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Summary Transcriptional regulation
- Instelling
- Universiteit Utrecht (UU)
Hoorcolleges van het regulome deel
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Bioinformatica & Genoomanalyse Evelien FloorTranscriptional regulation
Every cell holds a copy of all its DNA, its genome. The human body is made of approximately 10 13
cells. All originate from a single cell through repeated cell divisions. The most visible instructions in
our genome are genes. Genes explain exactly HOW to synthesize any protein. Proteins are the work
horses of every living cell. Different cells in our body hold copies of (essentially) the same genome.
Yet they express very different repertoires of proteins and non-coding RNAs.
The human genome encodes 20-25,000 genes (2% genome), and there are more than 1,000,000
genomic switches that control genes (>10%). A gene is a genomic substring that encodes HOW to
make a protein. A genomic switch is a genomic substring that encodes WHEN, WHERE & HOW MUCH
of a protein to make.
Transcriptional regulation
There are multiple ways to regulate the genome.
DNA methylation
In human the most common DNA
modification is methylation on the
cytosine. This results in repression of
gene activity like in heterochromatin.
Methylation does not change the
genetic code. DNA methylation and
histone modifications help to
compartmentalize the genome into
domains of different transcriptional
potentials.
Not every cytosine can be methylated,
4% of all cytosines are methylated. The only region that can be methylated is a cytosine followed by a
guanine (CpGs), 70-80% of all CpGs are methylated. CpGs are not distributed equally over the
genome, they are distributed in so called
CpG islands. Those islands are often
coupled with promoters.
DNA methylation is transferred to all
daughter cells. In case only one strand is
methylated, maintenance methylation
takes place and the other strand is
methylated too. In this way a muscle cell
daughter cell will become a muscle cell
instead of a blood cell.
Chromatin structure and function
Chromatin is the complex of DNA and proteins that comprise eukaryotic chromosomes. There are
two classes of chromatin proteins:
1. Histones
2. Non-histone proteins
Chromosome structure
Gene regulation proteins
A nucleosome is the basic unit of DNA packaging consisting of a
segment of DNA wound in sequence around eight histone protein cores.
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, Bioinformatica & Genoomanalyse Evelien Floor
146 base pairs of DNA are wrapped around an octamer of H2A, H2B, H3 and H4. Histone H1 is not
part of the core complex but stabilized the nucleosome.
Most important about histones are the histone tails. Those tails are
20-30 base pairs long and contain of residues that can be
methylated, ubiquitinated and acetylated by other proteins. Each
histone tail consists of different residues for regulation.
ChIP-seq
Chromatin immunoprecipitation allows mapping of protein-
DNA interactions in vivo. You will get information about a
protein and its localization on DNA, for instance transcription
factors or histone modifications. When you have a certain piece
of DNA the first thing that you have to do is covalently bind the
proteins to the DNA with formaldehyde. This will make sure
that the proteins won’t dissociate anymore. Then you
fragmentize the DNA into short pieces by sonication, some
pieces will now contain the protein of interest. Lastly, the
protein of interest is immunoprecipitated together with the
crosslinked DNA with a specific antibody. By heating the
fragments, decrosslinking of proteins and DNA will take place.
Afterwards it is possible to perform qPCR for a promoter or a
microarray essay. With qPCR only a couple of promoters can be
investigated while with microarray it can be genome wide.
Nowadays the method that is been commonly used is ChIP-seq.
After purification of the DNA fragments they are fragmentized for a second time. Then library
preparation will take place and PCR. Finally, the fragments are sequenced.
In the output
you can see two peaks, which are the places where the transcription factors bind. ChIP-seq is
genome wide and relatively cheap, it also gives a higher resolution.
ChIP-seq experimental design
Most experimental protocols involve a control sample that is
processed the same way as the test sample except that no
immunoprecipitation step or specific antibody is used. The
control sample shows how a failed experiment looks like and is
important to correct bias. It is also possible to do a no antibody
control, this is ChIP without a specific antibody. Another
possibility is no tag controle, this is ChIP in a cell not having a tag
on the analyzed protein.
The sequencing is possible in single-end and paired-end way.
Single-end sequencing is cheaper so more often used. It is
important that the experiments are done in duplo especially
when it is about identifying a new transcription factor.
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