Histone H2A and H2B Are Monoubiquitinated at AID-Targeted Loci

Background

Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.

Methodology/Principal Findings

Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt''s B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V_H and Sγ3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.

Conclusions/Significance

Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.     

Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis

Ubiquitination is one of many known histone modifications that regulate gene expression. Here, we examine the Arabidopsis thaliana homologs of the yeast E2 and E3 enzymes responsible for H2B monoubiquitination (H2Bub1). Arabidopsis has two E3 homologs (HISTONE MONOUBIQUITINATION1 [HUB1] and HUB2) and three E2 homologs (UBIQUITIN CARRIER PROTEIN [UBC1] to UBC3). hub1 and hub2 mutants show the loss of H2Bub1 and early flowering. By contrast, single ubc1, ubc2, or ubc3 mutants show no flowering defect; only ubc1 ubc2 double mutants, and not double mutants with ubc3, show early flowering and H2Bub1 defects. This suggests that ubc1 and ubc2 are redundant, but ubc3 is not involved in flowering time regulation. Protein interaction analysis showed that HUB1 and HUB2 interact with each other and with UBC1 and UBC2, as well as self-associating. The expression of FLOWERING LOCUS C (FLC) and its homologs was repressed in hub1, hub2, and ubc1 ubc2 mutant plants. Association of H2Bub1 with the chromatin of FLC clade genes depended on UBC1,2 and HUB1,2, as did the dynamics of methylated histones H3K4me3 and H3K36me2. The monoubiquitination of H2B via UBC1,2 and HUB1,2 represents a novel form of histone modification that is involved in flowering time regulation.     

Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.     

Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity

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.     

An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

The HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll‐setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up‐regulated the expression of drought‐related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought‐resistant transgenic plants.