76
C. Rochette-Egly
DNase-seq (DNase I hypersensitive sites sequencing) (Chen et al. 2018, Zeng and
Mortazavi 2012) combines digestion with DNaseI (which non-specifically digests
DNA in open chromatin) with NGS of the digested DNA fragments. FAIRE-seq
(Formaldehyde-Assisted Isolation of Regulatory Elements) (Giresi et al. 2007) starts
with formaldehyde crosslinking and DNA sonication. Then nucleosome free DNA
is separated by phenol–chloroform extraction and sequenced. ATAC-seq (Assay
for Transposase-Accessible Chromatin using sequencing) (Buenrostro et al. 2013)
probes open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open regions of the genome. The tagged DNA fragments are then
purified, amplified and sequenced. Interestingly, the results of these assays have
shown good correspondence with ChIP-seq data for specific factors. As an example,
in ECCs, the co-occurrence of FAIRE and RXRα binding sites revealed the direct
RAR-RXR target genes activated by RA or a RAR specific agonist (Chatagnon et al.
2015; Mendoza-Parra et al. 2016). Note that ATAC-seq has an important advantage in
that it requires a relative small amount of cells (5 × 10
4 ) compared to other methods
(1–10 × 10
6 cells) (Dirks et al. 2016).
Two methods based on chromosome conformation capture (3C) methods have
been developed to map genome-wide regulatory elements located far away from their
target promoters or on other genes or chromosome (Fig. 3.4). Unfortunately, these
techniques have not been used yet to investigate developmental RA-target genes.
Hi-C (Belton et al. 2012) relies on chromatin fixation and digestion with restriction
enzymes (to create pairs of cross-linked DNA fragments originating from distinct
genomic locations). The fragments are ligated to tagged adapters containing a recognition site for a restriction enzyme. Then, the juxtaposed regions are joined together,
followed by restriction enzyme digestion and sequencing of the purified paired-end
tags. ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) is
an extension of Hi-C (Zeng and Mortazavi 2012) with the enrichment of a factor
of interest by ChIP. Unfortunately the technique is limited by the requirement for
large amounts of starting material (>100 Mio. cells) (Dirks et al. 2016) and by the
quality/specificity of antibodies.
Integrative Approaches
The approaches described above have identified and characterized hundreds of RAregulated genes (RNA-seq) and the dynamic occupancy of DNA sites by RXR/RAR
heterodimers (ChIP-seq and FAIRE-seq). Integrative analysis of these data have
largely facilitated the identification of RAR/RXR selective target genes that are
regulated by direct RA-RAR/RXR interactions during cell differentiation. However,
as direct target genes represent only a fraction of the RA-regulated genes, it has been
suggested that the other RA-regulated genes are indirectly or secondarily regulated
genes. In other words, RAR/RXR heterodimers would initiate gene programs that
lead to the expression of TFs that would then amplify secondary gene programs that
determine cell fate and cell differentiation (Fig. 3.3). Accordingly, among the early
C. Rochette-Egly
DNase-seq (DNase I hypersensitive sites sequencing) (Chen et al. 2018, Zeng and
Mortazavi 2012) combines digestion with DNaseI (which non-specifically digests
DNA in open chromatin) with NGS of the digested DNA fragments. FAIRE-seq
(Formaldehyde-Assisted Isolation of Regulatory Elements) (Giresi et al. 2007) starts
with formaldehyde crosslinking and DNA sonication. Then nucleosome free DNA
is separated by phenol–chloroform extraction and sequenced. ATAC-seq (Assay
for Transposase-Accessible Chromatin using sequencing) (Buenrostro et al. 2013)
probes open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open regions of the genome. The tagged DNA fragments are then
purified, amplified and sequenced. Interestingly, the results of these assays have
shown good correspondence with ChIP-seq data for specific factors. As an example,
in ECCs, the co-occurrence of FAIRE and RXRα binding sites revealed the direct
RAR-RXR target genes activated by RA or a RAR specific agonist (Chatagnon et al.
2015; Mendoza-Parra et al. 2016). Note that ATAC-seq has an important advantage in
that it requires a relative small amount of cells (5 × 10
4 ) compared to other methods
(1–10 × 10
6 cells) (Dirks et al. 2016).
Two methods based on chromosome conformation capture (3C) methods have
been developed to map genome-wide regulatory elements located far away from their
target promoters or on other genes or chromosome (Fig. 3.4). Unfortunately, these
techniques have not been used yet to investigate developmental RA-target genes.
Hi-C (Belton et al. 2012) relies on chromatin fixation and digestion with restriction
enzymes (to create pairs of cross-linked DNA fragments originating from distinct
genomic locations). The fragments are ligated to tagged adapters containing a recognition site for a restriction enzyme. Then, the juxtaposed regions are joined together,
followed by restriction enzyme digestion and sequencing of the purified paired-end
tags. ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) is
an extension of Hi-C (Zeng and Mortazavi 2012) with the enrichment of a factor
of interest by ChIP. Unfortunately the technique is limited by the requirement for
large amounts of starting material (>100 Mio. cells) (Dirks et al. 2016) and by the
quality/specificity of antibodies.
Integrative Approaches
The approaches described above have identified and characterized hundreds of RAregulated genes (RNA-seq) and the dynamic occupancy of DNA sites by RXR/RAR
heterodimers (ChIP-seq and FAIRE-seq). Integrative analysis of these data have
largely facilitated the identification of RAR/RXR selective target genes that are
regulated by direct RA-RAR/RXR interactions during cell differentiation. However,
as direct target genes represent only a fraction of the RA-regulated genes, it has been
suggested that the other RA-regulated genes are indirectly or secondarily regulated
genes. In other words, RAR/RXR heterodimers would initiate gene programs that
lead to the expression of TFs that would then amplify secondary gene programs that
determine cell fate and cell differentiation (Fig. 3.3). Accordingly, among the early