Interplay between CTCF-binding and CTCF-lacking regulatory elements in generating an architectural stripe at the Igh...

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• Three-dimensional genome organization is critical for immunoglobulin heavy chain (Igh) gene regulation.
• Wild-type Igh alleles feature an architectural stripe from 3’ CTCF binding elements (3’CBE) interacting across the locus.
• The interplay of regulatory elements within the 3’Igh domain influences this stripe structure.

Purpose of the Study:

• To investigate how regulatory elements in the 3’Igh domain modulate chromatin structure and stripe formation.
• To understand the role of the intronic enhancer Eµ and CTCF binding elements in Igh locus organization.
• To explore the impact of specific regulatory elements on epigenetic features and VDJ recombination.

Main Methods:

• CRISPR-mediated gene editing to delete or invert regulatory elements (Eµ, IGCR1, 3’CBE).
• Analysis of epigenetic modifications (histone marks) and chromatin looping interactions.
• Assessment of VDJ recombination efficiency and gene transcription.

Main Results:

• The Eµ enhancer promotes stripe formation and tethers sub-topologically associating domains (sub-TADs).
• Replacing Eµ with an EF1α promoter partially restored epigenetic features and sub-TAD formation but not VDJ recombination.
• Loss of IGCR1 increased the stripe, while inverted 3’CBE redirected the stripe but maintained boundary functions.

Conclusions:

• Regulatory elements, particularly Eµ, play a key role in establishing the three-dimensional structure of the Igh locus.
• Specific elements modulate chromatin organization, influencing interactions and epigenetic states.
• The 3’Igh domain elements are essential for proper Igh gene regulation and locus architecture.