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Elucidating Mechanisms of RAG Endonuclease Mediated Feedback Inhibition of V(D)J Recombination

Award Number: R01AI172163
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 07/12/2022
PERIOD OF PERFORMANCE END DATE: 06/30/2027

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Elucidating Mechanisms of RAG Endonuclease Mediated Feedback Inhibition of V(D)J Recombination - PROJECT SUMMARY/ABSTRACT A hallmark of adaptive immunity is mono-allelic expression (allelic exclusion) of B and T cell antigen receptor proteins, which is thought to allow highly-specific immune responses and inhibit autoimmunity. Allelic exclusion is achieved via mono-allelic initiation of RAG1/RAG2 (RAG) nuclease-mediated V gene segment recombination, followed by protein from a V(D)J rearrangement signaling permanent feedback inhibition of V recombination on the other allele. A shortcoming of this control is that it needs time for completion of recombination, expression and signaling of protein, and changes on the second allele. The applicant demonstrated that RAG DNA double strand breaks (DSBs) induced during Vk-to-Jk recombination in pre-B cells signal via the ATM kinase to inhibit Rag1/Rag2 expression, Jk accessibility, and RAG cleavage of the other allele until after the first allele is repaired. He showed that ATM deficiency in mice increases bi-allelic Igk IgH, or TCRb expression. While these data imply that ATM enforces allelic exclusion by signaling DSB feedback inhibition of V recombination, they neither prove this model nor rule out a role for ATM-stimulated DSB repair. The applicant shows new data that ATM enforces Igk allelic exclusion by signaling via the NFkB essential modulator (NEMO) protein, with inhibition of Rag1/Rag2 transcription likely key for inter-allelic control of Vk recombination. The applicant shows that RAG DSBs during IgH D-to-J recombination in pro-B cells or TCRb D-to-J, TCRg, and/or TCRd recombination in DN thymocytes do not require ATM to repress Rag1/Rag2 expression. However, these RAG TCR DSBs signal via ATM to repress expression of a Vb region anti-sense long non-coding RNA, which the applicant shows is expressed only in DN cells where Vb and Db-Jb segments interact and rearrange over vast genomic distances. Based on these data, the applicant hypothesizes that RAG DSBs feedback inhibit V(D)J recombination through complementary mechanisms, including cell type-specific signaling pathways that repress Rag1/Rag2 transcription and antigen receptor locus-specific alterations that suppress V rearrangements. He proposes two independent aims to test fundamental aspects of his model. Aim 1 proposes to elucidate how RAG DSBs induced during different types of rearrangements in different cell types signal repression of Rag1/Rag2 transcription to coordinate initiation of V-to-(D)J recombination between alleles and thereby enforce allelic exclusion. Aim 2 proposes to determine the role of V region anti-sense long non-coding RNAs in promoting long-range V-to-(D)J rearrangements and serving as a DSB-responsive switch to transiently inhibit these rearrangements and thereby orchestrate allelic exclusion. The proposed work will employ powerful mouse models to rigorously elucidate mechanisms by which RAG DSBs trigger transient feedback inhibition of V recombination to help enforce allelic exclusion. The project will provide novel mechanistic insights into one understudied and one completely novel line of research in the field, the latter relevant to the biology all cells. Beyond advancing understanding of a hallmark of adaptive immunity, the findings could identify mechanisms important for suppressing autoimmunity and/or lymphoid malignancies.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $569,554 )
2025	2025	THE CHILDREN'S HOSPITAL OF PHILADELPHIA	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	4	7/29/2025	NON-COMPETING CONTINUATION	$569,554
														Subtotal = $569,554

Issue Date FY: 2024 ( Subtotal = $560,629 )
2024	2024	THE CHILDREN'S HOSPITAL OF PHILADELPHIA	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	3	5/31/2024	NON-COMPETING CONTINUATION	$560,629
														Subtotal = $560,629

Issue Date FY: 2023 ( Subtotal = $556,984 )
2023	2023	CHILDREN'S HOSPITAL OF PHILADELPHIA, THE	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	2	6/16/2023	NON-COMPETING CONTINUATION	$556,984
														Subtotal = $556,984

Issue Date FY: 2022 ( Subtotal = $517,160 )
2022	2022	CHILDREN'S HOSPITAL OF PHILADELPHIA, THE	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	1	7/12/2022	NEW	$517,160
														Subtotal = $517,160

Grand Total All Awards = $2,204,327

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Elucidating Mechanisms of RAG Endonuclease Mediated Feedback Inhibition of V(D)J Recombination

Award Number: R01AI172163

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 07/12/2022

PERIOD OF PERFORMANCE END DATE: 06/30/2027

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