The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs.

Two Successive Inversional Vß Rearrangements on a Single Tcrb Allele Can Contribute to the TCRß Repertoire.

Mammalian TCRß loci contain 30 Vß gene segments upstream and in the same transcriptional orientation as two DJCß clusters, and a downstream Vß (TRBV31) in the opposite orientation. The textbook view is upstream Vßs rearrange only by deletion and TRBV31 rearranges only by inversion to create VßDJCß genes. In this study, we show in mice that upstream Vßs recombine through inversion to the DJCß2 cluster on alleles carrying a preassembled Trbv31-DJCß1 gene. When this gene is in-frame, Trbv5 evades TCRß-signaled feedback inhibition and recombines by inversion to the DJCß2 cluster, creating αß T cells that express assembled Trbv5-DJCß2 genes. On alleles with an out-of-frame Trbv31-DJCß1 gene, most upstream Vßs recombine at low levels and promote αß T cell development, albeit with preferential expansion of Trbv1-DJß2 rearrangements. Finally, we show wild-type Tcrb alleles produce mature αß T cells that express upstream Vß peptides in surface TCRs and carry Trbv31-DJß2 rearrangements. Our study indicates two successive inversional Vß-to-DJß rearrangements on the same allele can contribute to the TCRß repertoire.

Genome Topology Control of Antigen Receptor Gene Assembly.

The past decade has increased our understanding of how genome topology controls RAG endonuclease-mediated assembly of lymphocyte AgR genes. New technologies have illuminated how the large IgH, Igκ, TCRα/δ, and TCRß loci fold into compact structures that place their numerous V gene segments in similar three-dimensional proximity to their distal recombination center composed of RAG-bound (D)J gene segments. Many studies have shown that CTCF and cohesin protein-mediated chromosome looping have fundamental roles in lymphocyte lineage- and developmental stage-specific locus compaction as well as broad usage of V segments. CTCF/cohesin-dependent loops have also been shown to direct and restrict RAG activity within chromosome domains. We summarize recent work in elucidating molecular mechanisms that govern three-dimensional chromosome organization and in investigating how these dynamic mechanisms control V(D)J recombination. We also introduce remaining questions for how CTCF/cohesin-dependent and -independent genome architectural mechanisms might regulate compaction and recombination of AgR loci.

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5' region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 5' RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 5' nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

Poor quality Vß recombination signal sequences stochastically enforce TCRß allelic exclusion.

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vß gene segments with a higher quality target elevates Vß rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRß chains derived from both Tcrb alleles. Thus, TCRß allelic exclusion is enforced genetically by the low quality of Vß recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.