---
title: "TFEA.ChIP: a tool kit for transcription factor enrichment analysis capitalizing on ChIP-seq datasets"
author: "Laura Puente-Santamaria, Luis del Peso"
date: "`r Sys.Date()`"
output: rmarkdown::html_vignette
vignette: >
  %\VignetteIndexEntry{TFEA.ChIP: a tool kit for transcription factor enrichment}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---
# Introduction
The identification of the transcription factor (TF) responsible for the coregulation of an specific set of genes is a common problem in transcriptomics. In the most simple scenario, the comparison of the transcriptome of cells or organisms in two conditions leads to the identification of a set of differentially expressed (DE) genes and the underlying assumption is that one or a few TFs regulate the expression of those genes. 
Traditionally, the identification of the relevant TFs has relied on the use of position weight matrices (PWMs) to predict transcription factor binding sites (TFBSs) proximal to the DE genes (Wasserman and Sandelin, 2004). The comparison of predicted TFBS in DE versus control genes reveals factors that are significantly enriched in the DE gene set. These approaches have been useful to narrow down potential binding sites, but can suffer from high rates of false positives. In addition, this strategy is limited by design to sequence-specific transcription factors (TF) and thus unable to identify cofactors that bind indirectly to target genes. 
To overcome these limitations, TFEA.ChIP exploits the vast amount of publicly available ChIP-seq datasets to determine TFBS proximal to a given set of genes and computes enrichment analysis based on this experimentally-derived rich information. Specifically TFEA.ChIP, uses information derived from the hundreds of ChIP-seq experiments from the ENCODE Consortium ^[ENCODE Project Consortium (2012) Nature 489, 57-74)] expanded to include additional datasets contributed to GEO database^[Edgar, R et al. (2002) Nucleic Acids Res. 30:207-10] ^[Barrett, T et al. (2013) Nucleic Acids Res. 41(Database issue):D991-5] by individual laboratories representing the binding sites of factors not assayed by ENCODE. 
The package includes a set of tools to customize the ChIP data, perform enrichment analysis and visualize the results. The package implements two enrichment analysis methods:

- Analysis of the association of TFBS and differential expression from 2x2 tables recording the presence of binding sites for a given TF in DE and control genes. The statistical significance of the association for each factor determined by a Fisher's exact test.
- GSEA analysis, based on the core function of the GSEA algorithm developed by the GSEA team at the Broad Institute of MIT and Harvard^[Subramanian, Tamayo, et al. (2005) PNAS 102, 15545-15550] ^[Mootha, Lindgren, et al. (2003) Nat Genet 34, 267-273]

TFEA.ChIP includes a TF-gene interaction database containing 1060 datasets from ChIP-Seq experiments testing 277 different human transcription factors from the ReMap 2018 repository^[Jeanne Chèneby, Marius Gheorghe, Marie Artufel, Anthony Mathelier, Benoit Ballester; ReMap 2018: an updated atlas of regulatory regions from an integrative analysis of DNA-binding ChIP-seq experiments, Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D267–D275, https://doi.org/10.1093/nar/gkx1092]. Due to space limitations, TFEA.ChIPs internal database only includes ChIP-seq experiments from the ENCODE project. All the plots included in this vignette have been generated using the full ReMap 2018 ChIP-seq collection. To download the full ReMap 2018 database, as well as other ready-to-use databases, visit https://github.com/LauraPS1/TFEA.ChIP_downloads . 

Although the package is mainly focused towards analyzing expression data generated from human cells, TFEA.ChIP includes the option to use datasets coming from experiments in mice, translating mouse gene names to their equivalent ID on the human genome.

# Analysis Example

TFEA.ChIP is designed to take the output of a differential expression analysis and identify TFBS enriched in the list of DE genes. In the case of the analysis of association, the only required input is a set of DE genes and, optionally, a set of control genes whose expression is not altered by the experimental conditions under study. For the GSEA analysis a ranked list of genes is required. This is supplied as a dataframe containing a column with gene names and a numerical column with the ranking metric, which typically are log-fold change or p-values for the gene expression changes in the two conditions under evaluation. 
For illustration purposes we will derive the input required for both analysis from a table containing the following field columns:

- Gene name (*Genes*). Internally the package uses Entrez IDs, but translating from Gene Symbols and ENSEMBL IDs is available.
- Log2 Fold Change (*Log2FoldChange*), indicating the difference in expression for each gene in the two experimental conditions being compared.
- p-value (*pvalue*) or adjusted p-value (*pval.adj*) for the difference in gene expression between the two conditions.

The output of popular packages, such as DESeq2, for detection of differentially expressed genes from the analysis of count data from RNA-seq experiments produce tables with this information. The *hypoxia_DESeq* and *hypoxia* datasets are the output of a differential expression analysis performed on an RNAseq experiment analyzing the response to hypoxia of endothelial cells^[Tiana, M et al. The SIN3A histone deacetylase complex is required for a complete transcriptional response to hypoxia. https://doi.org/10.1101/182691 ] deposited at the NCBI's GEO repository (GSE89831).

To extract the information from a DESeqResults object or a data frame the function *preprocessInputData* is available. 

Using the option *from.Mouse = TRUE* will convert mouse gene IDs to their equivalent human gene ID, thus taking advantage of the wider abailability of ChIP-seq experiments done on human cells. This strategy relies on the overlap between human and mouse transcription regulatory mechanisms. Nevertheless, we advise to be cautious using this approach, since extrapolating results from one organism to another is not always appropiate.


```{r eval=TRUE,echo=TRUE,message=FALSE}
library(TFEA.ChIP)
data( "hypoxia_DESeq", "hypoxia", package="TFEA.ChIP" ) # load example datasets
hypoxia_table <- preprocessInputData( hypoxia_DESeq )
head( hypoxia_table )

head( hypoxia )
hypoxia_table <- preprocessInputData( hypoxia )
head( hypoxia_table )

```

After running *preprocessInputData*, your dataset will be ready to use with the rest of the package; gene names will be in Entrez Gene ID format and the resulting table is sorted by log2(Fold Change). 

## Analysis of the association of TFBS and differential expression.

1.  Identification of DE genes


As indicated before, for this analysis, we must provide a list of genes are considered differentially induced and a list of control genes whose expression is not altered in the analyzed experiment. For that we will use the function *Select_genes*:
```{r eval=TRUE,echo=TRUE}
#extract vector with names of upregulated genes
Genes.Upreg <- Select_genes( hypoxia_table, min_LFC = 1 )
#extract vector with names of non-responsive genes
Genes.Control <- Select_genes( hypoxia_table,
    min_pval = 0.5, max_pval = 1,
    min_LFC = -0.25, max_LFC = 0.25 )
```

2. Translate the gene IDs to Entrez Gene IDs

In case the input dataset cannot be preprocessed or the user is interested in analyzing a particular set of genes that doesn't come from the input dataset, translating the IDs to Entrez Gene ID format is required. To that end its available the function *GeneID2entrez*:
```{r eval=TRUE,echo=TRUE,message=FALSE}
#Conversion of hgnc to ENTREZ IDs
GeneID2entrez( gene.IDs = c("EGLN3","NFYA","ALS2","MYC","ARNT" ) )
# To translate from mouse IDs:
# GeneID2entrez( gene.IDs = c( "Hmmr", "Tlx3", "Cpeb4" ), mode = "m2h" ) # To get the equivalent human gene IDs
# GeneID2entrez( gene.IDs = c( "Hmmr", "Tlx3", "Cpeb4" ), mode = "m2m" ) # To get mouse ENTREZ gene IDs
```

3.  Association analysis

In this step, we will construct a contingency table for each of the factors stored in the internal database categorizing the DE (DE_yes) and control (DE_no) genes according to the presence or absence of binding sites:


|      |TFbound_yes|TFbound_no|
|------|:---------:|:--------:|
|DE_yes|number y/y |number y/n|
|DE_no |number n/y |number n/n|

Then, we will apply Fisher's exact test to each contingency table to test the null hypothesis that factor binding and differential expression are independent. In addition, to the raw p-values the function also return the FDR-adjusted values to correct for multiple testing.

```{r eval=TRUE,echo=TRUE}
CM_list_UP <- contingency_matrix( Genes.Upreg, Genes.Control ) #generates list of contingency tables, one per dataset
pval_mat_UP <- getCMstats( CM_list_UP ) #generates list of p-values and OR from association test
head( pval_mat_UP )
```

In this example, all 1122 datasets in the internal database were used in the analysis. However, we can restrict the analysis to a specific subset of the database and/or a given set of transcription factors. To this end we can produce and index of the tables of interest with the function *get_chip_index* and pass this index as an additional argument to *contingency_matrix*. 
Finally, note that the list of control genes is optional. If not supplied, all human genes not present in the test list will be used as control. Thus, we could restrict the analysis to the datasets generated by the ENCODE project and use all non-DE genes as control:


```{r eval=TRUE,echo=TRUE}
chip_index <- get_chip_index( TFfilter = c( "HIF1A","EPAS1","ARNT" ) ) #restrict the analysis to datasets assaying these factors
chip_index <- get_chip_index( encodeFilter = TRUE ) # Or select ENCODE datasets only
CM_list_UPe <- contingency_matrix( Genes.Upreg, Genes.Control, chip_index ) #generates list of contingency tables
pval_mat_UPe <- getCMstats( CM_list_UPe, chip_index ) #generates list of p-values and ORs
head( pval_mat_UPe )
```
To know more about the experiments included in TFEA.ChIP's database or the conditions of a particular experiment, load the metadata table included using *data("MetaData", package = "TFEA.ChIP")*.

To summarize the results by transcription factor use *rankTFs*. This function performs Wilcoxon rank-sum test or GSEA to test whether ChIPs belonging to the same TF are, as a group, significantly enriched / depleted in the results of the analysis. Be aware that in the case of transcription factors whose behavior is dependent on cellular context, integrating the results of all the related ChIPs might conceal its enrichment in a particular set of experimental conditions.

```{r eval=TRUE, echo=TRUE, fig.width=8, fig.height=4}
TF_ranking <- rankTFs( pval_mat_UP, rankMethod = "gsea", makePlot = TRUE )
TF_ranking[[ "TFranking_plot" ]]
head( TF_ranking[[ "TF_ranking" ]] )
```

4. Plot results

The table of results generated by *getCMstats* can be parsed to select candidate TF. The function *plot_CM* uses the package *plotly* to generate an interactive plot representing the p-value against the odd-ratio that is very helpful to explore the results.

```{r eval=FALSE,echo=TRUE}
plot_CM( pval_mat_UP ) #plot p-values against ORs
```
![Snapshot of a plot generated with plot_CM.](CMplot1.png)

In fact, the exploration of this graph shows a strong enrichment for several HIF datasets, as expected for the *hypoxia* dataset. This can be clearly shown by highlighting the datasets of interest:


```{r eval=FALSE,echo=TRUE}
HIFs <- c( "EPAS1","HIF1A","ARNT" )
names(HIFs) <- c( "EPAS1","HIF1A","ARNT" )
col <- c( "red","blue","green" )
plot_CM( pval_mat_UP, specialTF = HIFs, TF_colors = col ) #plot p-values against ORs highlighting indicated TFs
```
![Snapshot of a plot generated with plot_CM.](CMplot2.png)

## Gene Set Enrichment Analysis.
    
1. Generate a sorted list of ENTREZ IDs

The GSEA analysis implemented in the TFEA.ChIP package requires as input a sorted list of genes. By default, the function *preprocessInputData* will sort genes according to log fold change in descending order. However, they could be sorted by any numerical parameter including p-value. If you want to generate your custom gene list with other parameters, remember to make sure the gene IDs are in Entrez Gene ID format or translate them with *GeneID2Entrez*

2. Select the ChIP-Seq datasets to analyze

By default, the analysis will include all the ChIP-Seq experiments available in the database. However, this analysis might take several minutes to run. To restrict the analysis to a subset of the database we can generate an index variable and pass it to the function *GSEA_run*. This will limit the analysis to the ChIP-Seq datasets of the user's choosing. This index variable can be generated using the function *get_chip_index* and allows the user to select the whole database, the set of ChIP-Seq experiments produced by the ENCODE project ("encode") or a specific subset of transcription factors (as a vector containing the TF names).

```{r eval=TRUE,echo=TRUE}
chip_index <- get_chip_index( TFfilter = c( "HIF1A","EPAS1","ARNT" ) ) #restrict the analysis to datasets assaying these factors
```

3. Run the GSEA analysis

The function *GSEA_run* will perform a GSEA-based analysis on the input gene list. This function is based on the R-GSEA R script bundle written by the GSEA team at the Broad Institute of MIT and Harvard ^[Subramanian, Tamayo, et al. (2005) PNAS 102, 15545-15550] ^[Mootha, Lindgren, et al. (2003) Nat Genet 34, 267-273]. The output of the analysis depends on the variable *get.RES*: 

* When **False**, the function returns a data frame storing maximum Enrichment Score and associated p-value determined for each dataset included in the analysis.

* When **True**, the function returns a list of three elements. The first element (*Enrichment.table*) is the enrichment data frame previously mentioned. The second element (*RES*) is a list of vectors containing the *Running Enrichment Scores* values (see GSEA documentation, http://software.broadinstitute.org/gsea/doc/GSEAUserGuideTEXT.htm#_Interpreting_GSEA_Results) for each of the sorted genes tested against each one of the analyzed ChIP datasets. The third element (*indicators*) is a list of vectors indicating if the sorted genes were bound by the factor analyzed in each ChIP dataset. 

```{r eval=TRUE,echo=TRUE,results='hide'}
GSEA.result <- GSEA_run( hypoxia_table$Genes, hypoxia_table$log2FoldChange, chip_index, get.RES = TRUE) #run GSEA analysis
```
```{r eval=TRUE,echo=TRUE}
head(GSEA.result[["Enrichment.table"]])
head(GSEA.result[["RES"]][["GSM2390642"]])
head(GSEA.result[["indicators"]][["GSM2390642"]])
```

The list of results can be restricted to a given set of transcription factors by setting the variable *RES.filter*.

4. Plotting the results

TFEA.ChIP includes two functions that use the package *plotly* to generate interactive html plots of your GSEA results: *plot_ES* and *plot_RES*.

  i) Plot Enrichment Scores with *plot_ES*
  
  We can choose to highlight ChIP-Seq from specific transcription factors plotting them in a particular color.

```{r eval=FALSE,echo=TRUE}
TF.hightlight <- c( "EPAS1","ARNT","HIF1A" )
names( TF.hightlight ) <- c( "EPAS1","ARNT","HIF1A" )
col <- c( "red","blue","green" )
plot_ES( GSEA.result, LFC = hypoxia_table$log2FoldChange, specialTF = TF.hightlight, TF_colors = col)
```
![Snapshot of a plot generated with plot_ES.](GSEAplot1.png)


  ii) Plot Runing Enrichment Scores with *plot_RES*. 
  This function will plot **all** the RES stored in the *GSEA_run* output. It is only recommended to restrict output to specific TF and/or datasets by setting the parameters *TF* and/or *Accession* respectively:
    
```{r eval=FALSE, echo=TRUE}
plot_RES( 
    GSEA_result = GSEA.result, LFC = hypoxia_table$log2FoldChange,
    TF = c( "ARNT", "EPAS1" ), Accession = c(
        "GSE89836.ARNT.HUVEC-C",
        "GSE89836.EPAS1.HUVEC-C" ) )
```
![Snapshot of a plot generatd with plot_RES.](GSEAplot2.png)



# Building a TF-gene binding database

If the user wants to generate their own database of ChIPseq datasets, the functions *txt2gr* and *makeChIPGeneDB* automate most of the process. The required inputs are:

- A Metadata table (storing at least, Accession ID, name of the file, and TF tested in the ChIP-Seq experiment). The metadata table included with this package has the following fields: "Name", "Accession", "Cell", "Cell Type", "Treatment", "Antibody", and "TF".

- A folder containing ChIP-Seq peak data, either in ".narrowpeak" format or the MACS output files "_peaks.bed" -a format that stores "chr", "start", "end", "name", and "Q-value" of every peak-.


1. Filter peaks from source and store them as a GRanges object

Specify the folder where the ChIP-Seq files are stored, create an array with the names of the ChIP-Seq files, and choose a format.
Set a *for* loop to convert all your files to GenomicRanges objects using *txt2GR*. Please note that, by default, only peaks with an associated p-value of 0.05 (for narrow peaks files) or 1e-5 (for MACS files) will be kept. The user can modify the default values by setting the alpha argument to the desired threshold p-value.

```{r eval=FALSE,echo=TRUE}
folder <- "~/peak.files.folder"
File.list<-dir( folder )
format <- "macs"

gr.list <- lapply(
    seq_along( File.list ),
    function( File.list, myMetaData, format ){
        
        tmp<-read.table( File.list[i], ..., stringsAsFactors = FALSE )
        
        file.metadata <- myMetaData[ myMetaData$Name == File.list[i], ]
        
        ChIP.dataset.gr<-txt2GR(tmp, format, file.metadata)
        
        return(ChIP.dataset.gr)
    },
    File.list = File.list,
    myMetadata = myMetadata,
    format = format
)
```
```{r eval=TRUE,echo=TRUE}
# As an example of the output
data( "ARNT.peaks.bed","ARNT.metadata", package = "TFEA.ChIP" ) # Loading example datasets for this function
ARNT.gr <- txt2GR( ARNT.peaks.bed, "macs1.4", ARNT.metadata )
head( ARNT.gr, n=2 )
```

2. [Optional] Dnase Hypersensitive Sites

By default (see step 3), TFEA.ChIP filters the ChIPseq peaks in each dataset to select those overlapping or near to (by default max. distance 10 nucleotides) regions in a reference associated to a gene. This reference can have any number of sources, such as gene coordinates, transcription starting sites, or regulatory regions defined in projects such as GeneHancer. 

As an example, we will build a reference with Dnase hypersensitive sites associated to overlapping genes using Encode's Master DNaseI HS:

  i) Load Encode's Master DNaseI HS and convert it to a Genomic Ranges object.
    
```{r eval=FALSE,echo=TRUE}
dnaseClusters<-read.table(
    file="~/path.to.file.txt",
    header = TRUE, sep="\t", stringsAsFactors = FALSE )
dnaseClusters<-makeGRangesFromDataFrame(
    dnaseClusters, ignore.strand=TRUE,
    seqnames.field="chrom", start.field="chromStart",
    end.field="chromEnd" )
```

  ii) Select the Dnase hypersensitive sites that are 1Kb or closer to a gene and assign a gene ID to every Dnase HS that remains.

```{r eval=FALSE,echo=TRUE}
library( TxDb.Hsapiens.UCSC.hg19.knownGene, quietly = TRUE )
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
Genes <- genes( txdb )

near.gene <- findOverlaps( dnaseClusters, Genes, maxgap = 1000 )

dnase.sites.list <- queryHits( near.gene )
near.gene <- subjectHits( near.gene )

gene_ids <- Genes[ near.gene ]$gene_id
DHS.database <- dnaseClusters[ dnase.sites.list ]
mcols(DHS.database)$gene_id <- gene_ids

```

These steps can be modified to generate a new GRanges object containing custom sites.

3. Assign TFBS peaks from ChIP dataset to specific genes

The function *makeChIPGeneDB* assigns the TFBS peaks in the ChIP datasets stored in *gr.list* to a gene. To this end, a ChIP peak overlaping a regulatory region region receive the gene label associated to said region. By default the function also assigns the gene name when the ChIP peak does not overlap a regulatory region but maps at less than 10 nucleotides from it. This behaviour can be modified by setting the argument *distanceMargin* to the desired value (by default distanceMargin = 10 bases).

The resulting ChIP-Gene data base is a list containing two elements:
  - Gene Keys: vector of gene IDs.
  - ChIP Targets: list of vectors, one per element in gr.list, containing the putative targets assigned. Each target is coded as its position in the vector 'Gene Keys'.
```{r eval=TRUE,echo=TRUE}
data( "DnaseHS_db", "gr.list", package="TFEA.ChIP" ) # Loading example datasets for this function
TF.gene.binding.db <- makeChIPGeneDB( DnaseHS_db, gr.list ) 
str( TF.gene.binding.db )
```

The function, accepts any Genomic Range object that includes a metacolumn with a gene ID (stored in the @elementMetadata@listdata[["gene_id"]] slot of the object) for each genomic segment. For example, asignation of peaks to genes can be done by providing a list of all the genes in the genome:

```{r eval=TRUE,echo=TRUE}
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
data( "gr.list", package="TFEA.ChIP") # Loading example datasets for this function
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
Genes <- genes( txdb )
TF.gene.binding.db <- makeChIPGeneDB( Genes, gr.list, distanceMargin = 0 )
str( TF.gene.binding.db )
```

In this case the information about Dnase hypersensitivity is disregarded and peaks are asigned to overlapping genes (or genes closer than *distanceMargin* residues).

4. Sustitute the default database by a custom generated table.

At the beginning of a session, use the function *set_user_data* to use your TFBS binary matrix and metadata table with the rest of the package.

```{r eval=FALSE,echo=TRUE}
set_user_data( binary_matrix = myTFBSmatrix, metadata = myMetaData )
```
