3’ Regulatory Regions (3’RR) in the human IgH locus show a complex mixture of insulator and enhancer properties

Friday, November 08, 2013 — Poster Session III
10:00 a.m. – 12:00 p.m.	FAES Academic Center (Upper-Level Terrace)	FDA/CBER	CHROM-4

Authors

F.C. Mills
R.M. Bernstein
V. Arudchandran
B. Cail
D. McLeod
Y. Sumathipala
E.E. Max

Abstract

Large 3' Regulatory Regions (3'RR) lie downstream from mammalian IgH (immunoglobulin heavy chain) loci. Recent studies show that the mouse 3'RR interacts with other IgH regulatory elements, playing a critical role in VDJ joining and class switching. We find complex features in the human 3'RR, which extend for ~ 25 kilobases downstream of both IgA1 and IgA2 genes. The 3'RR contains three B cell specific enhancers (HS3, HS12, HS4) followed by 5 CTCF sites. We demonstrate that the CTCF sites mediate enhancer blocking – characteristic of gene insulators – and that fragments containing the most 5’ CTCF site (AHS1) and penultimate 3’ CTCF site (AHS3) also have enhancer activity. AHS1 shows the strongest enhancer activity, stage-specific methylation, and binds vezf1 and NFkB, while AHS3 has repeated NFkB sites and shows the strongest NFkB binding. Testing of fragments across the 3’RR for insulator barrier function showed that a segment ~ 2 kb upstream from the CTCF region confers strong, persistent expression of a nearby reporter gene. This segment contains a cluster of predicted WT1 sites, whose function we are exploring. Therefore, sequences downstream of the known 3’ enhancers can function as positive genetic control elements, potentially responding to cellular signaling.