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Review
. 2012 Sep 7;287(37):30914-21.
doi: 10.1074/jbc.R111.309229. Epub 2012 Sep 5.

Uncovering transcription factor modules using one- and three-dimensional analyses

Xun Lan  1 , Peggy J Farnham, Victor X Jin
Affiliations
Review

Uncovering transcription factor modules using one- and three-dimensional analyses

Xun Lan et al. J Biol Chem. .

Abstract

Transcriptional regulation is a critical mediator of many normal cellular processes, as well as disease progression. Transcription factors (TFs) often co-localize at cis-regulatory elements on the DNA, form protein complexes, and collaboratively regulate gene expression. Machine learning and Bayesian approaches have been used to identify TF modules in a one-dimensional context. However, recent studies using high throughput technologies have shown that TF interactions should also be considered in three-dimensional nuclear space. Here, we describe methods for identifying TF modules and discuss how moving from a one-dimensional to a three-dimensional paradigm, along with integrated experimental and computational approaches, can lead to a better understanding of TF association networks.

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Figures

FIGURE 1.
FIGURE 1.
Experimental techniques to investigate TFBSs and chromatin interactions. A, schematic representation of major steps in one-dimensional ChIP-based high throughput methods used to identify TFBSs. Briefly, cells are treated with formaldehyde to cross-link the TFs to genomic binding sites, the genomic DNA is sheared, and bound fragments are selected by immunoprecipitation using an antibody to a TF of interest. The cross-links are then reversed, and the fragments are purified and applied to microarrays (ChIP-chip) or sequenced (ChIP-seq). B, assays used to study three-dimensional chromatin structure. ChIA-PET is similar to ChIP in that fragments bound to a TF of interest are immunoprecipitated. However, unlike ChIP assays, fragments brought into close proximity by DNA looping are ligated prior to the immunoprecipitation step. Hi-C is similar to ChIA-PET in that fragments in close proximity are ligated. However, Hi-C does not rely on immunoprecipitation by an antibody to a TF but rather uses biotin labeling of the ligation sites, followed by avidin-based purification. The fragments are then subjected to paired-end sequencing. The 3C, 4C, and 5C assays also detect pairs of genomic loci that are in close proximity in the three-dimensional space of the nucleus. Formaldehyde is used to cross-link spatially close chromatin regions, the DNA is digested with a restriction enzyme, and fragments within the cross-linked complexes are joined by ligation. In 3C, the joined regions are analyzed using PCR. In 4C, a second enzyme restriction digestion step is performed to shorten the hybrid fragments, which are circularized and subjected to inverse PCR; the products of inverse PCR are hybridized to a custom microarray. In 5C, a LMA step allows the ligation junctions of all the hybrid fragments in the 3C library to be analyzed using microarrays or next-generation sequencing. Note that this figure shows greatly simplified versions of the different technologies; for detailed descriptions, please see the original papers.
FIGURE 2.
FIGURE 2.
Pathway to identify one- and three-dimensional TF modules. The steps in identifying TF modules include the following: step 1, perform epigenomic profiling (histone ChIP-seq) and identify open chromatin (DNase-seq) in the cell type of interest; step 2, identify interacting chromosomal loci in the same cell type using the Hi-C method; step 3, use the epigenomic data to cluster the interacting chromosomal loci into distinct sets; step 4, either predict TF binding using a data base of TF consensus motifs (4A) or identify bound TFs using experimental ChIP-seq data (4B); step 5, integrate the chromatin structure information with the TF binding information; step 6, create one-dimensional (1D) and three-dimensional (3D) TF modules and TF association networks using computational methods such as the Apriori algorithm, a Bayesian approach, or a neural network; step 7, experimentally validate TF associations via methods such as 3C, fluorescent in situ hybridization (FISH), co-immunoprecipitation (Co-IP), immunohistochemistry (IHC), and immunofluorescence (IF).
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