NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors

Abstract

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.

Figure 1.

ChIP-seq of two components of the NF-Y complex in three cell types. (A) MACS peak analysis indicating peak numbers, mean peak lengths, and standard deviations at three different P-value thresholds for NF-YA and NF-YB ChIP-seq data sets in GM12878, HeLa S3, and K562. (B) Identification of the NF-Y DNA-binding site motif de novo from 12,655 K562 NF-YB peaks depicted as a sequence logo (Schneider and Stephens 1990). (C) Scatter plots of NF-YA, NF-YB, and input read counts at NF-YA or NF-YB sites in K562 showing correlation between data sets. (Blue shading) Correlation amongst NF-YA and NF-YB. (Orange shading) NF-YA or NF-YB correlation to input. (D) Venn diagrams depicting the overlap between NF-YB peak populations in GM12878, HeLa S3, and K562. Integers represent peak numbers called at the 10⁻⁹ P-value threshold. The percentages of peaks with CCAAT motifs are indicated (%). (E) ChIP-qPCR validation of NF-YB peaks unique to each cell type. (Error bars) Standard deviation of three biological replicates. “Pos. Ctrls” are loci known to be bound by NF-Y. “Neg Ctrls” are loci known to be devoid of NF-Y. Data represents a fold over background measurement compared with a non-NF-Y bound region (“GAPDH up”). (Solid and striped bars) ChIPs performed with NF-YB specific antibody and nonspecific rabbit IgG, respectively.

Figure 2.

Annotation of NF-Y peaks to genomic features. (A) Kernel density estimate of the distribution of the 5′-CCAAT-3′ and 5′-ATTGG-3′ sequences under NF-YA and NF-YB peaks in relation to the peak summit centered at 0 bp. Only the position of the best matching CCAAT motif within 100 bp of the peak summit is considered and plotted. (Solid and dashed lines) Raw and Gaussian smoothed data, respectively. (B) Annotation of K562 NF-YB sites to RefSeq gene features. (C) As in B, except chromatin state maps are used. (Prom) promoter; (enh) enhancer; (trxn) transcription. Numbering is from the chromatin state maps of Ernst et al. (2011). (D) Frequency distribution of K562 NF-YB peak summits at RefSeq TSSs showing a preferential location between −50 and −100 bp upstream of the TSS. (E) Gaussian kernel density estimate of the distribution of positive and negative strand 5′-CCAAT-3′ and 5′-ATTGG-3′ sequences at K562 NF-YB-bound RefSeq TSSs. Only the best motif per region is considered. Bandwidth is equal to the standard deviation of the smoothing kernel. (Gray arrows) Direction of transcription.

Figure 3.

NF-YB bound loci reside within five epigenetic domains. (A) K-means clustering of K562 NF-YB loci based on the distribution of histone PTM, RNA Pol II, NF-YB, and NF-YA ChIP-seq reads within a region spanning ±5 kbp from the summit of NF-YB peaks (centered at 0 bp). Clustering was carried out on transformed, rank normalized read counts. Raw read count intensity is depicted in red. The interpretation and classification of clusters into functional categories are shown at right. (B) NF-YB summits from clusters derived from A are annotated to genomic features: chromatin states, LTRs, dbTSS, RefSeq promoters, and FAIRE-seq regions. The percentage of peak summits within each cluster overlapping a specific feature is indicated. Overlap with LTRs is assayed within a window of ±250 bp from the ends of the LTR feature. RefSeq promoters are considered within a window of −2500:+500 bp from the TSS. A direct overlap with FAIRE-seq regions and chromatin states is used. Long poly(A) purified RNA reads were counted within a window of ±500 bp about the NF-YB peak summit, and the median value of that cluster is shown (n = size of cluster in peaks).

Figure 4.

NF-YB binds extensively to long terminal repeats. (A) The percentage of all K562 NF-YB peak summits that occupy the indicated feature. Core and proximal promoters are defined as −250:+50 bp and −2500:+500 bp from the TSS of RefSeq promoters, respectively. (B) Mapping of ChIP-seq reads from K562, GM12878, and HeLa S3 to Repbase consensus sequences showing an abundance of NF-Y ChIP-seq reads mapping to repetitive elements. Ratios reflect the enrichment of reads in the NF-YB ChIP sample as compared with input. Only Repbase entries with a read ratio ≥5 are shown. Orange shading indicates enriched repeats present in all cell lines. Green and red shading indicate the presence and absence, respectively, of a CCAAT motif match at P-value < 10⁻⁴ in the consensus sequence. (C) Frequency of overlap between NF-YB peak summits and the genomic locations of LTR families. Only LTR elements that overlap at least one NF-YB summit in each cell line are shown. The two most highly overlapping repeat families are indicated, LTR12 and MLTJ1. (D) Distribution of NF-YB bound LTRs from K562 and GM12878 at chromatin states. No chromatin state map is available for HeLa S3.

Figure 5.

NF-YB can occupy its motif in closed chromatin. (A) The percentage of genome-wide computationally discovered CCAAT motifs within each chromatin state, FAIRE-seq regions or the entire genome, that directly overlap NF-YB K562 sites plotted as a function of CCAAT motif quality (right axes). Also shown are the numbers of discovered CCAAT motifs as a function of quality (left axes). Numbering is derived from Ernst et al. (2011) and kept for consistency. (B) Distribution of CCAAT motif quality scores under NF-YB K562 peaks, called at three different P-values, a random genomic background sample set of 400 k 500-bp regions and K562 FAIRE-seq regions. (C) Similar to A, except motifs of different TFs are plotted as a function of motif quality. Only a subset of TFs is shown; see Supplemental Figure 13 for all TFs analyzed.

Figure 6.

NF-Y and FOS are closely coassociated at loci that lack JUN and the AP-1 motif. (A) Correlation between ChIP-seq read counts at NF-YB peak summits, within a window of ±500 bp, between NF-YB and NF-YA, FOS, JUN, or MYC in K562 cells. (B) Values represent the percentage of peak populations (left row) directly overlapping the peak population of a second factor (top column). All binding sites are called at a P-value < 10⁻⁹. FOS (n = 14404); JUN (n = 18480); MYC (n = 13693); NF-YA (n = 4726); NF-YB (n = 12655). (C) The number of ChIP-seq peaks at the indicated distance between adjacent peak summits is plotted. All peaks were called at a 10⁻⁹ P-value threshold in K562. (D) The top 1000 K562 FOS ChIP-seq sites, as ranked by site P-value, that directly overlap an NF-YB site (“FOS+NF-YB”) and the top 1000 that do not overlap an NF-YB site (10⁻⁵ P-value site list, “FOS-NF-YB”) are assayed for the distribution of the AP-1 motif in relation to the FOS peak summit centered at 0 bp. Plotted is the Gaussian kernel density estimate of the AP-1 motif using a bandwidth of 0.5 of the standard deviation of the smoothing kernel. The top three motifs discovered de novo from each FOS peak set, as above, are depicted with the percentage of FOS peaks containing a match to that motif indicated. (E) Representative view of a locus on chromosome 3 of the K562 ChIP-seq read counts from NF-YA, NF-YB, FOS, JUN, and MYC ChIPs, with an input control.

Figure 7.

NF-Y coassociates with many factors at promoters and enhancers. Illustration of the factors that significantly associate with NF-YB-bound strong promoters and enhancers. Only those factors that satisfy the following criteria are shown: greater than the median fold enrichment with respect to NF-YB-nonbound regions (enrichment indicated by circle size); greater than the median value of percent occupancy of NF-YB-bound regions (percentage occupied indicated by color); significantly coassociate with NF-Y (gray box, see Supplemental Fig. 14A). Factors enclosed within a yellow box are, additionally, the subset of factors that cluster with NF-YA and NF-YB (see Supplemental Fig. 14B). A black arrow indicates the start of a transcribed region. Two vertical slashes are used to represent being distal to a promoter area.

Figure 8.

Motif pairings with the CCAAT motif are stereo positioned. (A) The percentage of NF-Y sites that have an AP-1 motif at the specified distance from the best scoring CCAAT motif centered at 0 bp. NF-YB peaks overlapping LTRs are categorized as “predicted,” while the subset of NF-YB sites overlapping the respective ChIP-seq sites of FOS are categorized as “verified.” The negative strand plots are near identical mirror images of the positive strand plots and are not shown. (B) Similar to A, except that all genomic regions are considered. The percentage of NF-YB peaks that have a TATA element (TBP), E box (MYC, MAX, USF1), and E2F motif (E2F6) are plotted. All NF-Y peaks are categorized as “predicted,” while those NF-Y peaks overlapping the respective ChIP-seq peaks of the other TF are categorized as “verified.” Only the top 500 peaks in each category are plotted.