Nature: Dr. Ba, etc. reveals the important mechanisms of re-arrangement of the anti-weight chain V gene.

!---- major outbreak swept the world this year, not only pushed researchers to the forefront of developing vaccines and therapeutic antibodies, but also reaffirmed the public's popularization of professional terms such as "vaccine" "antibody testing" and neutralizing antibodies and the basic immunology knowledge behind themAs one of the important protein effect molecules of body fluid immunity, antibodies play an important role in the process of identifying and eliminating pathogenic bacteria such as bacteria and virusesThe antibody, which acts as a secreted immunoglobulin (Ig), is produced by B lymphocytes and is a Y-protein complex formed by two pairs of paired heavy chains (IgH) and light chains (IgL) by disulfur bondIgH and IgL have variable and constant regions, respectively, where variable regions are specifically identified and combined with antigensThe encoded gene of the variable region is produced by the V(D) J rearrangement (V(D) J recombination reactionIn the case of The IgH site, for example, the IgH site spans millions of base pairs (megabase, Mb) in the human and mouse genomes and consists of hundreds of V, more than a dozen Ds, multiple J gene elements, and numerous regulatory sequencesPrecursor B lymphocytes (progenitor B cell, In the process of pro-B) development, the V(D) J rearrangement reaction is used to catalyze the rupture of a D gene and a J gene element through the RAG incision enzyme to connect the fractured element through a non-homologoated recombinant end connection (NHEJ) path to form a DJH intermediate product, and then through the NHRAG and NHEJ catalytic fracture and connecting a VHgene to DJH intermediate product, resulting in a complete segment of The IgH Gene Coding VHHV(D) J re-emission reactions generate a large number of variable-zone encoded gene banks that are one of the important molecular bases that make up antibody diversityHow the numerous V, D, j gene components in the V(D) J rearrangement reaction are identified and cut by RAG incise asses to participate in the generation of a very diverse library of variable-zone coding gene stakes is a long-standing and fascinating core issueAlthough many studies have revealed a number of factors that affect V(D)J rearrangement from an (epigenetic) genetic perspective, it is not clear exactly how this rearrangement process occursIn recent years, the Frederick WAlt Fellows Laboratory at the Howard Hughes Institute of Medicine (HHMI), Harvard Medical School (HMS) and Boston Children's Hospital (BCH) has worked to show that v(D) J rearrangement is most likely performed through a linearized model of RAG chromatin scanning, in contrast to the long-suspected rearrangement process based on random diffusionThe laboratory pioneered the discovery that RAG had linearized "tracking" and cutting genomes with the activity of off-target sequences in a specific direction, and that the activity range was consistent with the genomic ring domain (CTCF-binding element, CBE) formed by the convergence-convergence-binding-binding elementSubsequently, the laboratory further proposed a linearized model of RAG chromatin scanning that may be based on the chromatin ring extrusion mechanism, which can well explain the rearrangement process of near-end VH-to-DJH and the important role of CBE in the process, by studying the function of a class of CBE located next to the near end VH gene of the IgH site D in miceAnother subsequent article published by the lab showed through a series of experiments that the model also explains physiologically removable D-to-JH rearrangement, and further demonstrates that chromatin ring extrusion plays an important role in the rearrangement processHowever, questions about the working scanning of RAG chromatin and, more importantly, how the many distant V genes separated from Mb are rearranged remain unclearOn July 27, 2020, Frederick W Alt and a team of Professor Rafael Casellas of the National Institutes of Health (NIH) published an online study entitled "CTCF Orchestrates Long-range-driven V(D) J recombinal scanning" in the journal Nature in the form of Accelerated Article Preview (Dr Ba is the first author and co-author of this article, Dr Yan Jiangman, co-author of this article) This article reveals the linear lysing ring extrusion of the adhesive protein cohesin to drive the linear migration of igH sites to provide rag-scanning substrates, and the important role of CTCF in regulating remote VH rearrangement under this mechanism, thus providing a new insight into a long-standing core problem in this field To study the driving force of RAG chromatin scanning, the researchers speculated that cohesin may be an important factor To prove this point, the researchers selected the v-Abl pro-B cell line of the immortalized mice The cell line is induced to survive long-term and stable at the G1 stage of the cell cycle, and can activate the RAG-mediated D-to-JH rearrangement in large quantities, activate the near end in a small amount and almost cannot activate the far-end VH-to-DJH rearrangement Previous studies in the cell line have confirmed that d-to-JH and near-end VH-to-DJH were mediated by RAG scans, so what happens about removing cohesin from the cell line? Using the growth hormone-induced protein degradation element (auxin-induced degron, AID) strategy, the researchers constructed the AID degradation system (Rad21-degron), an important factor for the cohesin complex, in the cell line, and quickly degraded Rad21 by adding auxin Subsequently, the researchers confirmed through ChIP-seq that the whole genome, including IgH site cohesin, had completely disappeared, while the transcription activity of the IgH site and the transcription or expression of known chromatin interoperability and V(D) J re-ecrimination of essential genes were not significantly changed The researchers went on to find that almost all chromatin ring-like domains at the corresponding IgH site had disappeared through 3C-HTGTS, previously developed by Dr Ba, in line with previous findings in other types of cells that cohesin is necessary for chromatin ring extrusion to form a ring-shaped domain Interestingly, the researchers further analyzed changes in Rad21 degradation before and after D-to-JH and near-end VH-to-DJH, and found that Rad21 degradation eliminated almost all near-end VH-to-DJH rearrangements, dramatically reducing almost all D-to-JH rearrangements, except for the D52 components located inside the rag-rich V(D) J rearrangement center (recombination center, RC) Previous studies have shown that DQ52 can rearrange due to its unique position in RC, which can occur by diffused close to RAG, so its rearrangement does not rely entirely on the cohesin-mediated ring extrusion process In order to better explore the effects of the absence of the cohesin, the study in the Rad21-degron system through CRISPR/Cas9 to further knock out the IGCR1 component, previously found that the IGCR1 missing RAG scan enhanced to the IgH near end VH region resulting in a sharp increase in the rearrangement of the near-end VH, especially the VH81X gene, so on this basis the absence of the cohesin? The researchers found that Rad21 degradation still eliminated almost all enhanced near-end VH-to-DJH rearrangements, while dramatically reducing almost all D-to-JH rearrangements, and again, only DQ52 rearrangements were still possible Correspondingly, Rad21 degradation also eliminates all chromatin ring structures at the IgH site, including the heavily enhanced ring interaction between RC and near-end VH due to the absence of IGCR1 Together, these results indicate that cohesin is most likely to mediate the D-to-JH and near-end VH-to-DJH rearrangement processes performed by RAG scans through its mediated chromatin ring extrusion In addition to the near-end VH gene, how do hundreds of far-end VH genes approach RAG and occur in long-distance rearrangement? It has long been speculated that the far-end VH may be rearranged through an Inherent "physical point contraction" process of IgH In this model, the far-end VH site approaches and surrounds the rearrangement center of the RAG enrichment with some unknown mechanism, so that each VH gene is randomly diffused towards the RAG and re-arranged The model has been in the hypothesis stage due to the lack of clear mechanism support The researchers speculated that, in contrast to the random diffusion model, the d and near-end VH gene rearrangement sitcoms, the far-end VH gene may also have been rearranged by a linear RAG chromatin scanning process that approached RAG So how do you prove it? The researchers adopted a clever strategy The researchers first made a bold guess: in the VH region, in addition to hundreds of VH genes, there are a large number of CTCF binding element CBEs; near-end VH adjacent CBE in the RAG scanning process in addition to the addition of the addition of the adjacent VH for the RAGaccessibility to enhance its re-arrangement capacity, but also additional blocking rags Further scanning of other near-end VHs upstream of it weakens its rearrangement potential, and while the specific functions of CBE between many remote VH genes are not yet known, are these CEs similar to near-end CBEs that can gradually block the LAGA upstream scanning process, thus affecting the re-emission potential of the entire far-end VH? Further combined with their findings, it showed that compared with normal precursor B cells in mice, the derived v-Abl cell line can only perform a small amount of near-end VH rearrangement, not remote VH rearrangement; The reason for this has been unknown, if this is due to the blocking effect of many CBE-to-ring extrusion-mediated RAG linear scans, can the suppression or removal of all these CBEs reactivate the rearrangement of the far-end VH? To test this, the researchers constructed a CTCF-degron degradation system in the v-Abl cell line After adding auxin to rapidly degrade CTCF at the overall level, ChIP-seq confirms that CTCF degradation is eliminated or significantly reduced the combination of the Chf itself and the majority of CBE sites in the genome, including IgH; The non-homogeneous binding changes of CTCF at chromatin level after induced degradation may reflect CTCF binding activity, local chromatin environment or other unknown factors of the different CBE sites themselves Further GRO-seq confirmed that CTCF degradation did not significantly affect the IgH rearrangement center and VH especially remote VH transcription, nor did it affect the transcription of any factors known to be involved in chromatin interactions and V(D) J rearrangement, indicating that CTCF degraded cells still have the potential of VH especially far-end VH rearrangement The researchers then found through 3C-HTGTS that, very interestingly, CTCF degradation strongly responded to the loss of the rag in the v-Abl cell line and the chromatin ring structure in the almost entire far-end VH region, and was highly similar to the interaction in normal precursor cells in mice, indicating that CTCF-degraded GH site chromatin cyclogens resumed to the far-end VH region So the corresponding, the far-end VH also replied to the rearrangement capacity? The answer is yes! The researchers further examined the V(D) J rearrangement changes and found that CTCF degradation significantly, and sometimes significantly activated, the rearrangement capacity of the vast majority of VHs, including the remote VH, and was not 100% consistent with normal precursor B cells in mice, and the v-Abl cell line of CTCF degradation still exhibited a very similar VH rearrangement frequency and pattern overall Correspondingly, ctcF-degraded v-Abl cell lines significantly increased in the relative ratio of VHDJH and DJH rearrangement and were very close to the values in normal mouse precursor B cells, indicating that VH re-ranking was also greatly enhanced at the overall level In addition, the researchers also analyzed the RAG-mediated off-target cutting activity and found that only in THE CELLS degraded by CTCF high frequency cut off-target site with a specific direction of the entire VH region, further supporting the conclusion that CTCF knockout allowed THE RAG scan to act on the entire VH region to mediate the remote VH rearrangement In addition, the researchers further studied the relationship between the mid-range VH rearrangement of other intermediate cell lines produced during the establishment of the CTCF-degron system and the level and potential activity of THE CTCF C end insertion mediated degradation of the AID-GFP element significantly reduced the level of CTCF protein, and the corresponding far-end VH began to occur Rearrangement; The non-auxin-treated CTCF-degron system has leaky CTCF degradation, which further reduces CTCF protein levels and the corresponding far-end VH re-arrangement is higher; further auxin treatment almost degrades the overall CTCF protein level, and the corresponding far-end VH rearrangement is more intense These results show that remote VH rearrangement is sensitive to changes in CTCF protein levels and potential activity, i.e the reduction of the latter has an important positive effect on activating RAG scanning far-end VH mediated its rearrangement Finally, through a more detailed analysis of various histological data, the researchers believe that the rearrangement of VH during CTCF activity reduction may be affected by residual CTCF binding sites and VH transcription levels, thus revealing that different strategies may have been adopted to ensure the rearrangement potential of each VH in different VHs in evolutionary mice Based on the above experimental findings and analysis, the researchers finally put forward a model of VH gene rearrangement in the rag scanning mediated by chromatin ring extrusion by cohesin and CTCF, and concluded that there was a direct regulation of CTCF/CBE block activity during the early development of mouse B cells, or by regulating the activity of other ring factors such as cohesin to indirectly overcome the resistance effect of CTCF/CBE to allow the cyclic-mediated cyclation process to drive the whole H-B Overall, the study boldly assumed and designed and adopted the clever.