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The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [3C-HTGTS]

ABSTRACT: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [3C-HTGTS]

PROVIDER: PRJNA534314 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9944790_1.fastq.gz	Fastqsanger.gz
	SRR9944790_2.fastq.gz	Fastqsanger.gz
	SRR9944800_1.fastq.gz	Fastqsanger.gz
	SRR9944800_2.fastq.gz	Fastqsanger.gz
	SRR9944791_1.fastq.gz	Fastqsanger.gz

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Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.

Project description:In a B lymphocyte immunoglobulin heavy chain locus (IgH), a developmentally assembled V(D)J exon encoding an antibody variable region lies upstream of exons encoding a m constant region (Cm), allowing generation of mIgH chain transcripts and IgM-class antibodies1. Mouse IgH class switch recombination (CSR) replaces Cmwith one of 6 sets of constant region exons (CHs) that lie 100-200kb downstream1. Each CH is flanked upstream by a promoter, non-coding I-exon, and long repetitive switch (S) region1,2. Cytokines/activators induce specific I-promoter transcription and activation-induced cytidine deaminase (AID)2,3. AID is transcriptionally-targeted to initiate DNA breaks in Sm and activated downstream acceptor S regions, which are joined in deletional orientation to complete CSR4,5. 3’IgH regulatory region (3’IgHRR) enhancers control upstream I promoters and, thereby, CSR via linear competition involving I promoter/3’IgHRR interactions6-11. Here, we report that synapsis of regulatory elements, S regions and DSBs for CSR is achieved by chromatin loop extrusion. In naive B cells, 3’IgHRR enhancers and adjacent 3’IgH CTCF-binding elements (CBEs) interact via loop extrusion with the upstream Igh intronic enhancer (iEm)/Sm locale to generate dynamic 200kb 3’Igh basal loop. In CSR-activated B cells, induced transcription from I-promoters within this basal loop generates dynamic sub-loops that directionally align Sm and target S regions near the 3’IgHRR for CSR. In CH12F3 B lymphoma cells, inactivation of the constitutively active Ia-promoter abrogates looping and CSR to Sa, while activating transcription, looping, and CSR to upstream S regions. CBEs inserted upstream of Ia in convergent orientation with 3’IgH CBEs generate sub-loops that activate inversional Sa CSR. In I-promoter-deleted CH12F3 cells, this ectopic CBE-based sub-loop inactivates upstream S region CSR, while transcriptionally activating non-S region sequences adjacent to the inserted CBEs for S synapsis and CSR. Together, our findings implicate chromatin loop extrusion in the “unprecedented mechanism”5 by which Igh organization in cis promotes orientation-specific CSR DSB joining.

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The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [3C-HTGTS]

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