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Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06 Podcast
Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization

Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06 vor 10 Jahren

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Eukaryotic genomes make use of nucleosomes to considerably reduce
their packaging volumes. As a consequence, the underlying DNA is
rendered inaccessible. Cells make use of ATP-dependent remodeling
factors to disrupt histone-DNA contacts and bring about access to
the DNA. ACF1 is the largest regulatory subunit of two nucleosome
remodeling factors, namely ACF and CHRAC. These complexes assemble,
slide or evenly space nucleosomes on DNA with an ability to sense
the linker lengths. However, roles of ACF1 in organizing
nucleosomes in vivo and their physiological consequences are
largely unclear. To understand the roles of ACF1 on chromatin
organization, I compared nucleosome occupancy and transcription
profiles in wild-type and ACF1-deficient Drosophila embryos. To
further investigate and corroborate these chromatin changes, I
performed genomewide mapping of ACF1 using chromatin
immunoprecipitation. Nucleosome occupancy was mapped by subjecting
DNA obtained from MNase-digested chromatin to deep sequencing and
the occupancies were analyzed using advanced analog signal
processing methods. We found discontinuous and discrete patches of
regularly positioned nucleosomes in wild-type tissue, referred to
as ‘regularity regions’. These regions span actively transcribing
and silent chromatin domains and show associated variation in the
linker lengths across them. A subset of these regions located at
sides remote from the transcriptional start sites loses regularity
upon ACF1 deletion and show presence of a novel DNA sequence motif.
Analyzing nucleosome periodicity by autocorrelation function
revealed that nucleosome linker length is longer in ACF1-deficient
embryos. Despite profound quantifiable changes in the chromatin
organization the RNA expression analyses did not show any major
changes. Genomewide localization of ACF1 was studied using by
chromatin immunoprecipitation. We observed a strong enrichment of
ACF1 along active promoter regions, coinciding strikingly well with
another remodeling factor, RSF-1. However, careful analyses using
mutant tissues for both proteins demonstrated that the observed
enrichments were in fact false positive. We define 3100 genomic
sites as false positive ‘Phantom Peaks’ that tend to enrich in the
ChIP-seq experiments. By comparing publicly accessible profiles and
the Phantom regions, we showed that several ChIP-seq profiles of
the epigenetic regulators show strong enrichment along the Phantom
Peaks. In conclusion, we identify regions of regularly organized
nucleosomes across the genome and show that a subset localized in
silent chromatin regions is affected by ACF1 deletion. Moreover, we
identified a class of false positive ChIP-seq peaks at active
promoters. This list of Phantom Peaks can be used to assess
potential false positive signal in a ChIP-seq profile, especially
when mutant tissue is not available as a control.