Profiling RE1/REST-mediated histone modifications in the human genome

Abstract

Background: The transcriptional repressor REST (RE1 silencing transcription factor, also called NRSF for neuron-restrictive silencing factor) binds to a conserved RE1 motif and represses many neuronal genes in non-neuronal cells. This transcriptional regulation is transacted by several nucleosome-modifying enzymes recruited by REST to RE1 sites, including histone deacetylases (for example, HDAC1/2), demethylases (for example, LSD1), and methyltransferases (for example, G9a).

Results: We have investigated a panel of 38 histone modifications by ChIP-Seq analysis for REST-mediated changes. Our study reveals a systematic decline of histone acetylations modulated by the association of RE1 with REST (RE1/REST). By contrast, alteration of histone methylations is more heterogeneous, with some methylations increased (for example, H3K27me3, and H3K9me2/3) and others decreased (for example, H3K4me, and H3K9me1). Furthermore, the observation of such trends of histone modifications in upregulated genes demonstrates convincingly that these changes are not determined by gene expression but are RE1/REST dependent. The outcomes of REST binding to canonical and non-canonical RE1 sites were nearly identical. Our analyses have also provided the first direct evidence that REST induces context-specific nucleosome repositioning, and furthermore demonstrate that REST-mediated histone modifications correlate with the affinity of RE1 motifs and the abundance of RE1-bound REST molecules.

Conclusions: Our findings indicate that the landscape of REST-mediated chromatin remodeling is dynamic and complex, with novel histone modifying enzymes and mechanisms yet to be elucidated. Our results should provide valuable insights for selecting the most informative histone marks for investigating the mechanisms and the consequences of REST modulated nucleosome remodeling in both neural and non-neural systems.

Figure 1

RE1 and REST-mediated gene repression. The expression levels in CD4+ T-cells are shown as boxplots for all human genes (All genes), RE1 genes without REST (cRE1-REST and ncRE1-REST) and with REST (cRE1+REST and ncRE1+REST) in their promoters, and genes with RE1 motifs in the repetitive sequences of their promoters (RpRE1-REST and RpRE1+REST). Conversely, the genes with REST in their promoters are also separated into two groups, one with (REST+DJ-RE1) and the other without (REST-RE1) RE1s annotated in a previous study [19]. An asterisk indicates groups significantly (P < 0.001) different from all human genes with respect to their expression scores.

Figure 2

Dynamics of nucleosomes near the promoter and non-promoter cRE1 modulated by REST binding. The y-axis shows the normalized number of sequence tags (in a 10 bp window) from the sense strand (red) and antisense strand (green). The x-axis shows the distance to the center of canonical RE1s (blue box).

Figure 3

H3K9ac and H4K8ac profiles in RE1 promoters. The profiles of these acetylations were generated and plotted for four groups of genes with different colors (black, blue, red, and cyan), defined by the presences of cRE1, ncRE1, and REST in their promoters. The 'REST On & Exp Up' (red lines) refers to the group of genes with cRE1 and REST but an expression score >300. The profiles of modifications for these RE1 genes are shown with solid lines. For each of the four groups, a control was constructed by randomly selecting (5×) genes with the same expression levels but with neither RE1 nor REST in their promoters (see Materials and methods). The profiles of these controls are shown with dashed lines and colors matching to their targeted group. For the convenience of visual comparison, the zoom-in profiles for the four RE1 groups and their controls are re-drawn in the bottom panels. The color scheme and line style in the bottom panels apply to Figures 5-7. The x-axis shows the distance to transcription start sites with a unit representing 200 bp, and the y-axis shows the normalized counts of ChIP-Seq tags.

Figure 4

The P-values of paired t-test for comparing profiles between cRE1 promoters without REST and cRE1 with REST (or ncRE1 with REST, or cRE1 with REST and an expression value > 300). The data for increased and decreased levels of modifications upon REST binding are shown in red and green, respectively. Numbers are -log(10) transformation of P-values. An asterisk indicates histone modifications whose P-value from the comparison of RE1 genes is <0.0001 and at least ten times smaller than that from contrasting the corresponding control groups.

Figure 5

H3K4 profiles in RE1 promoters. The profiles are drawn in the same style as the bottom panels of Figure 3. The y-axis applies to a RE1 group and its control (dashed lines).

Figure 6

H3K36me3 and H3K27me3 profiles in RE1 promoters, drawn in the same style as Figure 3. The y-axis applies to a RE1 group and its control (dashed lines).

Figure 7

H3K9 and Pol II profiles in RE1 promoters. The profiles are drawn in the same style as the bottom panels of Figure 3. The y-axis applies to a RE1 group and its control (dashed lines).

Figure 8

A schematic diagram illustrating the major components involved in REST-mediated local chromatin remodeling and their relationships to our findings. (a) RE1 is initially covered by a nucleosome. (b) A yet-to-identified cellular mechanism initiates nucleosome repositioning with the assistance of BRG1, resulting in the exposure of the RE1 motif and the subsequent occupation of it by REST. The exact sequential order is not clear to date. (c) With the assistance of mSin3 and coREST, the RE1-bound REST complexes then recruit histone deacetylases (HDACs) to promote histone deacetylations, histone methylases (G9a, PRC2) to increase methylations on H3K9 and H3K27, and histone demethylases (LSD1, SMCX) to reduce methylations on H3K4. The presence of PRC2 in REST complexes is unknown but suggested by our analysis, so we have drawn a dashed line around it. Our data also strongly suggest that REST can recruit additional histone methylases and demethylases (represented by question marks) to target other lysine residues of histones, which display RE1/REST-dependent changes in the current study. The enumeration of all the histone modifying enzymes in the REST complexes will enhance our comprehension of how the complicated histone modifications are established; then, more investigations will be needed to decipher how these modifications cross-talk and orchestrate the regulation of RE1 genes.