Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

Meiji Kit-Wan Ma,Adam G West,Alan Hair,Carol Heath,Jeannie T Lee

doi:10.1371/journal.pgen.1002175

Abstract

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3′ boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5′ boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.

Highlights

There is growing consensus that the non-random chromosomal arrangement of genes in higher eukaryotes enables the sharing of specific chromatin environments that facilitate co-regulation.Recent genomic profiling of histone modifications, chromatin factors and nuclear proximity in Drosophila and mammalian cells have revealed prevalent organization of genes into domains, or neighborhoods, of common chromatin state [1,2,3,4,5]
We have studied two chromatin boundary elements that flank a condensed chromatin region located between the chicken FOLR1 and b-globin genes, respectively
We propose that chromatin boundaries at many gene loci employ H2B ubiquitination to restrict the encroachment of repressive chromatin

Summary

Introduction

There is growing consensus that the non-random chromosomal arrangement of genes in higher eukaryotes enables the sharing of specific chromatin environments that facilitate co-regulation.Recent genomic profiling of histone modifications, chromatin factors and nuclear proximity in Drosophila and mammalian cells have revealed prevalent organization of genes into domains, or neighborhoods, of common chromatin state [1,2,3,4,5]. Genes taken out of their natural chromosomal environment become deregulated in a variety of human genetic diseases [6]. This so-called chromosomal position effect underlies the variable expression of transgenes depending on their site of integration [7]. Fixed chromatin boundaries can be established by DNA sequence elements called insulators, which function to protect genes from inappropriate signals emanating from their surrounding environment [9,10,11,12]. A 275 bp core of the HS4 element has two separable activities that functionally define insulators: it can block the action of an enhancer element on a linked promoter when positioned between the two and it can act as a barrier to chromosomal position effect silencing [16,17,18]. HS4 requires a USF1/USF2 binding site (FIV) and three VEZF1 binding sites (FI, FIII and FV) for its barrier activity, which control histone modifications and DNA methylation, respectively [21,22,23,24]

Methods

Results

Discussion

Conclusion