SUPPLEMENTARY INFORMATION

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11 W W W. N A T U R E. C O M / N A T U R E 1 1

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29 SUPPLEMENTAL DISCUSSION Many different considerations, starting with the finding that V H to DJ H rearrangement is the regulated step in the context of order, lineage-specifity and allelic exclusion 5,55,56, led to speculations that the V H -D intergenic region might contain elements involved in the regulation of V H to DJ H recombination 6,7,16,17,29,37. Several prior studies generated findings in support of such a regulatory element or elements in the V H -D intergenic region 7,29,30. In one study 7, a distal V H was moved several megabases from its normal distal location and inserted just upstream of the distal D HFL16.1 segment. This V H now rearranged directly to the adjacent D locus and also rearranged in thymocytes. The deregulation of this ectopically inserted V H led to the conclusion that the chromosomal position of a V H determines its activation and inactivation as a substrate for V(D)J recombination 7. Based on this finding, it was also speculated that there may be a potential V(D)J recombination regulatory element in the V H -D intergenic region 7. In a second study, a germline mutation was generated in mice that deleted the entire approximately 100kb region from just upstream of V H81X to just downstream of D 30 FL16.1. This large deletion, which juxtaposed V HQ to the D H locus, led to very high levels of antisense D transcripts that initiated within the D locus, ran "upstream" through the D locus and continued through the now closely juxtaposed proximal V H s. Generation of these long antisense transcripts was correlated with increased D to J H rearrangement and proximal V H to DJ H rearrangement in T cells. One interpretation of the phenotype of the 100kb V H -D deletion was that this large deletion eliminated a negative element that suppresses anti-sense D transcription and also physically juxtaposed V H s to the Ds, allowing the activated anti-sense D transcription to activate proximal V H rearrangement 30. Notably, the IGCR1 deletion (or CBE mutations) in our current study did not lead to increased antisense D H transcription or increased D segment rearrangement. Thus, our current findings, combined with those of the 100kb V H -D deletion study, suggest that there may be additional elements to be found in the V H -D intergenic region, or elsewhere, that control germline D H transcription. The IGCR1 deletion and IGCR1 CBE mutations were also distinct from the 100kb V H -D deletion in several other aspects. Thus, our currently described 29

30 IGCR1/CBE mutations led proximal germline V H s to be activated primarily for sense germline transcripts and premature rearrangement at distances of 100kb or more upstream of the D locus. In addition, the 100kb V H -D deletion did not disturb B cell development 30, in marked contrast to the dramatic impairment in B cell development observed for IGCR1/CBE mutations that we now describe. In this regard, the 100kb deletion likely deleted a number of different elements, including V H81X and its downstream CBE. Potentially, the 100kb V H -D deletion, by eliminating IGCR1 plus a large amount of additional sequence, may have allowed compensation for some of the more severe phenotypes we observe by mutating just IGCR1 or its CBEs, for example by eliminating dominant V H81X utilization or by allowing formation of aberrant new loops involving other CBEs within the IgH locus due to elimination of the V H81X CBE (see below). Based on the ability of the IGCR1 CBEs, like several other assayed CBEs, to function as silencing elements in vitro, these CBEs recently were speculated to serve an Igh regulatory role as insulators 29,33. Our current findings firmly demonstrate Igh V(D)J recombination regulatory roles for the IGCR1 CBEs based on the dramatic effects of mutating these elements, in the absence of any change at all in the linear proximity of the V H and D portions of the Igh locus. Notably, a study published while our current manuscript was in revision also demonstrated formation of loops between the IGCR1 and 3'IghRR locales in pro-b cells and found that knocking-down CTCF with RNAi led to some disruption of these loops 37. While one cannot unequivocally interpret results from a CTCF knock-down as reflective of the role of a particular CBE (because of the broad distribution of CBEs across the genome), the results of these CTCF knock-down experiments, combined with the results of our current CBE mutational studies, together strongly support the notion that the IGCR1 CBE functions we now describe are mediated via CTCF binding to the CBEs. The finding of the unusual number and location of CBEs throughout the Igh locus, including downstream of all proximal V H RSs or within upstream V H "PAIR" elements 32,33, has led to further speculation that these elements may serve positive functions in recruiting Igh V H s for recombination to DJ H intermediates 7,9,16,17,57. In this regard, we note that IGCR1 CBEs are in opposite sequence orientation and that CBEs near V H s and CBEs in the 3'IghCBE cluster, respectively, also are in opposite orientations (JASPAR database). Thus, given potential 30

31 orientation-dependent CBE activity 46,47, it is tempting to speculate that the two IGCR1 CBEs might serve differential roles in suppression and/or activation of V H to DJ H rearrangement. Clearly, there is still much to be learned about Igh V(D)J recombination regulation by further analyses of the function of the fascinating organization of V H, IGCR1, and 3'IghCBEs. SUPPLEMENTARY REFERENCES 54. Yusufzai, T.M. & Felsenfeld, G. The 5'-HS4 chicken beta-globin insulator is a CTCFdependent nuclear matrix-associated element. Proc Natl Acad Sci U S A 101, (2004). 55. Reth, M., Petrac, E., Wiese, P., Lobel, L. & Alt, F.W. Activation of V kappa gene rearrangement in pre-b cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J 6, (1987). 56. Alt, F.W., Blackwell, T.K., DePinho, R.A., Reth, M.G. & Yancopoulos, G.D. Regulation of genome rearrangement events during lymphocyte differentiation. Immunol Rev 89, 5-30 (1986). 57. Lucas, J.S., Bossen, C. & Murre, C. Transcription and recombination factories: common features? Curr Opin Cell Biol (2010). 31