Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo

Abstract

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dbeta, Jbeta, and Jalpha segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jalpha61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.

Figure 1

Nucleosome organization from D_β1 to J_β1.3. Real time PCR of mononucleosomes prepared from DN thymocytes of Rag^−/− mice carrying E_β-deleted, PD_β1-deleted or wild type alleles. Overlapping primer sets amplified 100–140 bp fragments (horizontal lines) spanning a 1.8 kb region encompassing PD_β1, D_β1, J_β1.1, J_β1.2, and J_β1.3. Data (top) are representative of 4 independent experiments and are the mean ±SE of triplicate PCRs in which the ratios of mononucleosome to genomic DNA are expressed relative to the values for a known positioned nucleosome in the IL-12 promoter. Numbering is according to nucleotide position in GenBank accession number MMAE000665, with data plotted at the midpoint of each amplicon. Letters identify specific positioned nucleosomes in the graph (top) and cartoon summary (bottom). Shading of nucleosomes in the cartoon summary indicates their relative abundance.

Figure 2

Nucleosome organization at D_β2. Real time PCR of mononucleosomes prepared from DN thymocytes of Rag^−/− mice carrying E_β-deleted, PD_β1-deleted or wild type alleles. Overlapping primer sets amplified 100–140 bp fragments (horizontal lines) spanning a 0.8 kb region encompassing D_β2, 5′PD_β2 and 3′PD_β2. Data (top) are representative of 3 independent experiments and are the mean ±SE of triplicate PCRs as described in the Fig. 1 legend. Numbering is according to nucleotide position in GenBank accession number MMAE000665. Nucleosome shading in the cartoon summary (bottom) is as described in Fig. 1.

Figure 3

Summary of nucleosome positioning relative to RSSs. Deduced positioning of nucleosomes from E_β- and E_α-deficient alleles.

Figure 4

Nucleosome organization from D_β1 to J_β1.3 as a function of DNA sequence. A. Nucleosome organization from D_β1 to J_β1.3 as predicted by computer modeling. Numbering is according to nucleotide position in GenBank accession number MMAE000665. The determined in vivo nucleosome organization of the D_β1 to J_β1.3 region on E_β-deficient alleles (Determined) is compared to predicted histone octamer occupancy (Occupancy), P values for nucleosome start sites (Start sites), and predicted nucleosome positions (Prediction) making use of an algorithm based on nucleosome positioning in the chicken genome. B. Nucleosome organization from D_β1 to J_β1.3 following in vitro assembly of nucleosomes. Real time PCR of mononucleosomes prepared from in vitro assembled chromatin is compared to in vivo data obtained from thymocytes of Rag2^−/− mice carrying E_β-deleted alleles (from Fig. 1). The In vitro data are the mean ±SE of two independent experiments in which relative amplification efficiency was determined by comparison to a genomic DNA standard (arbitrary units). In vivo data are normalized as in Fig. 1 and the two y-axes are arbitrarily scaled to allow comparison of the two profiles.

Figure 5

Nucleosome organization at J_α61. Real time PCR of mononucleosomes prepared from DP thymocytes of Rag^−/− × Tcrb tg mice carrying E_α-deleted, TEA-deleted, TEA-T or wild type alleles. Overlapping primer sets amplified 100–140 bp fragments (horizontal lines) spanning a 0.6 kb region encompassing J_α61. Data (top) are the mean ± SE of three experiments as described in the Fig. 1 legend. Numbering is according to nucleotide position in GenBank accession number M64239. Nucleosome shading in the cartoon summary (bottom) is as described in Fig. 1..