The paper by Skok and colleagues  in this issue of Cell Reports explores the role of nuclear organization of T cell receptor a (Tcra) alleles in their recombination. This topic will undoubtedly be of interest to a broad range of researchers, including those interested in regulation of V(D)J recombination, nuclear organization and genome regulation, and genome instability.
Of all the long-range interactions amongst genetic loci in the nucleus, none is more complex and convoluted than recombination of the antigen receptors (T cell receptors a, b, g, d, immunoglobulin heavy and kappa/lambda light chains). Not only must distal segments of these enormous loci find each other across distances of up to 2.5Mb on the same allele in 3D nuclear space, but also the two alleles have to coordinately regulate their fate to ensure that only one at a time is recombined and activated while the other is silenced. Furthermore, since recombination introduces DNA double-strand breaks, it must be correctly targeted to bona fide antigen receptor (AgR) recombination sites, or genomic instability will ensue.
Numerous seminal studies from the Skok and other groups have shown that this requires myriad epigenetic mechanisms, including relocation from the nuclear periphery, intra-locus DNA looping, non-coding transcription, DNA demethylation, histone modifications, and monoallelic recruitment to pericentromeric heterchromatin (PCH). Together these findings have provided a conceptual picture of exquisitely orchestrated allelic choreography, but they have lacked nuclear context with respect to recent discoveries of how the nucleus is organized, which the new Skok paper begins to address.
Chromosomes occupy discrete, constrained lineage-specific locations within the nucleus termed chromosome territories (CTs) that are largely immobile. Active genes tend to reside on the outer edge of CTs, and higher-order looping enables movement of these genes away from their CTs. This correlates with open chromatin and active transcription, while repressed genes remain inside the territory [2,3]. Active genes often loop out from CTs to engage with transcription factories,(metastable subnuclear foci of RNA Polymerase II in which genes are transcribed) .
The Skok lab has a long history of ground-breaking findings concerning the nuclear organization of AgR genes. Indeed while Dr Skok was a postdoc in Amanda Fisher’s lab, she was the first to show that immunoglobulin (Ig) alleles undergo monoallelic silencing by recruitment to PCH . In the present paper, Skok and colleagues aim to understand how dynamic nuclear structures contribute to V(D)J recombination of the Tcra locus in T cells. The Skok, Schatz, and several other labs showed that many markers of transcription, including active histone modifications and non-coding RNA transcription , along with RAG recombinase binding (all prerequisites for successful recombination) were highly enriched at the 3′ end of AgR loci, dubbed ‘recombination centers’ . Here, Skok and colleagues use several genetic mouse models and sophisticated DNA-RNA immunoFISH techniques to visualize Tcra alleles and tease out the intricate, dynamic, and unique movements of this AgR locus in single T cells. Using the DNA damage-specific gamma histone H2AX as a marker, they found that DNA double strand breaks always occur at the 3′ end of the Tcra locus, and importantly on only one Tcra allele. Thus, the activity of the RAG recombinase is strongly targeted both to this region of active chromatin, and to only one allele at a time.
What favors this directionality of RAG targeting? Since CTs constrain gene activity, the authors asked where the Tcra is located with respect to its CT during recombination. They find that 15% of Tcra alleles are looped outside of the CT, while 50% are at the active outer edge. This higher-order looping out occurs via the 3′ end of the locus, while the 5′ end remains embedded in the CT. These findings explain why active chromatin marks and RAG target to the 3′ end of Tcra.
Importantly, this is not only the first report of higher-order looping out of chromatin that is associated with a process other than transcription, but also the first example of higher-order looping out of a single allele from chromosome territories. The latter may very well be the first step in asynchronous V(D)J recombination, which is important to ensure both the production of only one recombined allele and a reduced frequency of DNA double-strand breaks. Monoallelic looping out of only one AgR allele would certainly provide the stochastic head-start that one allele needs to be the first to recombine. This is particularly striking given that this is the Tcra allele, which is ultimately one of the few AgRs that does not undergo allelic exclusion. As Skok and colleagues clearly show here, the timing of looping out of its two alleles is strictly coordinated.
The authors explore the mechanism and find that the RAG-1 recombinase is required for this higher-order looping, which is substantially reduced in a Rag–/– background. This is surprising because this is one of the first reports that a ‘trans-acting factor’ mediates higher-order looping out from CTs. More importantly, why and how would a recombinase regulate higher-order looping out from CTs? They do not answer the ‘how’ completely, but link it to a previous paper that has both excited and puzzled those in the field. The Skok lab previously found that a significant proportion of homologous Igh (and Igk) alleles physically colocalized (“paired”) in the nucleus before recombination, and that this was dependent on the RAG-1 protein, but not on its catalytic activity. Furthermore they found that DNA double-strand breaks occurred on only one of these paired alleles. The second allele was recruited to PHC in a manner dependent on the DNA damage response protein ATM, which was recruited to the site of the break and prevented biallelic breaks and genome instability .
I heard Dr Skok present this work at a Keystone meeting in Taos, and saw dozens of intrigued but puzzled scientists, many leaders in this field, gathered around her poster afterwards, proverbially scratching their heads. Why did the Igh alleles pair? Well, it was easy to propose a hypothesis: Physical proximity would certainly help the two homologous alleles to coordinate their recombination timing such that only one would be fully recombined and activated, while the other was silenced. Considering the fact that homologous chromosomes do not tend to be close together, how do they pair, and how does the RAG recombinase help to make the pairing happen? In the current paper, some questions are answered, but the mystery also deepens and widens.
First Skok and colleagues show that the same process happens in the recombining Tcra in T cells. A proportion of Tcra alleles pair, mediated by RAG-1, and after the first DSB, the second allele is recruited to PCH, through the action of ATM. Thus common epigenetic mechanisms regulate cleavage and accessibility of the AgRs. The authors favor the idea that locus pairing regulated by RAGs is a central mechanism. This interpretation comes with caveats, the most important of which is that the frequency of monoallelic recombination and recruitment to PCH was similar for unpaired and paired alleles. The authors acknowledge these caveats but suggest that pairing is likely to be transient and thus alleles that appear unpaired may have previously been paired. This is a reasonable perspective, but will require further experiments to confirm.
Transcription also appears to play a role in monoallelic looping out from CTs. Using RNA-FISH with probes that cover the entire length of Tcra, the Skok group show that monoallelic looping out of 3′ end of Tcra occurs more frequently on alleles that are (monoallelically) transcribing at the 3′ end. This is intriguing, although Skok and colleagues admit that due to the limitations of RNA-FISH sensitivity they can’t be absolutely certain that transcription only occurs at the 3′ end of one allele. Nevertheless, they can say that it occurs with higher (i.e., detectable) frequency at the 3′ end of one allele. Thus there is substantial  evidence for a role for monoallelic transcription in mediating higher order looping out of Tcra alleles, which in turn leads to monoallelic recombination. Interestingly, transcription alone is not sufficient since there is reduced looping in Rag–/– T cells even though they are transcribing Tcra.
These studies answer the question “where from?”, but raise an intriguing question: “where to?” Does all this looping out have a destination? The authors stop short of drawing together their findings of recombination associated with transcription to speculate about whether the looping out has a specific destination (e.g., a particular nuclear substructure). Even heterologous genes that are regulated by the same transcription factor loop out from different chromosomes to engage in the same transcription factory . Homologous AgR alleles undergoing V(D)J recombination arguably have an even greater imperative for coordinated transcription than most homologous transcribing genes. We have previously shown that the 3′ end of the Igh locus is highly transcribed in B cells and is thus constantly in a transcription factory, which facilitates both normal expression and predisposes it to aberrant DNA breaks and chromosomal translocations . We have therefore proposed that recombination itself occurs in specialized transcription factories . We are pleased that the Skok paper supports our model since it demonstrates that recombining loci undergo higher order looping out from CTs in a similar manner to transcribing genes.
The current paper by Skok and colleagues raises several other intriguing and important questions. Is mono-allelic looping out a widespread mechanism regulating AgR recombination? How does RAG-1 regulate both monoallelic higher-order looping out and pairing of AgR alleles? To gain a definitive picture of what is undoubtedly a highly dynamic mechanism, a key next step will be to visualize the RAG enzymes in the nucleus of living cells.
Overall, the present paper by Skok and colleagues represents an excellent and substantial body of work that provides several novel mechanistic insights into the nuclear organization of Tcra recombination, which is also a topic of great interest to researchers in gene regulation. It is currently an area of intense effort to understand the role of chromosome territories and dynamic looping out from territories in regulation of gene expression, and there are differing views on the prevalence and/or importance of higher-order looping. The finding that looping out is also integral to recombination is a major step in our understanding, and adds substantial support to the model that looping out from chromosome territories is required for specialized genome functions.
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