Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation

Area of Science:

• Immunology
• Developmental Biology
• Genetics

Background:

• Superenhancers regulate gene expression through distinct elements acting cooperatively or temporally.
• The Irf8 superenhancer controls classical dendritic cell (cDC1) development, with specific elements functioning at different stages.
• The +41-kb Irf8 enhancer is vital for pre-cDC1 specification, and the +32-kb enhancer supports cDC1 maturation.

Purpose of the Study:

• To investigate the mechanistic relationship between the +41-kb and +32-kb Irf8 enhancers in cDC1 development.
• To determine the role of the Gm39266 long noncoding RNA in Irf8 enhancer function.
• To elucidate how the +41-kb enhancer regulates the activity of the +32-kb enhancer.

Main Methods:

• Generation and analysis of compound heterozygous Δ32/Δ41 mice lacking specific Irf8 enhancers.
• CRISPR/Cas9-mediated deletion of Gm39266 lncRNA promoters.
• Premature polyadenylation to block transcription across the +32-kb enhancer.
• Assessment of chromatin accessibility and transcription factor binding (BATF3).

Main Results:

• Mice lacking both +32-kb and +41-kb Irf8 enhancers showed normal pre-cDC1 specification but a complete absence of mature cDC1s.
• The +41-kb enhancer is essential for the activity of the +32-kb enhancer in a *cis*-dependent manner.
• Gm39266 lncRNA transcription and blocking transcription across the +32-kb enhancer did not impede cDC1 development.
• Chromatin accessibility and BATF3 binding at the +32-kb enhancer rely on a functional +41-kb enhancer.

Conclusions:

• The +41-kb Irf8 enhancer acts upstream to control the activation of the +32-kb Irf8 enhancer.
• This regulatory mechanism is independent of the Gm39266 long noncoding RNA.
• The +41-kb enhancer ensures proper cDC1 maturation by enabling the function of the +32-kb enhancer through chromatin modifications.