---
title: Finding Candidate Binding Sites for Known Transcription Factors via Sequence Matching
output:
  BiocStyle::html_document
date: 6 Oct 2017
vignette: >
  %\VignetteIndexEntry{Finding Candidate Binding Sites for Known Transcription Factors via Sequence Matching}
  %\VignetteEngine{knitr::rmarkdown}
author:
- name: Bioconductor Maintainer
  affiliation: Roswell Park Cancer Institute, Elm and Carlton St, Buffalo, NY 14263
  email: maintainer@bioconductor.org
abstract: >
  The binding of transcription factor proteins (TFs) to DNA promoter
  regions upstream of gene transcription start sites (TSSs) is one of
  the most important mechanisms by which gene expression, and thus many
  cellular processes, are controlled.  Though in recent years many new
  kinds of data have become available for identifying transcription
  factor binding sites (TFBSs) -- ChIP-seq and DNase I hypersensitivity
  regions among them -- sequence matching continues to play an important
  role.

  In this workflow we demonstrate Bioconductor techniques for finding
  candidate TF binding sites in DNA sequence using the model organism
  Saccharomyces cerevisiae.  The methods demonstrated here apply equally
  well to other organisms.
---

# Version Info
```{r, echo=FALSE, results="hide", warning=FALSE}
suppressPackageStartupMessages({
library('generegulation')
})
```
<p>
**R version**: `r R.version.string`
<br />
**Bioconductor version**: `r BiocManager::version()`
<br />
**Package version**: `r packageVersion("generegulation")`
</p>

# Introduction

Eukaryotic gene regulation can be very complex.  Transcription factor
binding to promoter DNA sequences is a stochastic process, and
imperfect matches can be sufficient for binding.  Chromatin
remodeling, methylation, histone modification, chromosome interaction,
distal enhancers, and the cooperative binding of transcription
co-factors all play an important role.  We avoid most of this
complexity in this demonstration workflow in order to examine transcription
factor binding sites in a small set of seven broadly co-expressed
Saccharomyces cerevisiae genes of related function.  These genes
exhibit highly correlated mRNA expression across 200 experimental
conditions, and are annotated to Nitrogen Catabolite Repression (NCR),
the means by which yeast cells switch between using rich and poor
nitrogen sources.

We will see, however, that even this small collection of co-regulated
genes of similar function exhibits considerable regulatory complexity,
with (among other things) activators and repressors competing to bind
to the same DNA promoter sequence.  Our case study sheds some light on
this complexity, and demonstrates how several new Bioconductor
packages and methods allow us to

 * Search and retrieve DNA-binding motifs from the MotifDb package
 * Extract the DNA sequence of the promoter regions of genes of interest
 * Locate  motifs in the promoter sequence

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

# Installation and Use

To install the necessary packages and all of their
dependencies, evaluate the commands


```{r echo=FALSE}
## grImport is temporarily not available for Mac as a binary
if (Sys.info()['sysname'] == "Darwin" && 
    (!"grImport" %in% rownames(installed.packages())))
{
if (!"BiocManager" %in% rownames(installed.packages()))
     install.packages("BiocManager")
install("grImport", type="source")
}
```

```{r echo=FALSE}
## move along, nothing to see here

if (Sys.getenv("NODE_NAME") == "master")
{
    seqLogo <- function(x)
    {
        fs=10
        seqLogo::seqLogo(x, xfontsize=fs, yfontsize=fs)
    }
}


```




```{r eval=FALSE}
## try http:// if https:// URLs are not supported
library(BiocManager)
BiocManager::install(c("MotifDb",  "GenomicFeatures", 
           "TxDb.Scerevisiae.UCSC.sacCer3.sgdGene",
           "org.Sc.sgd.db", "BSgenome.Scerevisiae.UCSC.sacCer3",
           "motifStack", "seqLogo"))
```


Package installation is required only once per R installation.  When
working with an organism other than S.cerevisiae, substitute the three
species-specific packages as needed.

To use these packages in an R session, evaluate these commands:


```{r echo=FALSE}
suppressPackageStartupMessages(library(MotifDb))
suppressPackageStartupMessages(library(S4Vectors))
suppressPackageStartupMessages(library(seqLogo))
suppressPackageStartupMessages(library(motifStack))
suppressPackageStartupMessages(library(Biostrings))
suppressPackageStartupMessages(library(GenomicFeatures))
suppressPackageStartupMessages(library(org.Sc.sgd.db))
suppressPackageStartupMessages(library(BSgenome.Scerevisiae.UCSC.sacCer3))
suppressPackageStartupMessages(library(TxDb.Scerevisiae.UCSC.sacCer3.sgdGene))
```

```{r}
library(MotifDb)
library(S4Vectors)
library(seqLogo)
library(motifStack)
library(Biostrings)
library(GenomicFeatures)
library(org.Sc.sgd.db)
library(BSgenome.Scerevisiae.UCSC.sacCer3)
library(TxDb.Scerevisiae.UCSC.sacCer3.sgdGene)
```

These instructions are required once in each R session.

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

# Biological Background

The x-y plot below displays expression levels of seven genes across 200 conditions, from a
compendium of yeast expression data which accompanies 
<a href="http://www.biomedcentral.com/1471-2105/5/18">Allocco et al</a>, 2004,
"Quantifying the relationship between co-expression, co-regulation and gene
function":

<center>
<img src="compendium.png" width="100%" alt="compendium.png">
</center>

<br>

Allocco et al establish that 

<blockquote>
In S. cerevisiae, two genes have a 50% chance of having a common transcription
factor binder if the correlation between their expression profiles is equal to
0.84. 
</blockquote>

These seven highly-correlated (> 0.85) NCR genes form a connected subnetwork within the
complete co-expresson network derived from the compendium data (work not shown).
Network edges indicate correlated expression of the two connected genes across
all 200 conditions.  The edges are colored as a function of that correlation:
red for perfect correlation, white indicating correlation of 0.85, and 
intermediate colors for intermediate values.  DAL80 is rendered as an octagon to
indicate its special status as a transcription factor.  We presume,
following Allocco, that such correlation among genes, including one
transcription factor, is a plausible place to look for shared
transcription factor binding sites.

<br>
<a name="network"></a>
<center>
<img src="dal80-subnet.png" width="100%" alt="dal80-subnet.png">
</center>
<br>

Some insight into the co-regulation of these seven genes is obtained
from <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=19380492">Georis et al</a>, 2009,
"The Yeast GATA Factor Gat1 Occupies a Central Position in Nitrogen Catabolite
Repression-Sensitive Gene Activation":

<blockquote>
Saccharomyces cerevisiae cells are able to adapt their metabolism according to
the quality of the nitrogen sources available in the environment. Nitrogen
catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen
sources when rich ones are available. NCR-sensitive expression is modulated by
the synchronized action of <b>four DNA-binding GATA factors</b>. Although the first
identified GATA factor, <b>Gln3</b>, was considered the major activator of
NCR-sensitive gene expression, our work positions <b>Gat1</b> as a key factor for the
integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared
to be the limiting factor for NCR gene expression, (ii) GAT1 expression was
regulated by the four GATA factors in response to nitrogen availability, (iii)
the two negative GATA factors <b>Dal80</b> and <b>Gzf3</b> interfered with Gat1 binding to
DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also
provides mechanistic insights into the mode of action of the two negative GATA
factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition
at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression
occurred at three possible levels: Dal80 represses GAT1 expression, it competes
with Gat1 for binding, and it directly represses NCR gene
transcription. (<i>emphasis added</i>)
</blockquote>

Thus DAL80 is but one of four interacting transcription factors
which all bind the GATA motif.  We will see below that DAL80 lacks the
GATA sequence in its own promoter, but that the motif is well-represented in the
promoters of the other six. 

In order to demonstrate Bioconductor capabilities for finding binding
sites for known transcription factors via sequence matching, we will
use the shared DNA-binding GATA sequence as retrieved
from one of those factors from MotifDb, DAL80.


<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

# Sequence Search

Sequence-based transcription factor binding site search methods answer
two questions:

  * For a given TF, what DNA sequence pattern/s does it preferentially bind to?
  * Are these patterns present in the promoter region of some gene X?

A gene's <i>promoter region</i> is traditionally (if loosely) defined
with respect to its transcription start site (TSS): 1000-3000 base
pairs upstream, and 100-300 basepairs downstream. For the
purposes of this workflow, we will focus only on these cis-regulatory
regions, ignoring enhancer regions, which are also protein/DNA binding
sites, but typically at a much greater distance from the TSS.
An alternative and more inclusive "proximal regulatory region" may be appropriate
for metazoans: 5000 base pairs up- and down stream of the TSS. 

Promoter length statistics for yeast are available from  <a
href="http://www.ncbi.nlm.nih.gov/pubmed?term=19258451">Kristiansson et al</a>, 2009:
"Evolutionary Forces Act on Promoter Length: Identification of Enriched   Cis-Regulatory Elements"

<blockquote>
  Histogram of the 5,735 Saccharomyces cerevisiae promoters used in this study. The median promoter length is 455 bp and 
  the distribution is asymmetric with a right tail. Roughly, 5% of the promoters are longer than 2,000 bp and thus not 
  shown in this figure. 
</blockquote>

<center>
<img src="scerPromoterLength.png" width="100%" alt="scerPromoterLength.png">
</center>
<br>

* The "normal" location of a promoter is strictly and simply upstream of a
  gene transcript's TSS.

* Other regulatory structures are not uncommon, so a comprehensive search
  for TFBSs, especially in mammalian genomes, should include downstream
  sequence as well.

For simplicity's sake we will use a uniform upstream distance of 1000 bp, and 0
bp downstream in the analyses below.


<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>


# Minimal Example

Only eight lines of code (excluding  <code>library</code>
statements) are required to find two matches to the JASPAR DAL80 motif in the promoter of DAL1.

```{r}
library(MotifDb)
library(seqLogo)
library(motifStack)
library(Biostrings)
library(GenomicFeatures)
library(org.Sc.sgd.db)
library(BSgenome.Scerevisiae.UCSC.sacCer3)
library(TxDb.Scerevisiae.UCSC.sacCer3.sgdGene)

query(MotifDb, "DAL80")   
pfm.dal80.jaspar <- query(MotifDb,"DAL80")[[1]]
seqLogo(pfm.dal80.jaspar)
dal1 <- "YIR027C"
chromosomal.loc <- 
  transcriptsBy(TxDb.Scerevisiae.UCSC.sacCer3.sgdGene, by="gene") [dal1]
promoter.dal1 <- 
  getPromoterSeq(chromosomal.loc, Scerevisiae, upstream=1000, downstream=0)
pcm.dal80.jaspar <- round(100 * pfm.dal80.jaspar)
matchPWM(pcm.dal80.jaspar, unlist(promoter.dal1)[[1]], "90%")
```

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>


# Sample Workflow: an extended example

We begin by visualizing DAL80's TF binding motif using either of two Bioconductor
packages: seqLogo, and motifStack.  First, query MotifDb for the PFM (position
frequency matrix):

```{r}
query(MotifDb,"DAL80")
```

There are two motifs.  How do they compare?  The seqlogo package has been the
standard tool for viewing sequence logos, but can only portray one logo at a
time.  

```{r}
dal80.jaspar <- query(MotifDb,"DAL80")[[1]]
dal80.scertf <-query(MotifDb,"DAL80")[[2]]
seqLogo(dal80.jaspar)
seqLogo(dal80.scertf)
```
    
With a little preparation, the new (October 2012) package motifStack can 
plot both motifs together.  First, create instances of the <code>pfm</code> class:

```{r, dev="jpeg"}
pfm.dal80.jaspar <- new("pfm", mat=query(MotifDb, "dal80")[[1]], 
                        name="DAL80-JASPAR")
pfm.dal80.scertf <- new("pfm", mat=query(MotifDb, "dal80")[[2]], 
                        name="DAL80-ScerTF")
plotMotifLogoStack(DNAmotifAlignment(c(pfm.dal80.scertf, pfm.dal80.jaspar)))
```


Of these two, the JASPAR motif has more detail, but the ScerTF motif is more
recently published.  ScerTF has a reputation for careful yeast-specific
curation.  We will use the ScerTF version.  

Georis et al mention that DAL80 "competes with Gat1 for binding" -- suggesting
that they would have highly similar motifs.  MotifDb has 3 entries for GAT1:

```{r}
query(MotifDb, "gat1")
```

Plot the three together:

```{r, dev="jpeg"}
pfm.gat1.jaspar = new("pfm", mat=query(MotifDb, "gat1")[[1]], 
                       name="GAT1-JASPAR")
pfm.gat1.scertf = new("pfm", mat=query(MotifDb, "gat1")[[2]], 
                       name="GAT1-ScerTF")
pfm.gat1.uniprobe = new("pfm", mat=query(MotifDb, "gat1")[[3]], 
                       name="GAT1-UniPROBE")
plotMotifLogoStack(c(pfm.gat1.uniprobe, pfm.gat1.scertf, pfm.gat1.jaspar))
```


The GAT1-JASPAR motif is very similar to DAL80's GATAA motif, and thus consistent
with the Georis claim that GAT1 and DAL80 compete for binding. The GAT1-ScerTF and
GAT1-UniPROBE motifs are similar, but differ in length.  They are reverse
complements of the canonical <b>GATAA</b> motif.

To match motifs in a promoter, these steps are required:

  * Retrieve the binding motif (the position frequency matrix, or PFM) of a given
  transcription factor
  * Retrieve the promoter regions for a set of candidate targets
  * Identify the sequence matches of the binding motif in the the genes'
  promoter regions

The three search motifs, one for DAL80, and two for GAT1, must be transformed
before then can be matched to DNA sequence.
MotifDb returns a position frequency matrix (a <b>PFM</b>) with all columns summing to
1.0, but the Biostrings matchPWM method expects a position count matrix (a <b>PCM</b>) with integer values.
Transform the frequency matrix into a count matrix using the somewhat arbitrary
but functionally reliable scaling factor of 100:

```{r}
pfm.dal80.scertf <- query(MotifDb, "dal80")[[2]]
pcm.dal80.scertf <- round(100 * pfm.dal80.scertf)

pfm.gat1.jaspar <- query(MotifDb, "gat1")[[1]]
pcm.gat1.jaspar <- round(100 * pfm.gat1.jaspar)

pfm.gat1.scertf <- query(MotifDb, "gat1")[[2]]
pcm.gat1.scertf <- round(100 * pfm.gat1.scertf)
```

Create a list of the seven genes from the DAL80 co-regulated subnetwork
(displayed <a href=#network>above</a>).  Lookup their systematic names,
which will be needed immediately below.

```{r}
genes <- c("DAL1", "DAL2", "DAL4", "DAL5", "DAL7", "DAL80", "GAP1")
orfs <- as.character(mget(genes, org.Sc.sgdCOMMON2ORF))
```

Obtain the coordinates of the transcripts for the orfs. 

```{r}
    grl <- transcriptsBy(TxDb.Scerevisiae.UCSC.sacCer3.sgdGene, by="gene") [orfs]
```

These coordinates, returned in a GRangesList object, specify the start
location (chromosome and base pair) of every known transcript for each
gene.  With this information, GenomicFeatures::getPromoterSeq
calculates and returns the DNA sequence of the promoter:

```{r}
promoter.seqs <- getPromoterSeq(grl, Scerevisiae, upstream=1000,
                                downstream=0)
```

We will next search for a match of the motif to the first of these sequences,
the promoter for DAL1.  Note that here, and below, we use a 90% "min.score" when
we call matchPWM.  This high minimum match score seems reasonable
given the relative absence of variability in DAL80's PFM:

```{r}
pfm.dal80.scertf
```

The <b>GATAA</b> pattern is a very strong signal in this motif.

Note that some restructuring is needed for us to use the results of
<b>getPromoterSeqs</b> as an argument to <b>matchPWM</b>.  We call the
<b>getPromoterSeq</b> method with a GRangesList, which contains a
unique set of genomic ranges, representing transcript coordinates, for
each of several genes.  The corresponding result is a
<b>DNAStringSetList</b> in which there is one DNAStringSet
(essentially a list of DNAStrings) for each gene in the input list.
Both of these variables are therefore lists of lists, in which the
outer list is named with genes, and the inner lists are per-transcript
coordinates or DNA strings.

Since we need DNA strings without that overarching by-gene-name structure,
we call <b>unlist</b> to strip off that structure, leaving us with the desired DNAStringSet:

```{r}
print (class(promoter.seqs))
promoter.seqs <- unlist(promoter.seqs)
print (class(promoter.seqs))

matchPWM(pcm.dal80.scertf, promoter.seqs[[1]], "90%")
```

The close proximity of these two GATAA hits suggests that dimeric
DAL80, or some other GATAA-binding dimer,  may bind DAL1.

All of the matches in the promoters of all seven genes to one binding
motif may be found at once with this command:

```{r}
pwm.hits <- sapply(promoter.seqs, 
                      function(pseq) 
                         matchPWM(pcm.dal80.scertf, pseq, min.score="90%"))
```

And we can summarize the motif hits for each of the three motifs (dal80.scertf, gat1.jaspar,
gat1.scertf) by creating a data.frame of counts, by gene and motif.
First, determine the hits:

```{r}
dal80.scertf.hits <- sapply(promoter.seqs, function(pseq) 
                            matchPWM(pcm.dal80.scertf, pseq, min.score="90%"))
gat1.scertf.hits  <- sapply(promoter.seqs, function(pseq) 
                            matchPWM(pcm.gat1.scertf, pseq, min.score="90%"))
gat1.jaspar.hits  <- sapply(promoter.seqs, function(pseq) 
                            matchPWM(pcm.gat1.jaspar, pseq, min.score="90%"))
```

Now count their lengths:

```{r}
dal80.scertf <- sapply(dal80.scertf.hits, length)
gat1.jaspar  <- sapply(gat1.jaspar.hits,  length)
gat1.scertf  <- sapply(gat1.scertf.hits,  length)
```

Finally, create a data.frame from this information:

```{r}
tbl.gata     <- data.frame(gene=genes, dal80.scertf, gat1.jaspar, gat1.scertf)
```

The simple <b>dal80.scertf</b> 5-base motif has the most hits.  The
more complex 8-base <b>gat1.jaspar</b> mtoif has fewer hits:  perhaps
it is over-specified.  The 'other'(non-GATAA) motif of GAT1 obtained from ScerTF has fewer matches in
the promoters of these genes than do the GATA motifs.  The non-GATAA motif hits
may in fact, be not much different from chance, as could be revealed by sampling
the distribution of motif hits in the promoters of randomly selected genes.
Such analyses will be left as an exercise for the reader.  

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

# Biological Summary

This dataset and our exploration has revealed a number of GATAA
binding sites within these tighly co-regulated NCR genes, but leaves
unanswered questions, some of which are: 

  * GAT1 is reported to have two (or more) quite different binding motifs.  Is
    this due to its having two or more distinct binding domains?  Are they each
    functional, but only in different conditions?

  * The gene expression of the negative regulator DAL80 is highly correlated
    with the expression of genes it is known to repress.  We would
    expect the opposite relationship between a negative regulator and
    its targets. Why doesn't abundant DAL80 prevent the
    expression of the other six genes?

  * The DAL80/ScerTF motif and GAT1/JASPAR motif are very closely
    related.  The match table, just above, shows quite different
    totals for the two motifs.  Does the find structure of the motif
    explain the difference?

One speculative explanation for the counter-intuitive DAL80 expression
is "nuclear sequestration", a mechanism by which a gene is expressed
but the mRNA is held in reserve for later use.  See <a
href="http://www.ncbi.nlm.nih.gov/pubmed?term=22383896">Lavut A, Raveh D</a>
2012.

That GAT1 has multiple binding motifs (we show two, SGD 
<a href="http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000001873">four</a>
is yet another indication of the incompletely understood complexity of gene regulation.


The four GATAA-binding regulators, two positive and two negative, and their many
downstream targets, some of whose binding sequences we have studied here, can
thus be seen to be parts of complex regulatory circuits whose full elucidation
has not yet been worked out.  Judicious integration of many other kinds of data,
careful laboratory work, and the right computational tools, will eventually
clarify them.

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

# Exploring Package Content

The packages used here have extensive help pages, and include vignettes
highlighting common use cases. The help pages and vignettes are available from
within an R session. After loading a package, type, for instance:

    help(package="MotifDb")
    ?query
 
Though it is quite simple, with only a few methods, it will be worthwhile understand the
MotifDb package in detail.  To access the vignette:

```{r eval=FALSE}
browseVignettes(package="MotifDb")
```

Finally, you can open a web page containing comprehensive help resources for all installed packages:

```{r eval=FALSE}
help.start()
```


<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>

```{r}
sessionInfo()
```

<p class="back_to_top">[ <a href="#top">Back to top</a> ]</p>
