Questions Study Lectures 14/15 Transcription and chromatin structure
Question 1 For each of the following circle the T if the statement is true and the F if the
statement is false.
T
F
Changes in chromatin structure that result in changes in ge ne expression can be
associated with covalent modification of both histone proteins and the DNA
itself. Histone acetylation/methylation, DNA methylation
T
F
Southern blot analysis of specific genes after micrococcal nuclease treatment
showed that genes that are transcribed in a specific tissue are completely free of
nucleosomes. The opposite result was observed. Genes that are transcribed are
still packaged into nucleosomes (the nucleosomes are transiently removed as
the polymerase passes then reassembled...the array is dynamic rather than
static).
T
F
Methylation of DNA in promoter regions facilitates the recruitment of histone
deacetylases to these same regions. DNA binding proteins that recognize me-C,
such as MeCP2, can be responsible for this.
T
F
Nucleosome remodeling complexes are used to pack nucleosomes into less
accessible chromatin structures that make it more difficult to express genes
Remodeling complexes such as SWI/SNF use ATP energy to move
nucleosomes, which can reveal hidden binding sites for transcription factors.
T
F
Bromo domain- containing proteins recognize modifications, such as methylation
on specific histone residues, which are associated with chromatin silencing.
Chromo domain proteins would do this
T
F
Binding of a pioneer transcription factor to an enhancer before packaging of
DNA in that area into nucleosomes can result in specific positioning of
nucleosomes in the surrounding chromatin to leave a nucleosome free region.
Initial binding of a key transcription factor can restrict incorporation of that
region into nucleosomes and make it easier for other transcription factors to
bind to that region later to activate gene expression.
Question 2. You have fractionated nuclear extracts from sea urchins. A specific set of these
fractions can be combined into a "transcription mix" that is sufficient to promote active
transcription of genes in purified chromatin from which histone H1 has been removed.
Subsequent experiment s showed that adding back histone H1 to the chromatin prior to adding the
transcription mix led to a 25-fold lower level of transcription. In contrast, if the transcription mix
was added first, subsequent addition of histone H1 had little or no effect on t ranscription.
Circle "T" or "F" to indicate whether each of the statements below is true or false,
respectively. Use both the information above, and your knowledge from the course
lecture and readings to answer the questions.
T
F
Factors in the transcription mix appear to interfere with histone H1 interaction
with the transcribed genes. Inhibition by H1 is blocked by transcription
factors so interaction must be at least partially blocked
T
F
In the above experiments, gene transcription is inhibited by the
presence of nucleosomes.
Transcription occurs on chromatin so nucleosomes dont inhibit
Question 3. You have identified a new nuclear hormone receptor in mice that binds the novel
hormone XA. You name it the XA receptor (XAR) because it is the only receptor in mouse
cells that binds the XA hormone. You are studying the r ole of XA and XAR in regulating
expression of genes using chromatin immunoprecipitation (ChIP) and analysis of the ChIP
products using a mouse promoter sequence microarray. Samples of a mouse liver cell line were
grown in the absence ( on the figure below) or presence (+ on the figure below) of XA
hormone. Chromatin was extracted, sheared and precipitated with either no antibody (mockprecipitated), with -NCOR antibody specific for NCOR (a co-repressor protein known to
recruit histone deacetylases (HDAC)), or with -HAT1 antibody specific for HAT1 (a histone
acetyl transferase). The amount of precipitated fragments of the promoter regions of three
genes, A, B, and C were measured relative to mock-precipitated material on the mouse promoter
microarays. The data are presented for each of these three genes in the histograms below with the
height of the bars extending above the baselines indicating the relative hybridization intensity of
fragments in the promoter regions. On the basis of these data, and your knowledge of
transcription regulation, answer the questions below the figure.
a) Recruitment of histone acetyltransferase (HAT) to a promoter region would be expected to
INCREASE / DECREASE (circle one) expression of a gene.
Very briefly describe the mechanism by which it would produce this effect?
Acetylation of the histone tails in nucleosomes in the promoter would open the
chromatin structure facilitating recruitment of other positive regulators and of
components of the basal apparatus.
b) For each of the following, circle A, B, or C according to which gene in the table above the
statement is true for. If it is true for more than one gene, then circle all of the genes for which it
is true. If it is true for none of the genes, circle N
A
B
C
N
This gene is likely to be repressed in the tested liver cell line independent
of hormone application. (NCOR bound w/ or w/o XA being present)
A
B
C
N
This gene is likely to be activated in the tested liver cell line independent
of hormone application. (HAT1 bound with or w/o XA being present)
A
B
C
N
This gene is activated in the presence of XA hormone and is repressed in
the absence of XA hormone. (HAT activates, NCOR represses)
A
B
C
N
XAR is bound to the promoter region of this gene independent of the
presence of hormone. (XAR binds NCOR in absence and HAT in
presence of XA, but is likely bound to Gene C in both cases)
Question 4. You would like to identify potential regulatory DNA sequences where regulatory
transcription factors might bind to activate expression of an ion channel gene specifically in
neural tissue. You have decided to look for DNAse I hyp ersensitive sites in the chromosomal
DNA upstream of the start of transcription of the gene. Below is a diagram of the gene region
and the locations of EcoRI restriction sites in that area that you can use for your experiment.
Hatched areas show DNA reg ions that you are considering using as hybridization probes for your
experiment.
-3000
-1000
A
EcoRI
-10 +1
B
EcoRI
+1500
C
D
EcoRI
Channel Gene
EcoRI
For the following questions, assume that the transcription factors that regulate specific gene
activation of this gene in neural tissue bind to recognition sites in the sequence at
approximately -400 on the diagram above.
(a) Which hybridization probe(s) would allow you to detect that hypersensitive region (Write
down all that could be used).
Probe or B Probe C would work
(b) Write down the main steps that you would follow in the procedure for your experiment.
Starting with chromatin from neural tissue, you would first digest with low amounts of DNAse
I, then purify the chromosomal DNA (remove proteins), digest with the EcoRI restriction
enzyme, size fractionate on a gel, transfer fragments to filter (Southern blot), hybridize with
labeled probe DNA sequences, visualize where probes are bound
(c) On the Southern blot diagram to the right, draw in
the approximate sizes expected for the hybridization
results for the indicated lanes on the gel, using the
probe(s) you selected in part (a).
Lane 1 neural tissue chromatin,
digested with EcoRI
1
2
3
4
Marker
-- 2000 bp
Lane 2 neural tissue chromatin,
digested with DNAse I then EcoRI
Lane 3 liver tissue chromatin,
digested with EcoRI
--1500 bp
Lane 4 liver tissue chromatin,
digested with DNAseI then EcoRI
--1000 bp
-- 800 bp
B
C
-- 600 bp
-- 400 bp
-- 200 bp
*Question 5. (MBOC 7-55) How are histone acetylases and chromatin remodeling complexes
recruited to unmodified chromatin, and how are they thought to aid in the activation of
transcription from previously silent genes?
Histone acetylases and chromatin remodeling complexes are recruited to specific regions of
chromatin by gene activator proteins that can bind to DNA in unmodified chromatin. Histone
acetylases and chromatin (or nucleosome) remodeling complexes are often found as subunits
in transcriptional coactivator proteins that can interact with the activation domains of
enhancer bound transcription factors. Once bound, histone acetylases add acetyl groups to
histone tails, altering their packing properties and providing binding sites for some specific
proteins. Similarly, chromatin remodeling complexes, once recruited, alter the local chromatin
structure. This facilitates the binding of additional activator proteins that cannot bind to
unmodified chromatin. Together, these changes in histone acetylation and chromatin
structure allow the transcription machinery to be assembled at specific promoters and to
initiate transcription.
Question 6. Cytosine residues in the sequences 5CpG (C-phosphodiester bond-G) are often
methylated in animal genomes. CpG methylation is involved in long -term silencing (repression)
of certain genes during mammalian development. It is thought that the methyl-CpG-binding
proteins, MeCp1 and MeCp2, interact specifically with methylated DNA and mediate
transcriptional repression. MeCp2 contains a transcriptional repression domain (TRD) that can
function at a distance to silence relatively large chromatin domains. It ha s recently been shown
that the TRD of MeCp2 associates with a corepressor complex containing multiple proteins.
Furthermore, transcriptional repression in vivo is prevented by the histone deacetylase inhibitor,
trichostatin.
Based on the results summarized above, and your knowledge of transcriptional regulation,
propose a model for how the methylation of CpG can lead to gene silencing.
The observation that transcriptional repression is prevented by inhibitors of histone
deacetylases (HDAC) indicates that the deacetylation of histones is the basis for
transcriptional silencing. This makes sense in that histone acetyltransferases (HAT) is a
known component of several transcriptional coactivators. Accordingly, it is not surprising to
find a histone deacetylase as a component of a corepressor.
Therefore, the model is that HDAC is recruited to methyl CpG via its interaction with MeCp2
that in turn binds directly to methyl CpG. Once recruited to a region of DNA containing
methyl CpG, HDAC catalyzes the deacetylation of core histones resulting in a condensation of
the chromatin. The condensed chromatin is refractive to the transcriptional machinery and
hence, silenced.
Question 7.
In eukaryotic cells transcriptional regulation must occu r in the context of genomic DNA
packaged in multiple levels of chromatin structure. The following experiments were carried out
to investigate the relationship between chromatin structure and the mechanism of action of
transcriptional activators. The test system includes a reporter gene driven by a yeast promoter
containing a GAL4 site. GAL4 is a yeast transcriptional activator. Nucleosomes were
reconstituted in vitro on a plasmid containing the reporter gene. Reconstitution was carried out
in the presence of wild type GAL4 (++), a mutant GAL4 in which the activation domain was
removed (no Act-D) or a mutant GAL4 in which the DNA binding domain was nonfunctional
(no DNA-BD).
GAL4 and the general transcription factors (GTF) were either added prior to the addition of
histones (0 time) or at the completion of the reconstitution reaction (1 hr). The reconstituted
plasmid templates were then examined for general nucleosome formation on the plasmid,
transcriptional activity of the reporter gene and DNase I sensitivity of the GAL4 binding site.
The following results were obtained.
Time of GAL4 and
GTF addition
0 time
1hr
Structure of GAL4
++ no Act-D no DNABD
++ no Act-D no DNA
BD
DNase I protection
of GAL4 site
+
+
-
-
-
-
Transcriptional activity
+
-
-
-
-
-
Nucleosome formation
+
+
+
+
+
+
on the plasmid
________________________________________________________________
Propose a model to account for the activity of GAL4 that is consistent with these results. State
clearly how the results were interpreted to arrive at your model.
These results are consistent with the idea that the association of GAL4 with its
upstream element promotes the assembly of a preinitiation complex on the promoter
and that the assembly of this complex must take place before nucleosomes are formed.
The formation of a preinitiation complex prevents the packaging of the promoter into
nucleosomes and consequently insures that it is accessible for transcription. This is
supported by the following observations.
i. GAL4 footprints on the upstream activator only if added before nucleosome
reconstitution. Consequently, nucleosome formation must preclude the
association of GAL4.
ii. This interaction is specific in that (1) it is dependent on a functional DNA binding
domain and (2) an activation domain is necessary for transcriptional activity
iii. The overall formation of nucleosomes on the plasmid is not influenced by the
presence of GAL4. It is, therefore, not a general inhibitor of nucleosome
formation.
*Questions adapted from Molecular Biology of the Cell: The Problems book, J. Wilson and T. Hunt, 5 th ed, 2008,
Garland Science
Study Questions - Copyright UC Regents Davis campus, 2005-11. All Rights Reserved. May not be redistributed without prior written consent of course instructor. For personal use by students in course only.