target: an R package to predict combined function of transcription factors

Implementation

We developed an open-source R/Bioconductor package target to implement BETA for predicting direct transcription factor targets from binding and expression data. The details of the algorithm were described here⁶. In addition, our implementation extends BETA to apply for transcription factor combinations (7). Briefly, we identify the transcription factor potential binding sites by ChIP-sequencing and gene expression under factor perturbation by microarrays or sequencing. Next, we score the peaks based on their distances to the transcription start sites. The sum of the scores of the individual peaks in a certain region of interest is the region’s regulatory potential. The signed statistics (fold-change or t-statistics) from the differential gene expression of the transcription factor perturbation reflect the transcription factor effects. The product of the ranks of the regulatory potential and the signed statistics is the final rank of the regions.

To predict the combined function of two transcription factors, two sets of data are required. The overlapping peaks are the potential binding sites. The product of the two signed statistics is the transcription factor function. When the two transcription factors agree in the direction of the regulation of a region where they both bind, they could be said to cooperate on this region. When the sign is opposite, they could be said to regulate that region competitively.

The package leverages the Bioconductor data structures such as GRanges and DataFrame to provide fast and flexible computation on the data⁸. Similar to the original python implementation, the input data are the identified peaks from the ChIP-Seq experiment and the expression data from RNA-Seq or microarrays perturbation experiment. The final output is the peaks associated with the transcription factor binding and the predicted direct targets. We use the terms “peaks” to refer to the GRanges object that contains the coordinates of the peaks. Likewise, we use the term “region” to refer to a similar object that contains the information on the regions of interest; genes, transcripts, promoter regions, etc. In both cases, additional information on the ranges can be added to the object as metadata.

Operation

The algorithm was implemented in R (>= 3.6) and should run on any operating system. Libraries required for running the workflow are listed and loaded below. Alternatively, a docker image is available with R and the libraries installed on an Ubuntu image: https://hub.docker.com/r/bcmslab/target_flow

# load required libraries
library(GenomicRanges)
library(Biostrings)
library(rtracklayer)
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
library(BSgenome.Hsapiens.UCSC.hg19)
library(org.Hs.eg.db)
library(tidyverse)
library(BCRANK)
library(seqLogo)
library(target)

Preparing the binding data

The ChIP peaks were downloaded in the form of separate bed files for each transcription factor. We first locate the files in the data/ directory and load the files using import.bed. Then the data is transformed into a suitable format, GRanges. The resulting object, peaks, is a list of two GRanges items, one for each factor.

# locate the peaks bed files
peak_files <- c(YY1 = 'data/Oth.Utr.05.YY1.AllCell.bed',
                  YY2 = 'data/Oth.Utr.05.YY2.AllCell.bed')

# load the peaks bed files as GRanges
peaks <- map(peak_files, ~GRanges(import.bed(.x)))

Preparing the expression data

The differential expression data were downloaded in tabular format. After locating the files in data/, we read the files using read_tsv and select and rename the relevant columns. The resulting object, express, is a list of two tibble items.

# locate the expression text files
expression_files <- c(YY1 = 'data/DataSet_01_18.tsv',
                        YY2 = 'data/DataSet_01_19.tsv')

# load the expression text files
express <- map(expression_files,
                 ~read_tsv(.x, col_names = FALSE) %>%
                   dplyr::select(2, 3, 7, 9) %>% #9
                   setNames(c('tf', 'gene', 'fc', 'pvalue')) %>%
                   filter(tf %in% c('YY1', 'YY2')) %>%
                   na.omit())

The knockdown of either transcription factor in HeLa cells seems to change the expression of many genes in either direction (Figure 1A&B). Moreover, the changes resulting from the separate knockdown of the transcription factors are correlated (r = 0.56, P < 0.0001) (Figure 1C). These observations suggest that many of the regulated genes are shared targets of the two transcription factors, or they respond similarly to their perturbation of either factor.

Figure 1. Differential expression between transcription factor knockdown and control HeLa cells.

Gene expression was compared between transcription factors knockdown and control HeLa cells. The fold-change and p-values of (A) YY1- and (B) YY2-knockdown are shown as volcano plots. (C) Scatter plot of the fold-change of the YY1- and YY2-knockdown.

# Figure 1
par(mfrow = c(1, 3))

# volcano plot of YY1 knockdown
plot(express$YY1$fc,
      -log10(express$YY1$pvalue),
      xlab = 'Fold-change (log_2)',
      ylab = 'P-value (-log_10)',
      xlim = c(-4, 4), ylim = c(0, 6))
title('(A)')

# volcano plot of YY2 knockdown
plot(express$YY2$fc,
      -log10(express$YY2$pvalue),
      xlab = 'Fold-change (log_2)',
      ylab = 'P-value (-log_10)',
      xlim = c(-4, 4), ylim = c(0, 6))
title('(B)')

# plot fold-change of YY1 and YY2
plot(express$YY1$fc[order(express$YY1$gene)],
      express$YY2$fc[order(express$YY2$gene)],
      xlab = 'YY1-knockdown (log_2)',
      ylab = 'YY2-knockdown (log_2)',
      xlim = c(-4, 4), ylim = c(-4, 4))
title('(C)')

Preparing genome annotation

express records the gene information using the gene Symbols. We mapped the Symbols to the Entrez IDs before extracting the genomic coordinates. To do that, we use the org.Hs.eg.db to convert between the identifiers. Next, we use the TxDb.Hsapiens.UCSC.hg19.knownGene to get the genomic coordinates for the transcripts and extend them to 100kb upstream and 200bp downstream from the transcription start sites.

# load genome data
symbol_entrez <- AnnotationDbi::select(org.Hs.eg.db,
                           unique(c(express$YY1$gene)),
                           'ENTREZID', 'SYMBOL') %>%
  setNames(c('gene', 'gene_id'))

# format genome to join with express
genome <- promoters(TxDb.Hsapiens.UCSC.hg19.knownGene,
             upstream = 100000, # (default) downstream = 200,
             columns = c('tx_id', 'tx_name', 'gene_id')) %>%
  as_tibble() %>% mutate(gene_id = as.character(gene_id))

The resulting object, genome, from the previous step is a tibble that shares the column gene_id with the expression data express. Now the two objects can be merged. The merged object, regions, is similarly a tibble containing genome and expression information of all common genes.

# make regions by merging the genome and express data
regions <- map(express,
                 ~inner_join(genome, symbol_entrez) %>%
                   inner_join(.x) %>%
                   makeGRangesFromDataFrame(keep.extra.columns = TRUE))

Predicting gene targets of individual transcription factors

The standard target analysis identifies associated peaks using associated_peaks and direct targets using direct_targets. associated_peaks calculates and transforms the distances between the peaks and TSSs. Then it assigns the peaks to the nearst transcript. direct_targets calculates the final gene ranks based on the distances and the change in gene expression. The inputs for these functions are the objects peaks and regions from the previous steps in addition to the column names for regions regions_col or the region and the statistics column stats_col, which is the fold-change in this case. The resulting objects are GRanges for the identified peaks assigned to the regions, ap, or the ranked targets. Several columns are added to the metadata objects of the GRanges to save the output.

# get associated peaks
ap <- map2(peaks, regions,
            ~associated_peaks(peaks=.x,
                                regions = .y,
                                regions_col = 'tx_id'))

# get direct targets
dt <- map2(peaks, regions,
            ~direct_targets(peaks=.x,
                              regions = .y,
                              regions_col = 'tx_id',
                              stats_col = 'fc'))

To determine the dominant function of a transcription factor, we divide the targets by the direction of the effect of transcription factor knockdown. We group the targets by the change in gene expression (regulatory potential). We use the empirical distribution function (ECDF) to show the fraction of targets with a specified regulatory potential or less. Because we use the ranks rather than the absolute value of the regulatory potential, the lower the rank, the higher the potential. Then, we compare the groups of targets to each other or to a theoretical distribution.

# Figure 2
par(mfrow = c(1, 3))

# plot distance by score of associate peaks
plot(ap$YY1$distance, ap$YY1$peak_score,
      xlab = 'Distance', ylab = 'Peak Score',
      main = '(A)')
points(ap$YY2$distance, ap$YY2$peak_score)

# make labels, colors and groups
labs <- c('Down', 'None', 'Up')
cols <- c('green', 'gray', 'red')

# make three groups by quantiles
groups <- map(dt,~{
  cut(.x$stat, breaks = 3, labels = labs)
})

# plot the group functions
pmap(list(dt, groups, c('(B)', '(C)')), function(x, y, z) {
       plot_predictions(x$score_rank,
                          group = y, colors = cols, labels = labs,
                          xlab = 'Regulatory Potential', ylab = 'ECDF')
       title(z)
    })

The scores of the individual peaks are a decreasing function of the distance from the transcription start sites— the closer the transcription factor binding site from the start site, the higher the score. The distribution of these scores is very similar for both transcription factors (Figure 2A). The ECDF of the down-regulated of YY1 is higher than that of up-and none-regulated targets (Figure 2B). Therefore, the absence of YY1 on its targets results in aggregate in their downregulation. If indeed these are true targets, then we expect YY1 to induce their expression. The opposite is true for YY2, where more high-ranking targets are up-regulated by the transcription factor knockdown (Figure 2C).

# Table 2
# test individual factor functions
map2(dt, groups,
      ~test_predictions(.x$rank,
                          group = .y,
                          compare = c('Down', 'Up')))

Figure 2. Predicted functions of YY1 and YY2 on their specific targets.

Bindings peaks of the transcription factors in HeLa cells were determined using ChIP-Seq. Distances from the transcription start sites, and the transformed distances of the (A) YY1 and YY2 peaks are shown. The regulatory potential of each gene was calculated using target. Genes were grouped into up, none, or down-regulated based on the fold-change. The empirical cumulative distribution functions (ECDF) of the groups of (C) YY1 and (C) YY2 targets are shown at each regulatory potential rank.

To formally test these observations, we use the Kolmogorov-Smirnov (KS) test. First, we compare the distributions of the two groups for equality. If one lies on either side of the other, then they must be drawn from different distributions. Here, we contrast the up and down-regulated functions for both transcription factors (Table 2). In both cases, the distributions of the two groups were significantly different from one another.

Predicting the shared targets of two transcription factors

Using target to predict the shared target genes and the combined function of the two transcription factors is a variation of the previous analysis. First, the shared/common peaks are generated using the overlap of their genomic coordinates, subsetByOverlaps. Second, Instead of one, two columns for the differential expression statistics, one for each transcription factor is needed; these are supplied to the argument stats_col in the same way. Here, common_peaks and both_regions are the main inputs for the analysis functions.

# merge and name peaks
common_peaks <- GenomicRanges::reduce(subsetByOverlaps(peaks$YY1, peaks$YY2))
common_peaks$name <- paste0('common_peak_', 1:length(common_peaks))

# bind express tables into one
both_express <- bind_rows(express) %>%
  nest(fc, pvalue, .key = 'values_col') %>%
  spread(tf, values_col) %>%
  unnest(YY1, YY2, .sep = '_')

# make regions using genome and expression data of both factors
both_regions <- inner_join(genome, symbol_entrez) %>%
  inner_join(both_express) %>%
  makeGRangesFromDataFrame(keep.extra.columns = TRUE)

# get associated peaks with both factors
common_ap <- associated_peaks(peaks = common_peaks,
                                  regions = both_regions,
                                  regions_col = 'tx_id')

# get direct targets of both factors
common_dt <- direct_targets(peaks = common_peaks,
                                regions = both_regions,
                                regions_col = 'tx_id',
                                stats_col = c('YY1_fc', 'YY2_fc'))

The output, associated_peaks, is similar to before. direct_targets is the same, but the stat and the stat_rank columns carry the product and the rank of the two statistics provided in the previous step.

We can also visualize the output in a similar way. The targets are divided into three groups based on the statistics product. When the two statistics agree in the sign, the product is positive. This means the knockdown of either transcription factor results in the same direction change in the target gene expression. Therefore, the two transcription factors would cooperate if they bind to the same site on that gene. The reverse is true for targets with oppositely signed statistics. The two transcription factors would be expected to compete on these targets for inducing opposing changes in the expression.

# Figure 3
par(mfrow = c(1, 2))

# plot distiace by score for associated peaks
plot(common_ap$distance,
     common_ap$peak_score,
     xlab = 'Distance',
     ylab = 'Peak Score')
title('(A)')

# make labels, colors and gorups
labs <- c('Competitive', 'None', 'Cooperative')
cols <- c('green', 'gray', 'red')

# make three groups by quantiles
common_groups <- cut(common_dt$stat,
                      breaks = 3,
                      labels = labs)

# plot predicted function
plot_predictions(common_dt$score_rank,
                   group = common_groups,
                   colors = cols, labels = labs,
                   xlab = 'Regulatory Interaction', ylab = 'ECDF')
title('(B)')

The common peak distances and scores take the same shape (Figure 3A). Furthermore, the two transcription factors seem to cooperate on more of the common target than any of the two other possibilities (Figure 3B). This observation can be tested using the KS test. The curve of the cooperative targets lies above that of none and competitively regulated targets (Table 3).

Figure 3. Predicted function of YY1 and YY2 on their shared targets.

Shared bindings sites of YY1 and YY2 in HeLa cells were determined using the overlap of the individual transcription factor ChIP-Seq peaks. (A) Distances from the transcription start sites, and the transformed distances of the shared peaks are shown. The regulatory interaction of each gene was calculated using target. Genes were grouped into cooperatively, none, or competitively regulated based on the product of the fold-changes from YY1- and YY2-knockdown. (B) The empirical cumulative distribution functions (ECDF) of the targets groups are shown at each regulatory potential rank.

# Table 3
# test factors are cooperative
test_predictions(common_dt$score_rank,
                   group = common_groups,
                   compare = c('Cooperative', 'None'),
                   alternative = 'greater')

# test factors are more cooperative than competitive
test_predictions(common_dt$score_rank,
                   group = common_groups,
                   compare = c('Cooperative', 'Competitive'),
                   alternative = 'greater')

Binding motif analysis

The users can perform any number of downstream analyses on the final output. For example, we could apply binding motif analysis to the groups of regulated targets. In this example, all the motif analysis itself is handled by the BCRANK package¹⁴. Here, we explain how to prepare the input from the shared peaks and target objects produced in the last step.

First, we extract the transcript IDs of the targets in their respective groups. Then the peaks assigned to these targets are ordered and sliced.

# group peaks by their assigned targets
peak_groups <- split(common_dt$tx_id, common_groups)

# reorder peaks and get top n peaks
peak_groups <- lapply(peak_groups, function(x) {
    # get peaks in x targets group
    p <- common_ap[common_ap$assigned_region %in% unique(x)]

    # order peaks by score
    p <- p[order(p$peak_score, decreasing = TRUE)]

    # get n top peaks
    p[seq_len(ifelse(length(p) > 50, 50, length(p)))]
})

The input for bcrank is a fasta file with the sequence of the regions to look for frequent motifs. We used the BSgenome.Hsapiens.UCSC.hg19 to extract the sequences of the common peaks in the competitive and cooperative target groups. The sequences are first written to a temporary file and feed to the search function.

bcout <- map(peak_groups[c('Competitive', 'Cooperative')], ~{
    # extract sequences of top peaks from the hg19 genome
    pseq <- getSeq(BSgenome.Hsapiens.UCSC.hg19, names = .x)

    # write sequences to fasta file
    tmp_fasta <- tempfile()
    writeXStringSet(pseq, tmp_fasta)

    # set random see
    set.seed(1234)

    # call bcrank with the fasta file
    bcrank(tmp_fasta, silent = TRUE)
})

The sequences in the search path of the regions of interest are shown in (Figure 4). In the competitively regulated regions, one sequence was more frequent than all other sequences. By contrast, no sequence was uniquely frequent in the regions of cooperative targets.

# Figure 4
par(mfrow = c(1, 2))

# plot the occurrences of consensus sequesnce in the regions
map2(bcout, c('(A)', '(B)'), ~{
     plot(toptable(.x, 1))
     title(.y)
})

Figure 4. Occurrences of consensus sequences in the ranked regions.

The number of occurances of the sequences in the search path in the regions of (A) competitively and (B) cooperatively regulated regions.

The most frequent motifs in the two groups are shown as seq logos using the seqLogo package (Figure 5).

# Figure 5
# plot the sequence of the predicted motifs
map(bcout, c('(A)', '(B)'), ~{
    seqLogo(pwm(toptable(.x, 1)))
    title(.y)
})

Figure 5. Predicted motifs of the cooperative and competitive binding sites.

The position weight matrices of the most frequent motifs in the (A) competitively and (B) cooperatively regulated regions were calculated and shown as sequence logos. y-axis represents the information content at each position. The size of each letter represents the frequency in which the letter occurs at that position.