Comprehensive lecture notes for the Regulation of transcription initiation module covered in MCB3025F. These notes cover all content taught in lectures as well as additional materials (powerpoints, textbooks) required to succeed. These notes were created by a student who achieved a distinction in t...
MCB3025F
Section 1: Thomas
The regulation of RNA polymerase II transcription at protein-coding genes
Module 4C: Research paper
Introduction
- The research aims to provide insights into the regulation of the gene coding for the
protein CDCA8
- This gene is of interest because previous research has implicated the CDCA8 as a
putative oncogenic gene i.e. a gene that may be involved in the transformation of a
normal cell into a cancerous cell
- Expression is highly expressed in undifferentiated human embryonic stem cells and
expression is significantly reduced upon differentiation
- Differentiation is a process whereby cells develop into a more specialized cell type
- Different ways to induce differentiation of cultured human embryonic stem cells e.g.
induce the formation of embryoid bodies which are aggregates of pluripotent cells that
are induced to differentiate by a change in culture conditions (figure EB)
- Induce differentiation through signaling molecules (figure RA)
- Figure 1A shows the results of an immunoblot analysis (western blot analysis)
Immunoblotting
- Proteins in the sample are first subjected to SDS polyacrylamide gel electrophoresis, SDS PAGE where individual
polypeptides present int eh sample are separated by size
- The gel is then removed from the assembly and placed onto a nitrocellulose membrane which binds proteins
- Gel on the nitrocellulose membrane is between filtered paper soaked in buffer and between anode and cathode in a blotting
apparatus where polypeptides are transferred from the gel onto the nitrocellulose membrane by applying an electric field
(process is called blotting)
- After blotting, the pattern is not visible unless proteins are stained (can be done by a variety of methods)
- The membrane is incubated with an antibody specific for the
protein of interest to test if the protein is present
- If present, the antibody will bind to the respective polypeptide on
the membrane and unbound antibodies are washes away
- the bound antibody and polypeptide are visualized by incubation
with a secondary antibody that is coupled to an enzyme
(horseradish peroxidase)
- The secondary antibody is specific for antibodies from a specific
species (e.g. anti-rabbit secondar antibody for rabbit primary
antibody)
- Horseradish peroxidase, when provided with the correct substrate, produces light (chemiluminescence) which can be
detected using chemiluminescence imaging or x-ray film
- Pre-stained molecular mass marker proteins are usually run in a separate lane in the SDS PAGE to confirm that the
polypeptide recognized comes from the protein in question
- A secondary antibody is used and not the primary antibody conjugated to horseradish peroxidase because one would have
to produce HRP-conjugated versions of all the different primary antibodies and more than one secondary antibody can bind
to a primary antibody present on the membrane which amplifies the signal
Back to research paper Figure 1
Panel A
- They induced differentiation of cultured human ESC by embryoid formation and
by retinoic acid treatment
- CDCA8 is the protein of interest, Oct4 is a protein known to be specifically
expressed in undifferentiated cells and GAPDH is a key enzyme in glycolysis so
should be expressed in any cell type and serves as an internal control
- Disappearance of Oct4 could be because the authors failed to load comparable
amounts of total protein (not true cause in lanes where Oct4 is undetectable,
GAPDH has a strong signal) or that there is a selective decrease in Oct4 levels upon differentiation
- embryoid body formation resulted in differentiation after 8 days and retinoic acid formation resulted in differentiation after
4 days
- CDCA8 expression drops when cells transition from an undifferentiated state to a differentiated state
,- Note that weaker signals cannot be used to taken as a measure for relative protein amounts as different antibodies for
different proteins detect their targets with different efficiency
- GAPDH signal is likely saturating (once you reach a certain exposure within a band, further exposure no longer leads to an
increase in signal because the exposed area if already completely black)
- Possible solution is to load less material onto the SDS PAGE
Panel B
- Determined the presence of mRNA for a number of proteins by RT-PCR
- Is an endpoint analysis
- They prepared total RNA from cells at different time points and used RT-PCR to amplify
the cDNA for the different genes over a fixed number of PCR cycles
- PCR products loaded onto an agarose gel, separated by size and visualized by ethidium
bromide staining
- DNA bands generated using specific primer pairs for the different mRNAs of interest
- RT-PCR signals for RUNX1, SOX17 and cdx2 are undetectable at time 0 but become detectable after EB formation and
RA treatment
- Genes are known to be induced during cell differentiation and corroborate the conclusion that cells differentiate under
conditions of EB formation and RA treatment
- Note that PCR amplifies DNA targets exponentially, after a certain number of cycles, this is no longer the case because
one of the components in the PCR reaction (the PCR primers) get used up and this point is primarily dependent on the
number of target molecules (cDNA in this case)
Transcription start site determination using RACE analysis
- Figure 2 shows an image of the result of agarose electrophoresis of RACE PCR products in lane one, P, and of a DNA size
marker in lane two, labelled M, containing DNA fragments of known size
- RACE produced at least 3 PCR products
- They sequenced these products and found thar they corresponded to 3 different mRNAs originating from TSS located at
positions -175, -194 and -200 upstream of the CDCA8 translation start site
- The -194 transcript retained the sequence of an intron suggesting that the transcription of the CDCA8 gene results in
alternatively spliced transcripts
Part 2
- Researches investigated DNA sequencies up- and downstream of the mapped TSS for promoter activity by performing
dual luciferase transfection assays
Figure 3
Panel A
Method
- Shows a schematic depicting the plasmid constricts that were
generated in order to determine promoter proximal DNA regions that
contribute to regulated CDCA8 transcription using dual luciferase
reporter assays
- CDCA8 positions were mapped to -175, -194 and -200 upstream of
the CDCA8 translation start site
- Tested promoter activity extending downstream of the mapped TSS
(53 nucleotide positions upstream to -2,093 upstream)
- This region was cloned and inserted into a commercially available
reporter plasmid called pGL3 upstream of the coding region for firefly luciferase
,- To determine regions important for CDCA8 promoter activity (the ability of the CDCA8 promoter to stimulate RNA Pol II
transcription), authors constructed a panel of pGL3 plasmids containing truncated versions of the CDCA8 promoter regions
from -1645 to -53, from -1123 to -53 etc.
- Included a construct containing only a piece of DNA spanning from position -53 to +93 relative to the start site of the
coding region
- This region is downstream of the mapped TSS and therefore should not have promoter activity
Panel B
- Constructs were transfected into two different cell types, undifferentiated
human embryonic stem cells and differentiated human embryonic stem
cells and were analyzed by immunoblotting and RT-PCR
- Presented results in form of a bar diagram
- X axis shows names of plasmid constructs containing different promoter
regions upstream of the coding region for firefly luciferase that were
transfected together with Rinella luciferase internal control
- Y axis shows quantitative data, relative luciferase activity
- Not absolute measurements of firefly luciferase activity but relative
activity
- pGL3, the empty plasmid vector containing only the firefly luciferase coding regions, without any promoter upstream is
the true negative control for the dual luciferase reporter assay
- The luciferase activity of each transfection is presented as the percent observed in pGL3 control vector transfection
- pGL3 control is a plasmid containing the SV40 enhancer upstream of the firefly luciferase reporter which is a very strong
promoter and so pGL3 control plasmid is a positive control for a very strong promoter
- pGL3-2041, the plasmid with the longest CDCA8 promoter region, gives a firefly luciferase readout of about 40%
compared to the control plasmid in undifferentiated stem cells and clearly has detectable promoter activity
- Promoter activity is far less active in differentiated stem cells compared to the control plasmid (8-fold lower)
- In all cases, much lower activity in differentiated than undifferentiated stem cells
- Look at the successive deletions of CDCA8 promoter regions
- Removal of 448 bp between -1593 and -2041 does not affect promoter activity to a significant extent
- Further deletion from -1593 to -1017 has little effect on promoter activity
- The magnitude of biological effects is not equivalent to their physiological significance
- Deletion of region to position 952 yields an interesting result
- Deletion of 119 bp from pGL3-1071 to pGL3-952 results in a noticeable increase of promoter activity
- Suggests that a binding site for a transcription repressor protein that negatively regulates CDCA8 promoter activity was
removed
- Further deletions of DNA regions resulted in a stepwise reduction of promoter activity compared to pGL3-952, until
activity became essentially undetectable suggesting that the deleted regions contain binding sites fir transcription regulatory
proteins that contribute to overall CDCA8 promoter strengths
- Q: which region is the most important for CDCA8 promoter activity?
- A: Between positions -269 and -143 as has greatest effect on promoter activity
Figure 6
Panel A
- Continuation of the work towards the identification of potential transcription factor binding sites that have the greatest
effect on CDCA8 promoter activity
- Panel A shows the potential binding sites identified by the authors using
online tools
- Introduced nucleotide substitutions to disrupt the sequence-specific
interactions with a DNA binding protein and a DNA region of interest to
see effect on promoter activity
- Most radical change is GC or CG base pair to an AT or TA base pair
and vice versa
- E.g. mutant E introduced two G to T substitutions in a sequence motif
predicted to be recognized by the transcription factor Y (nuclear factor Y)
Panel B
, - Bar diagram shows the result of the experiments comparing the mutant constructs derived from p GL#-269 with the
wildtype pGL3-269 plasmid in dual luciferase reporter assays
- All substitutions (except B), affect promoter activity to some degree
- Strongest was mutants E and G which reduced promoter activity from
100% to 10% or less providing strong evidence that these DNA regions are
important in the stimulation of CDCA8 promoter activity
- E recognized by nuclear factor Y and G by cyclic amp-responsive element-
binding protein 1 (CREB1)
Figure 3
- Panels C, D, E and F address the same point
- Already seen that CDCA8 promoter regions had
much higher efficiency in undifferentiated human
embryonic stem cells compared to differentiated
- Performed dual luciferase assays with a panel of
pGL3 constructs containing different fragments of
the CDCA8 proximal promoter region with other
cell lines
Panel C
- A panel of cancer cell lines
- Including MCF7 (breast cancer cell line), A549
(lung cancer cells), K562 (a leukemia cell line) and
HeLa (a cervical cancer cell line)
Panel D
- Same experiments carried out in noncancerous
cultured cells, including HUVEC human umbilical vein endothelial cells, HSF primary human skin fibroblasts, AEC
alveolar epithelial cells and HEFs human embryonic fibroblasts
- By comparing C and D, the CDCA8 promoter has consistently higher activity in cancer cell lines compared to non-
transformed primary cell lines
- Transformation of cells into cancerous states always coincides with some degree of dedifferentiation (loss of
differentiation) so the data are consistent with higher expression of CDCA8 in undifferentiated cells compared to
differentiated cells
Panel E
- Shows a comparison of dual luciferase activity using pGL3-952 the promoter construct with the highest activity in panel B
- Compares activity of this construct in undifferentiated human embryonic stem cells, differentiated human embryonic
fibroblasts and induced pluripotent stem cells
- Induced pluripotent stem cells can be obtained by reprogramming differentiated cell types to return to an undifferentiated
state
Panel F
- Performed RT-PCR to compare CDCA8 mRNA transcript abundance in undifferentiated cells, differentiated cells and
cancer cells
- Analysis in which CDCA8 cDNA obtained by reverse transcription in the different RNA isolates of the different cell types
is amplified with a specific number of PCR cycles
- In parallel, performed RT-PCR analysis of the house keeping gene GAPDH as an internal control
- PCR amplified DNA run on agarose gel containing PCR fragments of interest
- Observed that GAPDH, RT-PCR produced a very uniform signal across all cell lines, suggesting that total RNA amounts
used for the analysis were comparable
- The RT-PCR signal is clearly much weaker in differentiated cells compared to undifferentiated or cancer cells
- Confirms observations made in transient transfection experiments and further suggests that the transient transfection
results recapitulate the behavior of the natural gene in its physiological chromatin context
- Q: Why can promoter DNA regions have different activity when transfected into different cell types?
- A: Different cell types differ because they express a different complement of proteins and they differ in their gene
expression pattern, these differences in gene expression are to a significant extent caused by the expression of cell type-
specific gene regulatory proteins. Promoter regions are very complex and contain many different binding sites for sequence-
specific transcription regulators however these may only be expressed in some cell types and not in others
- When performing promoter studies using transient transfection, must take into account the nature of cultured cells
Module 5A: Analysis of protein-protein interactions
Molecular shape and electrostatic potential of B-DNA
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