Oncology 3
HC 2
3.1
Cancer is a genomic disease, with alterations at the cellular level being caused by changes in
gene expression. Mutations, along these lines, can change gene expression directly – if in
regulatory regions – or indirectly, if causing an altered epigenetic regulation. Epigenetic
events, after all, also affect gene expression. The activation of oncogenes and the
inactivation of tumor suppressor genes has shown how mutations can lead to cancer. Gene
expression anyway results or manifests as changes in transcription (and thus later on
translation). According to the central dogma, DNA is transcribed into RNA, which is then
translated into protein. Especially the transcription part here is interesting, as this is what
can be modulated on various levels and what thus leads to changes in protein levels
eventually.
Transcription is the process when an RNA copy is made from a gene. For this, not only the
protein-coding region of the gene is important, also the promoter site. This is the site for
initiation of transcription. It exists of a response element, to which transcription factors
adhere and tell the gene to be transcribed or not. RNA polymerase, the enzyme that
transcribes, also needs to bind to a specific promoter region; the TATA-box. After binding of
the polymerase and transcription factors, transcription can start. Aside from transcription
factors, there are also other further regulatory elements, but which reside at distant sites in
the genome – yet can activate (enhance) or repress (silence) transcription. Regulatory
proteins bind these sites, which are either up- or downstream, along with some co-factors.
All together, they curl up the DNA and allow for this distant site to still interact with the
direct promoter region just upstream of the gene.
Transcription factors’ activities are diverse, but all to ensure regulation. There are in total
3000 but all together they regulator 20 000 genes – the activity is versatile due to the
restricted expression per cell type of each factor. The transcription factors determine the
cell nature. Moreover, at some places in the cell is the transcription factor localized, it is not
interspersed throughout the whole cell. The Transcription factors contain a set of domains,
each with a specific role:
- All TFs have a DNA-binding domain. Examples include the zinc finger, helix-loop-
helix, helix-turn-helix and the leucine zipper motifs. Each of these are characteristic
protein conformations that in the end enable the protein to bind to DNA. These
amino acid side chains can interact with the DNA grooves, or some recognize specific
DNA sequences.
- All TFs have a transcriptional activation domain, to bind e.g. polymerase. These
transactivation domains can thus bind to other parts of the transcriptional apparatus
to help induce transcription.
- Some TFs have a dimerization domain. These have to work in pairs, so the
dimerization domains work to be able to facilitate protein-protein interactions
between two proteins. If there are three factors, they can alternatively bind one
another to give rise to six different dimers. If of the same factor, it is a homodimer, if
not, a heterodimer.
o An example is the AP-1 transcription family, which functions in signaling for
cell growth, differentiation and cell death. Dysregulation may thus be a cause
, of cancer. It consists of a dimer form the proteins of the Jun and Fos family.
Many different dimers are possible to form, and AP-1 is activated in response
to signals like growth factors, ROS or radiation – which dimer forms affects
the biological response. Jun is pro-proliferative, while Jun-B is anti-
proliferative. AP-1 has been associated with tumorigenesis, as its response
element is TPA which is a tumor promoter.
- Some TFs have a ligand-binding domain. Only if a ligand binds, can the TF function. In
absence of a ligand, for example, the gene is silenced.
o An example here are steroid hormones, which are lipid soluble and hence can
pass the cell membrane easily. They then bind to their intracellular
transcription factor, whereby they move to the nucleus and connect with the
DNA response element. An example is vitamin A (retinoic acid) which acts
through the receptor RAR – if vit A is absent, transcription is suppressed. The
domains act independently, as has been shown in an experiment with
chimeric genes; the thyroid hormone was connected to the ligand binding
domain of retinoic acid receptor, and in the presence of retinoic acid, thyroid
was transcribed.
All of the above show how a TF can be regulated: synthesis in particular cell types, covalent
modification (phosphorylation), ligand binding, cell localization, dimerization. Transcription
factor binding has been examined by various experimental methods:
- Electrophilic mobility shift assay: protein incubation with a (radio)labeled DNA
fragment containing the promoter sequence of interest products of incubation
reaction analyzed on a gel if protein and DNA is bound, the band moves more
slowly through the gel so is retained more.
- DNase foot-printing: a DNA-cleaving enzyme probes a promoter region if bound
to a protein, this protects the region from DNA-cleavage the DNA is end-labeled
and then analyzed in gel electrophoresis.
- ChIP-seq: have DNA bound to protein fragment it immunoprecipitate it with
antibodies specific for the bound proteins sequence.
- Reporter assays: cloning of the promoter of interest in front of a reporter, like
luciferase, which can – if the promoter is activated – report that it is activated.
3.2
The chromosomes are built up of DNA, RNA and protein; all together packaged to fit in the
nucleus. The high-level packaging in eukaryotic cells is enabled due to chromatin packaging,
which resembles beads on a string. The beads here are nucleosomes, which consist of
histones and DNA wrapped around it. The nucleosome is an octamer of eight histone
proteins (H2A, H2B, H3 and H4 x2) with around 147 bp of DNA wrapped around it. Two
histones are connected by the H1 linker molecule, and DNA is strung between the two. The
beads on a string are packaged into a short fiber, which in turn loops and interacts further,
giving next-level packaging.
Each histone, furthermore, contains domains for histone-histone and histone-DNA
interactions. These are also termed histone tails that can be post-translationally modified:
methylation, phosphorylation or acetylation. This enables the chromatin to, aside from
being a structural scaffold, to help in DNA regulation. The extent of compactness and
relaxation of the DNA around a histone determines which proportion can be transcribed.
HC 2
3.1
Cancer is a genomic disease, with alterations at the cellular level being caused by changes in
gene expression. Mutations, along these lines, can change gene expression directly – if in
regulatory regions – or indirectly, if causing an altered epigenetic regulation. Epigenetic
events, after all, also affect gene expression. The activation of oncogenes and the
inactivation of tumor suppressor genes has shown how mutations can lead to cancer. Gene
expression anyway results or manifests as changes in transcription (and thus later on
translation). According to the central dogma, DNA is transcribed into RNA, which is then
translated into protein. Especially the transcription part here is interesting, as this is what
can be modulated on various levels and what thus leads to changes in protein levels
eventually.
Transcription is the process when an RNA copy is made from a gene. For this, not only the
protein-coding region of the gene is important, also the promoter site. This is the site for
initiation of transcription. It exists of a response element, to which transcription factors
adhere and tell the gene to be transcribed or not. RNA polymerase, the enzyme that
transcribes, also needs to bind to a specific promoter region; the TATA-box. After binding of
the polymerase and transcription factors, transcription can start. Aside from transcription
factors, there are also other further regulatory elements, but which reside at distant sites in
the genome – yet can activate (enhance) or repress (silence) transcription. Regulatory
proteins bind these sites, which are either up- or downstream, along with some co-factors.
All together, they curl up the DNA and allow for this distant site to still interact with the
direct promoter region just upstream of the gene.
Transcription factors’ activities are diverse, but all to ensure regulation. There are in total
3000 but all together they regulator 20 000 genes – the activity is versatile due to the
restricted expression per cell type of each factor. The transcription factors determine the
cell nature. Moreover, at some places in the cell is the transcription factor localized, it is not
interspersed throughout the whole cell. The Transcription factors contain a set of domains,
each with a specific role:
- All TFs have a DNA-binding domain. Examples include the zinc finger, helix-loop-
helix, helix-turn-helix and the leucine zipper motifs. Each of these are characteristic
protein conformations that in the end enable the protein to bind to DNA. These
amino acid side chains can interact with the DNA grooves, or some recognize specific
DNA sequences.
- All TFs have a transcriptional activation domain, to bind e.g. polymerase. These
transactivation domains can thus bind to other parts of the transcriptional apparatus
to help induce transcription.
- Some TFs have a dimerization domain. These have to work in pairs, so the
dimerization domains work to be able to facilitate protein-protein interactions
between two proteins. If there are three factors, they can alternatively bind one
another to give rise to six different dimers. If of the same factor, it is a homodimer, if
not, a heterodimer.
o An example is the AP-1 transcription family, which functions in signaling for
cell growth, differentiation and cell death. Dysregulation may thus be a cause
, of cancer. It consists of a dimer form the proteins of the Jun and Fos family.
Many different dimers are possible to form, and AP-1 is activated in response
to signals like growth factors, ROS or radiation – which dimer forms affects
the biological response. Jun is pro-proliferative, while Jun-B is anti-
proliferative. AP-1 has been associated with tumorigenesis, as its response
element is TPA which is a tumor promoter.
- Some TFs have a ligand-binding domain. Only if a ligand binds, can the TF function. In
absence of a ligand, for example, the gene is silenced.
o An example here are steroid hormones, which are lipid soluble and hence can
pass the cell membrane easily. They then bind to their intracellular
transcription factor, whereby they move to the nucleus and connect with the
DNA response element. An example is vitamin A (retinoic acid) which acts
through the receptor RAR – if vit A is absent, transcription is suppressed. The
domains act independently, as has been shown in an experiment with
chimeric genes; the thyroid hormone was connected to the ligand binding
domain of retinoic acid receptor, and in the presence of retinoic acid, thyroid
was transcribed.
All of the above show how a TF can be regulated: synthesis in particular cell types, covalent
modification (phosphorylation), ligand binding, cell localization, dimerization. Transcription
factor binding has been examined by various experimental methods:
- Electrophilic mobility shift assay: protein incubation with a (radio)labeled DNA
fragment containing the promoter sequence of interest products of incubation
reaction analyzed on a gel if protein and DNA is bound, the band moves more
slowly through the gel so is retained more.
- DNase foot-printing: a DNA-cleaving enzyme probes a promoter region if bound
to a protein, this protects the region from DNA-cleavage the DNA is end-labeled
and then analyzed in gel electrophoresis.
- ChIP-seq: have DNA bound to protein fragment it immunoprecipitate it with
antibodies specific for the bound proteins sequence.
- Reporter assays: cloning of the promoter of interest in front of a reporter, like
luciferase, which can – if the promoter is activated – report that it is activated.
3.2
The chromosomes are built up of DNA, RNA and protein; all together packaged to fit in the
nucleus. The high-level packaging in eukaryotic cells is enabled due to chromatin packaging,
which resembles beads on a string. The beads here are nucleosomes, which consist of
histones and DNA wrapped around it. The nucleosome is an octamer of eight histone
proteins (H2A, H2B, H3 and H4 x2) with around 147 bp of DNA wrapped around it. Two
histones are connected by the H1 linker molecule, and DNA is strung between the two. The
beads on a string are packaged into a short fiber, which in turn loops and interacts further,
giving next-level packaging.
Each histone, furthermore, contains domains for histone-histone and histone-DNA
interactions. These are also termed histone tails that can be post-translationally modified:
methylation, phosphorylation or acetylation. This enables the chromatin to, aside from
being a structural scaffold, to help in DNA regulation. The extent of compactness and
relaxation of the DNA around a histone determines which proportion can be transcribed.
