Download Regulation of Gene Expression Outline Objectives are first and

Regulation of Gene Expression Outline
Objectives are first and then the outline:
Obj. 1: Alterations in genes will either inhibit their expression which could be good or bad depending on
what you are suppressing or expressing. First you can get:



Gene Loss- when you lose the part of a gene or it is entirely deleted. This is usually bad if it is an
important or required gene. If you loose the nuclei from an erythroblast in maturation you are
no longer going to make mRNA and therefore no proteins.
Gene Amplificiation- a gene is increased or induced to make too many proteins.
Ex. Double minutes are chromosome piece w. many copies. Its seen in tumor cells- u
overexpress oncogene.
DNA arrangement-combine and rearrange genes to allow variability and different proteins.
Ex. Our good-old Heavy chain DNAIt has over 200variable genes, 20diverse genes, and 6 J-joing genes located in a cluster.
They combine to allow the expression of a particular antibody.
Various combinations in the V, H, and J gene give a different or specific IG production.
Obj. 2: Methylation and Acetylation are ways to modify gene expression at the DNA level.

Methylation- is a base modification to the DNA.
Methylation of DNA- you add a methyl group to a CpG island or CG rich region and reduce that
genes transcription.
Euk. DNA has a promoter region that either allows the gene to be turned on by binding RNA
poly. or off depending on it is making the protein.
-The promoter region is usually found to have high regions of CG or CpG islands. Therefore,
these regions can be highly methylated and prevent the binding of transcription factors and
prevent transcription.
EXGenerally this can be good for example in non-erythroid cells the Globin gene is highly
methylated near its promoter to inhibit its protein production.

Histone Acetylation:
Recall: DNA is tightly bound to histone proteins. Histones are positively charged and bind tightly
to DNA.
-We can modify histones so that our DNA can be more readily accessed by transcription factors
for expression. We do this by adding acetyl coenzyme A (acetyl CoA). The Acetyl CoA bind to the
Lysine’s positive charge on the HISTONE; therefore this results in a weaker interaction between
the DNA and histone since the histone is more strongly interacting with Acetyl group.
Deacetylation- the opposite! you remove the Acetyl group to make the DNA more tightly bound
and decrease gene expression of a particular protein.
Both Acetylation and Methylation affect the Histone tails around DNA. They can alter the
chromation state.
Example:
1 If you have methylation + deacetylation = no expression
2. Methylation + Acetylation = gene expression ( usually lower though)
3. No methylation + Acetylation = gene expression which is higher than number 2 .
Obj 3: Explain Alternative Splicing
Introns -spliced out of the primary RNA transcript before they can be translated into protein
Variant mRNAs-generated b y skipping some introns, or by using a seq as an intron in one cell type and
as an exon in another cell type
Alternative promoters aka polyadenylation sites-used to generate variants at the beginning and end of
the mRNA and protein
Variant proteins =isoforms
Variant proteins are believed to be involved in hearing sound across a broad range of frequencies; more
on this in outline
Obj 4: Explain regulation of gene expression at the transcriptional level
All of #2 in outline
Obj. 5: Describe translational and post-translational regulatory mechanisms
I felt like these weren’t clear in the Powerpoint, but I emailed Dr. Brenner and she said that :
methylation, histone acetylation
were what fit into this category. Cheers!
A. Molecular basis for (most) epigenetic mechanisms: methylation of cytosines in the DNA
a. The C must be followed by a G (CpG) for this to happen- the methylation sequence CpG
is also CpG on the opposite strand.
b. Methylation of C’s near the promoter region of a gene prevents transcription. This
means a heavily methylated gene is permanently inactivated.
c. Each cell type and tissue has its own methylation pattern, keeping some genes
functional and others permanently inactivated. This provides cells with "memory": after
cell division, the daughter cells know what their type is.
d. How is the methylated state preserved? All of the cytosine methylations are renewed
with every DNA replication: an enzyme called hemimethylase recognizes a 5-methyl C
on the old strand, then methylates the corresponding C in the new strand.
e. The methylation pattern is (mostly) reset in the early embryo, allowing embryonic cells
to develop into any cell type.
f. Summary: Base modification- methylation of DNA at CpG islands= lack of transcription
-CG or CpG islands are generally found in the promoter regions of genes.
Methylcytosine binding proteins may prevent binding of Tfs
B. Histone Acetylation DNA struct in nucleosomes can be loosened (for TFs, etc) by this
 Process: acetyl groups are added to lysines, removing their positive charge
binding of DNA to histones is weakened DNA struct opens up, allow TF access
 Deacetylation tightens the chromatin struct, preventing transcription
throughout that region of the chromosome
Histone tails can be covalenently modified in ways that activate their interaction
with a variety of proteins
 Proteins affect state of chromatinenhancing or preveneting
gene expression
1. methylation + deacetylation = no expression
2. Methylation + Acetylation = gene expression ( usually lower
though)
3. No methylation + Acetylation = gene expression which is
higher than number 2
I.
Our Genomethe number of genes we have is irrelevant due to the fact that each gene can get spliced
which leads the expression of several proteins.
Then the various proteins can get altered thru phosphorylation, methylation, acetylation
and glycosylation.
The modified proteins then partake in various protein complexes.
a. Human roughly 30,000 genes; Ecoli 3,200 genes
II. Gene Regulation
A. Importance: Different tissues have identical DNA, but each tissue expresses different
genes in response to stimuli.
B. Three main Levels to Regulate Gene Expression
1. @ the DNA level:
a. loss of genes, gene amplification, and gene rearrangements, methylation of
bases, and acetylation of histones that may inhibit or permit the expression of
certain genes.
2. @ Transcription level:
a. initiation of transcription with specific transcription factors, alternative splicing
and polyadenylation sites, RNA editing & mRNA stability will effect gene
expression.
b. Initiation of transcription is the principle point at which gene expression is
regulated (TFs bind to the promoter and either repress or activate
transcription initiation)
3. @ Translational level and post-translational:
a. The initiation of translation
b. Protein degredation
1. Regulation @ the DNA level:
a) Gene loss- total or partial deletion of genes, resulting in the loss of capability to prod.
mRNA
b) Gene amplification- specific genes can be amplified to generate multiple copies of
the gene and over-production of the protein product
Ex: Double minutes- chromosomal fragments containing multiple copies of a
gene often seen in tumor cells, associated with overexpression of oncogene products can be integrated into another chrom
c) DNA rearrangements- to produce different proteins
Ex: rearrangement of genes for the variable region of immune-globulun heavy
chain –in antibody production by lymphocytes
d) Chromatin condensation
euchromatin
-active transcription occurs in diffuse chromatin called
-no transcription occurs in dense chromatin called
condensed aka heterochromatin
i) chromatin structure
-DNA organized into nucleosomes: about 200 b.p. wrapped around a protein
core
-pr core= 8 histones
*Histones are basic, contain positively charged amino acids, which are
attracted to and bind with the negative charges of DNA (from the backbone phosphate
groups)
-DNA tightly wrapped around histones is inaccessible to RNA polymerase one
important event in preparing a gene for transcription is “chromatin remodeling”
* chromatin remodeling-sliding the nucleosomes along the DNA to
expose the promoter region
*allows processes that require access to DNA to get that access
Ex: gene expression, DNA replication, problem because
nucleosome packaging does not allow molecules to bind to DNA
e) Base modification- methylation of DNA at CpG islands= lack of transcription
-CG or CpG islands are generally found in the promoter regions of genes.
Methylcytosine binding proteins may prevent binding of Tfs
f) Histone Acetylation


DNA struct in nucleosomes can be loosened (for TFs, etc) by this
Process: acetyl groups are added to lysines, removing their positive charge
binding of DNA to histones is weakened DNA struct opens up, allow TF access
Deacetylation tightens the chromatin struct, preventing transcription
throughout that region of the chromosome
Histone tails can be covalenently modified in ways that activate their interaction
with a variety of proteins
 Proteins affect state of chromatinenhancing or preveneting
gene expression
1. methylation + deacetylation = no expression
2. Methylation + Acetylation = gene expression ( usually lower
though)
3. No methylation + Acetylation = gene expression which is
higher than number 2 .
2. Regulation @ Transcription level:
a) Inducible Gene Expression
i) inducers-bind to receptors, which bind to specific response elements
thyroid hormones, steroid homrones)
(exs:
-Trans-acting factors- proteins encoded by a gene residing elsewhere in the
genome
-Cis elements- DNA sequences in the regulatory regions of genes
-the rate at which a gene is transcribed can be increased by the binding of
specific transcription factors (inducer-receptor) to their cognate DNA response elements (cis-acting)
ii) Assembly of basal transcription complex- and initiation of transcription
-TBP (TATA-binding protein) is a subunit of TFIID
-General transcription factors- required for the transcription of all
genes and are part of the basal transcription complex
-Specific transcription factors (aka transactivators)- gene specific
(ex: thyroid hormones, sterioid hormones-like inducer’s exs-so
my guess is that they are the same thing, and enhancer is aka)
Promote the assembly of the basal transcription complex either
alone or with coactivators and thereby increase the rate of
transcription
Ex’s of transactivation via hormones:
Transcriptional activation by glucocorticoid and thyroid hormones
-these receptors are intracellular
-Transcriptional activities at their respective genes are strongly
influenced (if not dependent) on the presence of the specific hormone
signal
DNA binding domain and transactivating domain are domains in
specific TFs
*transactivating domain interacts with transcriptional complex
to stimulate transcription (after DNA binding domain has bound
to DNA)
Without transact domain, even with present of DNA binding
protein, transcriptional activation does not occur.
Chimeric gene can be formulated to replace missing transact
domain
”These motifs are common protein forms found within the
DNA-binding domains of regulatory proteins. You won’t need
to identify them by sight, but you should recognize that a
DNA binding domain is being referred to if you encounter these motifs.”
b) transcriptional repressors-3 modes of action
normal: Activation- activator is able to bind to the positive regulatory
element and RNA pol. Normal transcription occurs.
1. Repression by competition- repressor binds to the regulatory element
(on DNA), so activator can’t bind Transcription inhibited
2. Repression by Quenching- repressor binds to the activation domain
of the activator Transcription inhibited (b/c the activator can’t interact with the transcriptional
complex)
3. Active Repression- repressor binds to the negative regulatory
elementno transcription
c) multiple regulatory elements
-binding of any single gene may be regulated by several regulatory
signals (both activation and inactivation)
- bind of more than one regulatory protein may result in synergism
d) regulation by external stimuli
-polypeptide hormones and growth factors can regulate gene
expression without entering cells
-binding their cell surface receptors activates a series of second
messengers inside the cell, ultimately affecting gene expression
-In addition to the regulation by external stimuli that use internal
receptors, gene expression can be affected via ligand-bound cell surface receptors such as this enzymelinked pathway
e) ALTERNATIVE RNA TRANSCRIPTION AND SPLICING
i) introns spliced out of the primary RNA transcript before they can be
translated into protein
ii) variant mRNAs- generated by skipping some
itnrons, or by using a sequence as an intron in
one cell type and as an exon in another cell
type
-alternative promoters(polyadenylation sites)- used to generate variants at the beginning and end of the
mRNA and proten
-variant proteins=isoforms
iii) Alternative splicing and hearing
-splicing of SLO pre-mRNA to produce calcium regulated
potassium channel
8 sites of alt. splicing; more than 500 possible
cominations; protein variants believed to be involved in hearing
sound across a broad range of frequencies
-hair cells of cochlea
III. Epigenetics
C. Definition: study of heritable changes in gene expression that operate outside of changes in DNA
itself
D. Description:
a. This can between parent and offspring, or between cells within a single organism.
i. Within an organism, epigenetic changes are the main reason why it isn’t easy to
take the nucleus from any random cell and use it to grow a whole new organism
(i.e. reproductive cloning)
E. Molecular basis for (most) epigenetic mechanisms: methylation of cytosines in the DNA
a. The C must be followed by a G (CpG) for this to happen- the methylation sequence CpG
is also CpG on the opposite strand.
b. Methylation of C’s near the promoter region of a gene prevents transcription. This
means a heavily methylated gene is permanently inactivated.
c. Each cell type and tissue has its own methylation pattern, keeping some genes
functional and others permanently inactivated. This provides cells with "memory": after
cell division, the daughter cells know what their type is.
d. How is the methylated state preserved? All of the cytosine methylations are renewed
with every DNA replication: an enzyme called hemimethylase recognizes a 5-methyl C
on the old strand, then methylates the corresponding C in the new strand.
e. The methylation pattern is (mostly) reset in the early embryo, allowing embryonic cells
to develop into any cell type.
F. An example of epigenetic effects: a small deletion of part of the long arm of chromosome 15
results in Prader-Willi syndrome if the single copy of chr 15 is inherited from the mother, and
Angelman syndrome if the chromosome is inherited from the father.
a. This chromosomal region has different methylation patterns depending on whether it
come from the sperm or the egg.
b. Prader-Willi is characterized by an uncontrollable appetite for food (among other
things).
c. Angelman has been called the Happy Puppet syndrome: jerky movements and happy,
laughing demeanor.