DS0408 -

Genetic and functional dissection of the role of the IgH 3’ regulatory region during class switch recombination – EpiSwitch-3RR

Submission summary

B lymphocytes have a remarkable capacity to somatically alter their genomes. Mature B cells have the unique ability to undergo class switch recombination (CSR) which specifically targets the constant genes of the immunoglobulin heavy chain (IgH) locus. CSR occurs between highly repetitive, transcribed switch sequences. Transcription across switch regions provides the substrate for the enzyme AID, which initiates DNA cleavage ultimately leading to double-strand breaks that are repaired by the classical and alternative non-homologous end joining repair pathways (NHEJ). The mechanisms that govern the targeting of AID are still unclear.
Switch transcription is regulated in a major part by a locus control region (LCR) called the 3’ regulatory region (3’RR) composed of four enhancers embedded in a highly repetitive palindrome. In stark contrast to known LCRs, the 3’RR displays a striking pattern: it mediates a silencing activity in B cell precursors, acts as a constitutive LCR in developing B cells and as an inducible LCR in mature B cells. The mechanisms underlying this developmental shift are unknown.

This programme aims to achieve a significant progress in our understanding of the basic mechanisms through which the 3’RR controls in vivo the expression of a complex locus featuring a fascinating interplay between development, transcription, epigenetics, DNA breaks, recombination and repair. The whole project relies on already available mutant mouse models and on an original combination of functional, genetic, and mechanistic studies. The programme brings together two partners with solid expertise and established record in molecular biology of B cell development, and has four inter-linked aims:

Aim 1: Stepwise activation of switch transcription
We will perform a detailed mechanistic analysis of how the long-range effect of the 3’RR unfolds at the level of a target promoter, i.e. the 3’RR-dependent mechanisms that control the cascade of molecular events from chromatin-remodeling and promoter opening to transcriptional pausing and elongation across switch regions. We will determine precisely which of these events are controlled by the constitutive or the inducible 3’RR and which are 3’RR-independent.

Aim 2: Long-range control of the IgH constant domain by the 3’RR
We ambition here to provide a comprehensive conceptual framework for the dynamics of the 3’RR during the late stages of B cell development. We will investigate the mechanisms by which the 3’RR effects its constitutive and inducible long-range activation of multiple promoters within dynamic chromatin domains, including the 3’RR-mediated looping/scanning, the potential competition between promoters, the role of transcriptional/architectural factors, and the status of the chromatin of the IgH constant domain.

Aim 3: Role of the palindromic architecture of the 3’RR
The aim here is to analyze if and how the palindromic structure of the 3’RR may affect its function. We will focus on the functional importance of the core enhancers and the inverted repeats for B cell development, transcription, epigenetic modifications, and AID-mediated DNA remodeling of the IgH locus. This long-time due analysis is expected to uncover how structural elements and local events within the 3’RR influence its long-range activity.

Aim 4: Role of the cross-talk between IgH enhancers in post-transcriptional events
We aim here to investigate whether and how the master regulatory elements of the IgH locus (Eµ enhancer and 3’RR) influence the interplay between alternative splicing and recombination/repair, in a lymphoid compartment-dependent manner, by focusing on the delta constant gene. The analysis will include the set-up of epigenetic marks, the differential recruitment of AID and classical and alternative NHEJ components.

Project coordination

Ahmed Amine Khamlichi (Centre National de la Recherche Scientifique/Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CRIBL CRIBL, CNRS UMR 7276
CNRS/IPBS Centre National de la Recherche Scientifique/Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089

Help of the ANR 576,999 euros
Beginning and duration of the scientific project: January 2017 - 42 Months

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