Supplementary Figure Legends. Supplementary Figure 1. Positioning of nucleosomes by different RSSs.

Transcription

1 Supplementary Figure Legends Supplementary Figure 1. Positioning of nucleosomes by different RSSs. A. Mapping of nucleosomes in the '' and 'END' positions on fragments carrying different RSSs. The RSSs with the nonamer sequences indicated were cloned in an equivalent position to the RSS in MAB5. Restriction enzyme digestions of the bp fragments obtained following micrococcal nuclease digestion of the reconstituted nucleosomes are shown. B. Schematic diagram of the nucleosome positions mapped in A. All RSSs with a consensus nonamer sequence (i.e. all except TCRγ5), cause nucleosome positioning over the RSS in the '' position and at the left end in the 'END' position. In the '' position, a fraction of nucleosomes remain in one of the favoured positions mapped in the absence of an RSS on MAB4. We suggest the extent of repositioning of the nucleosome over the RSS depends on the relative strength of adjacent positioning sequences. The non-rss positioning sequence in MAB4 appears to be relatively strong: in vitro some nucleosomes stay in the alternate position whereas in vivo the Vκ21c RSS is fully protected (Figure 5C). Supplementary Figure 2. Accessibility of the Jκ1 RSS in 103-BCL/2 cells. The pre-b cell line, 103-BCL/2 was grown at either the permissive temperature ( o C) or non-permissive temperature (39 o C) for 12 hours to induce recombination. Nuclei were prepared, digested with ScaI for 1 hour at 37 o C and 1

2 cutting at the RSS and adjacent neutral site was analysed as described. Only a very modest increase in accessibility at the RSS compared to the neutral site was observed under conditions where the cells were undergoing recombination. Supplementary Figure 3. Substitution of the nonamer from cryptic RSSs results in loss of nucleosome positioning over the RSS. Restriction enzyme digestions of the bp fragments obtained following micrococcal nuclease digestion of the MAB12-1, -2 and -3 nucleosome complexes shown in Figure 6A. Considerably more positions are detected than when the consensus RSS is present. Moreover, the smear on the native gel suggests that yet further nucleosome positions exist. Supplementary Figure 4. Time course of RSS accessibility and RAG induction following transfection of NIH3T3 cells. A. Maximal protection of the 23-RSS ScaI site from restriction enzyme cutting is achieved by 32 hours after transfection. Nuclei were prepared from cells harvested at the times indicated after transfection and were digested with an excess of ScaI. The extent of ScaI cutting was then determined by Southern blotting as described in Figure 5B. B. Induction of RAG1 expression. NIH3T3 cells were transfected with an auto-regulatory tetracycline-controlled transactivator (ptet-ttak; Shockett et al., 1995) and RAG1 under the control of a tetracycline-inducible promoter. 2

3 Tetracycline was withdrawn from the medium 36 hours after transfection and induction of RAG1 expression was measured via western blotting using anti- RAG1 antibodies (PharMingen). An arrow indicates the time of tetracycline withdrawal. 3

4 END END END END END END A Vκ1 (ACAAAAACC) Vκcy9 (ACATAAACC) Vκdv36 (ACAAAAACC) Vκ21c (ACAAAAACC) Vκ24 (ACAAAAACC) TCRγ5 (ACTGAAGAG) Supplementary Figure 1A

5 B : Vκ1 Vκcy9 Vκdv36 Vκ21c Vκ24 : TCRγ5 + END: Vκ1 Vκcy9 Vκdv36 Vκ21c Vκ24 END: TCRγ5 Supplementary Figure 1B

6 103-BCL/2 23RSS (Jκ1) non-rss kb 2.0 M 39 kb M parental cleaved % accessibility Supplementary Figure 2

7 MAB12-1 MAB12-2 MAB12-3 Supplementary Figure 3

8 A B pmab1-23rss RAG1 expression time of transfection kb 32 h 36 h 40 h 64 h time of transfection 32 h 36 h 40 h 64 h kd parental cleaved % accessibility Supplementary Figure 4