Genome-wide function of H2B ubiquitylation in promoter and genic regions

Abstract

Nucleosomal organization in and around genes may contribute substantially to transcriptional regulation. The contribution of histone modifications to genome-wide nucleosomal organization has not been systematically evaluated. In the present study, we examine the role of H2BK123 ubiquitylation, a key regulator of several histone modifications, on nucleosomal organization at promoter, genic, and transcription termination regions in Saccharomyces cerevisiae. Using high-resolution MNase chromatin immunoprecipitation and sequencing (ChIP-seq), we map nucleosome positioning and occupancy in mutants of the H2BK123 ubiquitylation pathway. We found that H2B ubiquitylation-mediated nucleosome formation and/or stability inhibits the assembly of the transcription machinery at normally quiescent promoters, whereas ubiquitylation within highly active gene bodies promotes transcription elongation. This regulation does not proceed through ubiquitylation-regulated histone marks at H3K4, K36, and K79. Our findings suggest that mechanistically similar functions of H2B ubiquitylation (nucleosome assembly) elicit different functional outcomes on genes depending on its positional context in promoters (repressive) versus transcribed regions (activating).

Figure 1.

K123ub modulates genic nucleosomal occupancy. (A) Heat maps of nucleosomal occupancies in wild-type (WT) and H2BK123A strains. Each row represents a genic nucleosomal array; arrays are sorted by array length and aligned by array midpoint (illustrated above the plot). Blue areas are the 5′ and 3′ NFRs. The color bar represents the nucleosomal occupancy from dark blue (zero) to green (genomic average) to dark red (twice the genomic average). (B) Heat maps of log₂ fold changes in nucleosomal occupancies in K123A over wild type, aligned by TSS and sorted by gene length. Each column represents a consensus nucleosome position relative to the TSS. (C) Composite distribution of nucleosomal tags (shifted to represent dyads) in wild type (gray fill) and the K123A mutant (line trace), aligned by the wild-type consensus position of the first nucleosome dyad for all genes (black), highly expressed genes (red), and lowly expressed genes (blue).

Figure 2.

Nucleosomal organization in K123ub-deficient mutants. (A) Heat maps of log₂ fold changes in nucleosomal occupancies in wild type (WT) or the indicated mutants relative to an independently derived wild type. (B) Composite distribution of nucleosomal tags in wild type and indicated mutants for highly expressed genes (bre1Δ was not tested).

Figure 3.

Nucleosomal organization in H3K4A and H3K79A mutants. Heat maps of nucleosomal occupancies in wild type (WT) and mutants are shown in the top panels. Heat maps of log₂ fold changes in nucleosomal occupancies in the indicated mutants relative to wild type are shown in the middle panels. Composite distribution of nucleosomal tags in wild type (gray fill) and indicated mutants (black traces, no fill) for all genes are shown in the bottom panel.

Figure 4.

K123ub and K36me modulates nucleosomal organization through distinct pathways. (A) Composite nucleosomal H3 (filled gray), H3K36me2 (red), and K36me3 (blue) tag distribution for all genes. (B) Log₂ fold changes in K36 methylated nucleosomal occupancies in K123A relative to wild type (WT) and aligned by the first nucleosome dyad. (C) Heat maps of log₂ fold changes in nucleosomal occupancies in K36A relative to wild type.

Figure 5.

K123ub differentially regulates transcription based on its genomic location. (A) Median transcription frequency for all genes (Holstege et al. 1998) and genes that are positively or negatively regulated by K123ub by >1.5-fold (see the Materials and Methods). (B) Log₂ fold changes in pol II occupancies in a K123A mutant strain over wild type (WT) for the indicated set of H2Bub-regulated genes, aligned by the TSS. (C) Composite nucleosomal H3 (filled gray) and K123ub (black trace) distribution for all genes. Similar plots were obtained using the data of Schulze et al. (2009; not shown). (D) Log₂ fold changes in ubiquitylation levels per base pair in promoter regions (−1 nucleosome) over coding regions (+1 to +5 nucleosomes) for the indicated set of genes.

Figure 6.

Increased nucleosome occupancy in a Δubp8 strain. (A) The median nucleosome turnover rate from Dion et al. (2007) for all nucleosomes that belong to highly or lowly expressed genes or ubiquitylation-enriched nucleosomes (top 500 nucleosomes ranked according to H2Bub/H3 ratio) was divided by the genome-wide median for all nucleosomes, then log₁₀-transformed. (B) Heat maps of nucleosomal occupancies in wild-type (WT) and Δubp8 strains. (C) Composite nucleosomal distribution in wild-type (filled gray) and Δubp8 (black trace) strains for divergently transcribed genes.