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A functional genomics approach to map transcriptional and post-transcriptional gene regulatory networksBhinge, Akshay Anant 15 October 2009 (has links)
It has been suggested that organismal complexity correlates with the complexity
of gene regulation. Transcriptional control of gene expression is mediated by binding of
regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify
the chromosomal targets of transcription factors (TFs) to delineate transcriptional
regulatory networks underlying gene expression programs. The development of ChIP-chip
technology has enabled high throughput mapping of TF binding sites across the
genome. However, there are many limitations to the technology including the availability
of whole genome arrays for complex organisms such human or mouse. To circumvent
these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment
(STAGE) methodology that is based on extracting short DNA sequences or “tags” from
ChIP-enriched DNA. With improvements in sequencing technologies, we applied the
recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput
sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found
that several microRNAs are potential E2F4 targets.
Binding of TFs to their respective chromosomal targets requires access of the TF
to its regulatory element, which is strongly influenced by nucleosomal remodeling. In
order to understand nucleosome remodeling in response to transcriptional perturbation,
we used ultra high throughput sequencing to map nucleosome positions in yeast that were
subjected to heat shock or were grown normally. We generated nucleosome remodeling
profiles across yeast promoters and found that specific remodeling patterns correlate with
specific TFs active during the transcriptional reprogramming.
Another important aspect of gene regulation operates at the post-transcriptional
level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress
translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be
regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc
and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts
enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon
pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how
the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. / text
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