukasz Śnieżewski,Sebastian D Fugmann

doi:10.1371/journal.pone.0106927

Agnieszka Łaszkiewicz, Paweł Kisielow + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0106927

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Abstract

Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter - thus identifying a new Ikaros target - but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed.

Highlights

NWC (‘‘Nad Wyraz Ciekawy’’, which translates from Polish to ‘‘extremely interesting’’) is the third evolutionarily conserved gene within the recombination-activating genes RAG-1 and Recombination activating gene-2 (RAG-2) locus [1] encoding a protein complex indispensable for the recombination of immunoglobulin and T-cell receptor minigenes [2,3,4,5]
Ikaros binds to NWC promoter We have recently shown that the promoter of NWC gene is activated by ZFP-143 transcription factor, which binds two inverted evolutionarily conserved sites of the promoter [8] spanning 210/237 and 274/2100 nucleotides relative to transcriptional start site
In the present study we have shown that two mechanisms contribute to the lymphocyte specific inactivation of NWC promoter: Ikaros induced repression and DNA methylation, gained through cis-antisense transcription driven by RAG-2

Summary

Introduction

NWC (‘‘Nad Wyraz Ciekawy’’, which translates from Polish to ‘‘extremely interesting’’) is the third evolutionarily conserved gene within the recombination-activating genes RAG-1 and RAG-2 locus [1] encoding a protein complex indispensable for the recombination of immunoglobulin and T-cell receptor minigenes [2,3,4,5]. We suggested that NWC transcription may negatively control RAG-1 and RAG-2 promoter activities in non-lymphoid cells owing to transcriptional interference caused by NWC transcription proceeding through RAG-2 promoter and RAG-1/ RAG-2 cis-regulatory elements localized upstream RAG-2 gene [9]. This hypothesis has not been verified so far, since due to the remaining activity of a secondary promoter [10], we have been unable to abrogate completely the transcription of NWC in mice, in which primary NWC promoter was deleted

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