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Raja Jothi, Suresh Cuddapah, Artem Barski, Kairong Cui,
Keji Zhao
Laboratory
of Molecular Immunology, National Heart Lung and Blood Institute, National Institutes
of Health,
Bethesda 20894, USA
Abstract
ChIP-Seq, which combines chromatin
immunoprecipitation (ChIP) with ultrahigh-throughput massively parallel sequencing,
is increasingly being used for mapping protein-DNA interactions in-vivo on a genome-scale.
Typically, short sequence reads from ChIP-Seq are mapped to a reference genome for
further analysis. Although genomic regions enriched with mapped reads could be inferred
as approximate binding regions, short read lengths (~25-50nt) pose challenges for
determining the exact binding sites within these regions. Here, we present SISSRs
(Site Identification from Short Sequence Reads),
a novel algorithm for precise identification of binding sites from short reads generated
from ChIP-Seq experiments. The sensitivity and specificity of SISSRs are demonstrated
by applying it on ChIP-Seq data for three widely studied and well-characterized
human transcription factors: CTCF, NRSF, and STAT1. We identified 26184, 5813, and
73956 binding sites for CTCF, NRSF, and STAT1 proteins respectively, which is 32%,
299%, and 78% more than that inferred previously for the respective proteins. Motif
analysis revealed that an overwhelming majority of the identified binding sites
contained the previously established consensus binding sequence for the respective
proteins, thus attesting for SISSRs’ accuracy. SISSRs’ sensitivity and precision
facilitated further analyses of ChIP-Seq data revealing interesting insights, which
we believe will serve as a guidance for designing ChIP-Seq experiments to map in
vivo protein-DNA interactions. We also show that tag densities at the binding sites
are a good indicator of protein-DNA binding affinity, which could be used to distinguish
and characterize strong and weak binding sites. Using tag density as an indicator
of DNA binding affinity, we have identified core residues within the NRSF and CTCF
binding sites that are critical for a stronger DNA binding.
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