Ikaros and RAG-2-Mediated Antisense Transcription Are Responsible for Lymphocyte-Specific Inactivation of NWC Promoter

et al. (2014) Ikaros and RAG-2-Mediated Antisense Transcription Are Responsible for Lymphocyte-Specific Inactivation of NWC Promoter. PLoS ONE 9(9): e106927. doi:10.1371/journal.pone.0106927 Ikaros and RAG-2 -Mediated Antisense Transcription Are Responsible for Lymphocyte-Specific Inactivation of NWC Promoter Agnieszka aszkiewicz. 0 ukasz Bzdzion. 0 Monika Kasztura 0 ukasz S niez_ewski 0 Sylwia Janik 0 Pawe Kisielow 0 Magorzata Cebrat 0 Sebastian D. Fugmann, Chang Gung University, Taiwan 0 Laboratory of Molecular and Cellular Immunology, Department of Tumor Immunology, Institute of Immunology and Experimental Therapy , Wrocaw , Poland 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. - Funding: This work was supported by the Polish Ministry of Science and Higher Education/National Science Centre (grant N N401 049138). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. 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 [25]. The popular hypothesis on the origin of RAGs proposes that the transposon containing one of or both RAG genes infected a germ cell of an ancestor of jawed vertebrates (or deuterostomes), ultimately allowing for the development of lymphocytes [6,7]. The first exon of NWC gene and its promoter are located in the intron preceding the coding exon RAG-2 gene and these two genes are convergently transcribed [1] (Fig. 1A). The promoter of NWC gene is active in non-lymphoid cells and exhibits bidirectional activity, which can drive the transcription of both NWC and RAG2 transcripts in some non-lymphoid cells [8]. Based on this observation, we have recently proposed that the bidirectional activity of NWC promoter could facilitate the integration and survival of RAG transposon in the ancestral genome [8]. Previously, 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. The primary NWC promoter is associated with a CpG island which is unmethylated in non-lymphoid cells and becomes methylated in immature T- and B- lymphocytes, which coincides with the promoters inactivation [11]. In lymphocytes the function of NWC promoter is taken over by RAG-1 promoter, which results in the expression of RAG-1/NWC hybrid transcripts [1]. The methylation of NWC promoter is not accompanied by other changes in chromatin organization, i.e. changes in posttranslational modifications of histone H3 [11] that are commonly associated with transitions between transcription permissive and repressive chromatin configuration and usually precede DNA methylation. Blocking DNA methylation with 5-azacytidine partially restores the activity of NWC promoter in lymphocytes [11], proving the primary role of DNA methylation in controlling its activity. The activation of NWC promoter is mediated by ZFP143 transcription factor which binds to its two conserved elements, also possessing consensus binding sites for Ikaros transcription factor [8]. Ikaros is an essential transcription factor required for lympocyte development. It is expressed in lymphoid cells, haematopoietic stem cells and some myeloid cells. Ikaros deficiency impairs the development of lymphoid and myeloid cell lineages [12]. Ikaros can be involved both in gene activation and repression and its activity occurs at different levels: by direct competition with the activator proteins for common binding sites at the target promoter [13], by restructuring chromatin through targeting different types of chromatin remodelling factors [1416] such as SWI/SNF (activator) or NuRD deacetylase (repressor) as well as by bridging the target genes destined for inactivation with centromeric foci, thereby facilitating their assembly into pericentromeric heterochromatin [17]. Ikaros target genes include RAG-1 and RAG-2 genes, which are tightly controlled throughout lymphocyte development. High and coordinated expression of RAG genes is regulated by the activity of several cis-elements localized mainly upstream RAG-2 gene [1821]. Investigating the role of Ikaros in regulating V(D)J recombination in B-cell lineage Reynaud and collegues [22] showed that Ikaros binds directly to regulatory elements of RAG locus in pro-B cells, namely with the Ep, D3, Erag enhancers and RAG-1 promoter but not to RAG-2 promoter. These authors also compared the histone modification status of regulatory elements in RAG22/2 vs Ikfz2/2 pro-B cells and concluded that activation of RAG transcription by Ikaros is accompanied by histone-H3 acetylation. Here we demonstrate that NWC represents a new target of Ikaros activity within RAG locus. We show that binding of Ikaros to NWC promoter downregulates NWC expression, but is unable to cause promoter methylation which is established by antisense transcription driven by the activity of RAG-2 promoter. We discuss how these two mechanisms: binding of Ikaros and antisense transcription may act in concert to inactivate NWC promoter. 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. We noticed that these regions also contain consensus binding sites for Ikaros transcription factor (TGGGAA) [12], which overlap with the ZFP-143 binding sequences (Fig. 1A). This observation rais (...truncated)