The BRCT domain containing protein PTIP has been linked to both gene transcription and DNA repair (26
). In this report, we utilize a B-cell-specific deletion to address the role of PTIP in CSR, a process that requires both transcription regulation and DNA recombination (35
). The loss of PTIP significantly attenuates CSR in B cells after treatment with LPS/IL-4, a stimulus that promotes switching to the IgG1 and IgG2b isotypes. This defect in switching is most likely due to a transcriptional block, as the GLTs corresponding to the new constant regions are suppressed. Consistent with the role for PTIP in assembling a histone methyltransferase complex, localized H3K4me3 at the GLT promoters is also reduced in PTIP−
B cells. These data are consistent with a recent report from Daniels et al. (10
), which also demonstrated an effect on CSR in PTIP mutant B cells. However, we have taken this observation further and now demonstrate a specific link to Pax5 and to higher-order chromatin-remodeling events that are likely to determine transcription from the switch region promoter.
In response to cytokine stimulation and receptor cross-linking, the GLTs that correspond to specific isotype constant regions are transcribed and are required for the initiation of CSR. In our PTIP−
B cells, the failure to switch to IgG1 is most likely due to a transcriptional defect of the γ1 GLT. Activation of the γ1 promoter appears to be mediated by NF-κB (20
) and STAT6 (21
) binding near the GLT start site. We have shown significant increases in H3K4me3 at the GLT promoter regions in response to LPS/IL-4 stimulation. For IgG1, this increase is dependent upon PTIP and is coincident with PTIP and Pax5 localization to the γ1 promoter region in vivo
. Given that this promoter does not contain functional Pax5 binding sites, based on our in vitro
assays, we propose that Pax5 bound at the 3′ enhancer site hs4 is brought to the γ1 promoter by chromatin looping in a PTIP-dependent manner. Upon stimulation of B cells, Pax5 chromatin immunoprecipitates to the γ1 promoter, while the ChIP signal at the 3′ enhancer is actually decreased over time. This is likely to reflect limited cross-linking such that upon DNA looping, Pax5 is linked to either the 3′ enhancer or the γ1 promoter complex but not both. Thus, our model would predict that upon stimulation, PTIP helps stabilize Pax5 binding at the 3′ regulatory region and brings this region to the promoter of the GLTs, perhaps through interactions with proteins bound at the promoter, such as STAT6 or NF-κB (). This is consistent with the inability to see higher-order DNA associations between hs4 and the γ1 or γ2b promoters by using the chromatin conformation capture assay in PTIP-deficient B cells. Given the phospho-serine binding domains of PTIP, it is possible that stimulation phosphorylates Pax5 and the promoter-bound proteins to facilitate PTIP recruitment to chromatin. The need for stimulation in HEK293 cells is not evident for PTIP-Pax interactions, because Pax protein overexpression already results in a strong basal level of phosphorylation (4
Fig. 8. Model of PTIP-dependent chromatin interactions. In untreated B cells, Pax5 binds to the hs4 site with lower affinity. Upon LPS/IL-4 stimulation, PTIP stabilizes Pax5 binding and promotes chromatin looping to either the Iγ1 or Iγ2b transcription (more ...)
Our model would predict that CSR and the GLTs are also dependent on the 3′ enhancer, which contains 4 DNase I-hypersensitive sites. Indeed, there is significant experimental evidence that the 3′ enhancer is essential for CSR and for regulating the GLTs. An initial deletion spanning 3.5 kb of the 3′ enhancer inhibited switching to all isotypes except IgG1 (9
). Subsequent deletion of the DNase-hypersensitive sites hs3A and hs1,2 did not show significant attenuation of CSR (32
). However, deletion of hs3b and hs4 significantly affected IgG3 and IgA isotypes and corresponded to a decrease in transcription from the GLT promoters (29
). Further definition of the 3′ enhancer revealed that deletion of the entire 28-kb sequence containing all four hypersensitive sites affected GLTs from most, but not all, switch region promoters, with the α promoter being the least affected (11
). Interestingly, in a mouse line carrying an IgH bacterial artificial chromosome (BAC) transgene inserted near the endogenous IgH locus, the endogenous 3′ enhancer could rescue some of the CSR defects by rearranging the transgenic VDJ with the endogenous Cγ. These data, argue for an essential role of the 3′ enhancer in regulating GLTs. This point is underscored by the observation that the 3′ enhancer is able to directly interact with upstream promoter elements through potential chromatin looping mechanisms (17
The data presented here argue for a model in which Pax5, bound to the 3′ enhancer, complexes with the different upstream GLT promoter elements, depending upon which stimulus and which isotype are selected for switching. This model may be analogous to that of the locus control region of the globin genes, which has the ability to regulate multiple genes in cis
, but only at one gene at a time through direct DNA looping mechanisms (14
). Alternatively, the H3K4me3 marks may be prerequisites for interactions between Pax5 and GLT promoters, independent of PTIP localization to the actual DNA sequences. In other words, the failure to maintain H3K4me3 levels could prevent higher-order chromatin conformations.
Mammals have multiple H3K4 methylation protein complexes, including the KMT2A-D (MLL1 to -4) and the KMT2F/G (hSet1A/B) complexes that are homologous to the COMPASS complex first described in yeast (25
). To date, the best studied of these is KMT2A (MLL1), which regulates Hox gene expression and is responsible for H3K4me at about 5% of all promoters in mouse embryo fibroblasts (37
). Thus, the remaining promoters are likely to interact with different KMT2s. However, PTIP has only been associated with the KMT2C/D complexes (7
). In cell culture assays, PTIP links the H3K4 methyltransferase complex to sequence specific DNA binding proteins (28
), such as Pax2 and Pax5. The PTIP germ line mouse mutant phenotype is more severe and earlier in embryonic development than any single Pax gene mutation or KMT2 gene mutation (6
), suggesting that PTIP may interact with multiple DNA binding proteins and methyltransferase complexes. This is consistent with the effects of PTIP deletion on global levels of H3K4 methylation in mouse embryos (6
), stem cells (19
), and Drosophila
). In our B-cell-specific PTIP knockout, the KMT2A/B complexes that do not contain PTIP appear unable to rescue the H3K4 methylation defect at the GLT promoters, in all likelihood because they lack the locus-specific DNA binding component to target their activity to the IgH locus promoters. Precisely how this locus specificity is achieved remains to be determined, but the data in this report clearly indicate a role for PTIP in recruiting H3K4me3 activity to the GLTs within the IgH locus.
Since the discovery of distal enhancers, how such enhancer elements communicate with more proximal promoters is poorly understood. In this report, we have used the IgH locus as a model to investigate the role of PTIP in transcription regulation and CSR, events that depend on both proximal and distal chromosomal elements. PTIP is part of a KMT2C/D H3K4 methyltransferase complex and is essential for development and lineage specification in mice and flies. The data presented provide the first evidence that PTIP is important for recruiting a KMT2 complex to an endogenous transcription start site and for the communication between a promoter and a distal enhancer. This communication is likely to require Pax5 and results in epigenetic modifications such as H3K4 methylation at the transcription unit. Still, many questions remain unanswered, as additional proteins are likely to be involved for regulating and stabilizing such chromatin interactions.