2. Specificity of gene expression:
“different folks, different
A cell typically
strokes”
expresses
only a fraction
of its genes,
and the
different
types of cells
in multicellular
organisms
arise because
different sets
of genes are
expressed.

3. Eukaryotic Gene Regulation
Levels of eukaryotic gene
regulation:
 Genome (amplification or
rearrangement of DNA
segments, chromatin remodeling:
decondensation/ condensation
and DNA methylation).
 Transcription.
 Processing (and nuclear export)
of RNA.
 Translation (and targeting) of
protein.
 Posttranslational events (folding
and assembly, cleavage, chemical
group modifications and
organelle import/secretion).
 Degradation of mRNA and
proteins http://highered.mcgraw-
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4. GENOMIC CONTROL
 Yeast mating-type
switching depends upon
the swapping of genetic
cassettes to alter the
DNA sequence.
 Saccharomyces
cerevisiae has mating-
types (sexes): α or a.
 Chromosome III contains
three separate copies of
the mating-type
information.
 The HML α and HMR a
loci contain complete
copies of the α and a  The cell's actual mating type is determined
forms but the by the allele present at the MAT locus.
transcription of these  When a cell switches mating types, the α or
loci is inhibited by the a DNA at the MAT locus is removed and
products of gene the SIR replaced by a DNA "cassette" copy of the
gene. alternative mating type DNA.

5. Active genes are
specifically
attached to the
nuclear matrix by
AT-rich DNA
sequences called
matrix-associated
regions (MARs),
which are
recognized by an
enzyme-
topoisomerase
that unwind the
DNA helix

6. Different
chromatin
remodeling
complexes disrupt
and reform
nucleosomes. The
same complex
might catalyze
both reactions.
The DNA-binding
proteins could be
involved in gene
expression, DNA
replication, or
DNA repair.

7. Normal chromatin
blocks the
association of
transcription
factors and
polymerase II with
the DNA. Chromatin
can enter a "silent"
state or can be
converted to a
"poised" state
through histone
acetylation.

8. Transcriptional activation by
histone
acetylation. p300/CBP can
interact with a variety of
transcriptional regulators
such as pCAF (a histone
acetyltransferase) and TBP
(which recognizes the
promoter). During
transcription, they are all
assembled at the promoter
region. Histone acetylation
by p300/CBP, pCAF and
TAFII250 facilitates the
transcription.
Transcriptional repression
by histone
deacetylation. The
repressor Mad/Max dimer
interacts with SIN3 (or N-
CoR, SMRT, etc.) which
recruits histone deacetylase
(HDs) to repress
transcription.

9.  CpG islands (CG clusters) occur in 40,000 regions
near 5’ ends of genes.
 They are typically
unmethylated; are thought
to indicate the positions of
‘housekeeping’ genes.
 DNA methylase methylate CG regions on DNA.
creating a “5th base" in DNA, 5-methylcytosine.
 This base is made enzymatically after DNA is
replicated, at which time
about 5% of the cytosines
in mammalian DNA are
converted to 5-methyl
cytosine.

10.  Both methylation and acetylation provide means
for maintaining the state of activity of
developmental genes even after the original
signals for activation or repression have
disappeared
 Examples: globin promoters in RBC are
unmethylated, globin promoters are highly
methylated in other cells.

11. How is methylation involved in
repressing genes?
 Methylated DNA is preferentially
bound by histone H1, the
histone that associates
nucleosomes into higher-order
folded complexes.
 Histone deacetylases aggregate
in methylated regions of
DNA, causing them to bind
tightly to DNA, and form a stable
nucleosome.
 Acetylation of histones reduces
their affinity to DNA and cause
the nucleosomes to disperse.
 This opens up chromatin
structure for binding with the
transcription complex.

12. How DNA methylation patterns are faithfully inherited.
During replication, the DNA template strand retains the
methylation pattern, while the newly synthesized strand
does not. However, the enzyme DNA cytosine-5-
methyltransferase has a strong preference for a
methylated strand, and when it sees a methyl-CpG on one
side of the DNA, it methylates the new C on the other side.

13.  Differential methylation in germ cells is
implicated in genomic imprinting, the marking
of a gene as
either maternal or
paternal.
 Paternal 15q- causes
Prader-Willi syndrome
(mild mental retardation,
obesity, small gonads,
short stature)
 Maternal form causes
Angelman syndrome (severe mental
retardation, seizures, lack of speech,
inappropriate laughter).

14. In human females, random X-inactivation is due
to unmethylation of the Xist gene, which
produces a XIST transcript that coats the X-
chromosome. This produces a XIST-Barr body
complex that inactivates the chromosome.

15.  Since DNA methylation is not encoded in the DNA
sequence itself, it is called an EPIGENETIC modification.
 DNA methylation is therefore a form of cellular memory.

16.  Epigenetic marks are ERASED during gametogenesis and
reset to ensure appropriate gene activity. Old marks are
purged and new tags are established in a process called
PROGRAMMING.
 However, some methylated epigenomes can escape
purging during gametogenesis.
 These genes are inherited by mature gametes and are
carried into the next generation as GENOMIC IMPRINTS.
In a pregnant mother, 3
generations are directly
exposed to the same
environmental
conditions at the same
time. Germ cells thus
carry the effects of
gorgeous lola’s lifestyle!

17.  Bisphenol A in baby’s
bottle is linked to obesity.
 Causes demethylation
that reprograms the
hypothalamus resulting
to increased appetite
 Supplementing the
mothers' diets with
methyl-donating
substances (folic acid, Mice are GENETICALLY
vitamin B12) and identical but
genistein in soy products EPIGENETICALLY
counteract the reduction different.
in DNA methylation
caused by bisphenol A. The yellow mouse is
exposed to bisphenol A

18. TRANSCRIPTIONAL
CONTROL
Different subsets of
genes are
transcribed in
different tissues.
Tissue specific
gene regulation is
responsible for
these differences.
DNA microarrays
are used to profile
gene expression
patterns in various
cells.

19. Differences between the populations of mRNA sequences in the
cytoplasm of different kinds of cells reflect corresponding differences in
nuclear RNA populations which result from differential transcription of
genes. cDNA probes for tissue-specific transcripts are prepared by
eliminating from the tissue's mRNA population the molecules also found
in another tissue and reverse-transcribing the remaining mRNA.

20.  The core promoter is where the general transcription factors
and RNA polymerase assemble for the initiation of
transcription.
 Presence of basal transcription factors is not sufficient for
the activation of the promoter. DNA-binding proteins regulate
genes either by repression or activation.
 Regulatory sequences (enhancers or silencers) serve as
binding sites for these proteins, whose presence on the DNA
affects the rate of transcription initiation.

21. A Model for Enhancer Action
An enhancer is brought
close to the core promoter
by a looping of the DNA.
The influence of an
enhancer on the promoter
is mediated by regulatory
transcription factors
called activators.
The activator proteins bind to the
enhancer elements, forming an
enhanceosome. Bending of the
DNA brings the enhanceosome
closer to the core promoter. TFIID
is in the promoter's vicinity. The
DNA-bound activators interact with
specific coactivators that are part
of TFIID. This interaction facilitates
the correct positioning of TFIID on
the promoter. The other general
transcription factors and RNA
polymerase join the complex, and
transcription is initiated.

22. http://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf

23. Enhancers are modular. There are various DNA
elements that regulate temporal and spatial gene
expression, and these can be mixed and matched.
Whether the gene is transcribed or not will
depend on the combination of transcription
factors present.

24. The gene for the protein
albumin is associated
with an array of
regulatory DNA
elements. Cells of all
tissues contain RNA
polymerase and the
general TFs, but the set
of RTFs available varies
with the cell type. Liver
cells contain a set of
RTFs that includes the
factors for recognizing
all the albumin gene
control elements. When
these factors bind to the
DNA, they facilitate
transcription of the
albumin gene at a high
level. Brain cells have a
different set of
RTFs, which does not
include all the ones for
the albumin gene. In
brain cells, the
transcription complex
can assemble at the
promoter, but not very
efficiently. The result is
that brain cells
transcribe the albumin
gene only at a low level.

25.  The DNA response sequences that bind transcription
factors are often comprised of inverted repeat elements.
 Reading the sequence of the glucocorticoid response
element in the 5’- 3’ direction from either end yields the
same DNA sequence (5’-AGAACA -3’).
 The thyroid hormone element contains the same
inverted repeat sequences as the estrogen element but
the 3 bases
that separate
the 2 copies
of the
sequence in
the estrogen
element are
absent.

26. The glucocorticoid receptors activate Binding the steroid
gene transcription. Cortisol, a causes the release of an
hydrophobic steroid hormone, can inhibitory protein and
diffuse through a plasma membrane activates the
then bind to the intracellular glucocorticoid receptor
glucocorticoid receptor. molecule's DNA binding
site. The glucocorticoid
receptor molecule then
enters the nucleus and
binds to a glucocorticoid
response element in
DNA which causes a 2nd
glucocorticoid receptor
molecule to bind to the
same response element.
The resulting
glucocorticoid receptor
dimer activates
transcription of the
target gene.

28.  Transcription factors are proteins that bind to
the enhancer or promoter regions and interact
such that transcription
occurs from only a small
group of promoters in any
cell.
 Most have:
a.DNA- binding domains
that bind to promoter
or enhancer regions of DNA of specific genes
b.trans-activating domain that binds RNA polymerase II
or other transcription factors to regulate amount of
mRNA that the gene produces
c.protein-protein interaction domain that allows the
transcription factor's activity to be modulated by TAFs
or other transcription factors.

29. Gene
regulatory
proteins
contain
structural
motifs that
can read
DNA
sequences

30. Different base pairs in DNA can be recognized from
their edges without the need to open the double helix.
Regulatory proteins make contact with major groove .

31. Determining DNA
sequence recognized
by a regulatory protein

32. A DNA fragment is labeled at
one end with 32P. The DNA is
cleaved that makes
random, single stranded cuts.
After the DNA denaturation, the
resultant fragments from the
labeled strand are separated on
a gel and detected by
autoradiography. The pattern of
bands from DNA cut in the
presence of a DNA-binding
protein is compared with that
from DNA cut in its absence.
When protein is present, it
covers the nucleotide at its
binding site and protects their
phosphodiester bonds from
cleavage. As a result, those
labeled fragments that would
otherwise terminate in the
binding site are missing, leaving
a gap in the gel pattern called a
"footprint.“

33. Several structural motifs are commonly found in the DNA-binding domains
of regulatory transcription factors. The parts of these domains that directly
interact with specific DNA sequences are usually α helices (or recognition
helices) which fit into DNA's major groove. The helix-turn-helix motif
contains 2 α helices joined by a short flexible turn. The zinc finger motif has
an α helix and a 2-segment, antiparallel ß sheet, all held together by the
interaction of 4 cysteine residues (or 2 cysteine & 2 histidine residues) with
a zinc atom. Zinc finger proteins normally contain a number of zinc fingers.

34. The leucine zipper motif contains an α helix that has a regular
arrangement of leucine residues that interacts with a similar region
in a 2nd polypeptide to coil around each other. The helix-loop-helix
motif contains a short and long helix connected by a polypeptide
loop that interacts with a similar region on another polypeptide to
create a dimer, e.g. human TF MAX, lac and trp repressor.

35. The homeodomain is a
helix-turn-helix DNA-
binding domain
containing 3 α helices
encoded by a 180 bp
homeobox. This was
originally found in
homeotic genes which
control the
establishment of
pattern in an organism
such as the spinal
column in vertebrates.

36. Superclass: Basic Domains Families
 AP-1(-like) components: Jun,
Fos, GCN4, CRE-BP/ATF
Leucine zipper factors
 CREB: CREB, ATF-1,
(bZIP)
 C/EBP-like factors and 4 other
families
Helix-loop-helix factors  Myogenic transcription factors
(bHLH) and 8 other families
Helix-loop-helix / leucine  Cell-cycle controlling factors:
zipper factors (bHLH-ZIP) Myc, Max, and 1 other family
NF-1
RF-X
bHSH

37. Superclass: Zinc-coordinating DNA-binding domains
 Steroid hormone receptors
Cys4 zinc finger of  Thyroid hormone receptor-like factors:
nuclear receptor type RAR, RXR, Vitamin D receptor, PPAR,
Knirps
 4 families: WT-1 in the kidney, Krox20 in
Cys4 zinc fingers
the rhombomeres of the hindbrain
 Ubiquitous factors: TFIIIA, Sp1
 Developmental / cell cycle regulators:
Cys2His2 zinc finger Krüppel, Hunchback
domain  3 other families involved in cytoskeletal
organization, organ development and
oncogenesis
Cys6 cysteine-zinc
 Metabolic regulators in fungi
cluster
Zinc fingers of
 2 families
alternating composition

38. Superclass: Helix-turn-helix
 Homeodomain only: Antp, Ubx,
Engrailed, Eve
 POU domain factors: Oct-1, Oct-2
Homeodomain (activate IgG genes)
 2 other families implicated in
craniocaudal segmentation of
the body.
 Pax1-9: Pax6 in the eye, Pax3 in the
Paired box
developing somite
Fork head / winged  Developmental regulators: Forkhead
helix  2 other families: HNF3
Heat shock factors
 Myb
Tryptophan clusters
 2 other families
TEA domain

39. Superclass: β-Scaffold Factors with Minor Groove Contacts
 Rel/ankyrin: NF-kappaB1
RHR (Rel homology region)
 2 other families
STAT
p53
 Regulators of differentiation:
MADS box Agamous, AP1, Deficiens, AP3
 2 other families
beta-Barrel alpha-helix TFs
TATA-binding proteins  TBP
 TDF-SRY, TCF factors
HMG
activated by the Wnt pathway
Heteromeric CCAAT factors
Grainyhead
Cold-shock domain factors
Runt

40. Superclass: Other Transcription Factors
Copper fist proteins
HMGI(Y)
 Rb - retinoblastoma
Pocket domain
 1 other family
E1A-like factors  E1A
 AP2
AP2/EREBP-related factors
 2 other families
The most recent update of the transcription factor
database lists 2785 entries. Many of these are
homologous proteins from different species,
nevertheless this number is indicative of the vast
number of transcription factors now known that
regulate the expression of eukaryotic genes. Majority
function as ligands in cell signaling pathways.

42.  Alternative splicing makes it possible for the
same gene to produce several different mRNAs,
by splicing together different combinations of
exons from the primary transcript.
 Different proteins formed from the same gene
are called splicing isoforms.

44.  Genes coding for the human
antibody heavy chains are
created by DNA rearrangements
involving multiple types of V, D
and J segments.
 Initially, the DNA of the immune
cells is arranged as tandem
arrays of V, D and J regions
 DNA excision randomly removes
several D and J segments to
place individual D and J
sequences side by side.
 A second random excision
removes several V and D
segments to join a V section to
the others to form a VDJ
segment.
 After transcription, the
sequences separating the VDJ
segment from the C segment are
removed by RNA splicing.

45. The antibody protein immunoglobulin M (IgM) exists in 2 forms, as secreted IgM and
membrane-bound IgM. These molecules, encoded by a single gene, differ in their heavy
chain’s carboxyl ends. The IgM gene has 2 possible poly(A) addition (termination) sites
and a number of exons that can produce 2 alternative forms. The plasma membrane-
bound form contains a transmembrane anchor which is encoded by exons 5 and 6. If a
splice junction within exon 4 is used, exons 5 and 6 (carrying the anchor) are added to
generate the IgM heavy chain. The secreted product is produced when the exon 4 splice
is not made and these transcripts are terminated just after exon 4.

46. Developing RBC
synthesize hemoglobin
(4 globin polypeptides
and a heme prosthetic
group).
The heme-controlled inhibitor (HCI)
protein regulates hemoglobin synthesis in
response to the presence of heme. When
present, heme binds to inactivate the HCI.
When heme is absent, the HCI is active.
Active HCI functions as a kinase that
catalyzes the phosphorylation of eIF2, a
key TF. P-eIF2 is inactive; it cannot
When heme is
combine with methionyl tRNA and GTP to
present
form the translation initiation complex. In
translation of
the absence of heme, translation of all
the mRNA
mRNA in the cell is inhibited. The effect is
proceeds. Newly
on globin synthesis because globin mRNA
made globins
constitutes most of the developing RBC’s
combine with
mRNA.
heme to form
hemoglobin
molecules.

47. Translation of ferritin is activated in the presence of iron.
Translation is inhibited by binding of the IRE-binding protein
to the hairpin structure of an iron response element (IRE) in
the 5’UTR leader sequence of ferritin mRNA. When iron
binds to IRE-binding protein, it contorts into a conformation
that does not recognize the IRE. When iron is available,
ribosomes can assemble on the mRNA and proceed to
translate ferritin. The hairpin does not interfere with the
ribosome activities.

48. Degradation of the transferrin receptor mRNA (required for
iron uptake) is also regulated by the allosteric IRE-binding
protein. Transferrin receptor mRNA has an IRE in its 3’ UTR.
When intracellular [iron] is low, the IRE-binding protein
remains bound to the IRE which 1) protects the mRNA from
degradation and 2) allowing more transferrin receptor
protein to be synthesized. When intracellular [iron] is
high, iron binds to the IRE-binding protein, it releases the
IRE and the mRNA can be degraded.

49. By RNA interference, short RNA's can
lead to silencing the expression of
genes that contain complementary
sequences in their mRNA. A complex
of dsRNA is cleaved into short
fragments of 21-22 bp in length by the
ribonuclease Dicer. The fragments are
siRNA's (short interfering RNA's). The
siRNA's bind to the RISC (RNA-induced
silencing complex). One of the strands
of siRNA is degraded. The remaining
single-stranded siRNA , complexed
with the RISC can then bind to
complementary mRNA. If a perfect or
near perfect match, the mRNA is
cleaved. In addition, the RISC-siRNA
complex can enter the nucleus, binds
the genomic sequence and initiates a
DNA methylation based chromatin
condensation inactivation of the gene.

50. microRNAs (miRNAs) are gene
products that are 21-22
nucleotides in length. The 10
miRNAs are transcribed, form hair-
pin structures and are cleaved by
Drosha to make precursor
microRNAs (70 nucleotides in
length). The pre-miRNAs are
exported to the cytoplam where
they are cleaved by Dicer into the
21-22 nucleotide mature
microRNA's. The miRNA's form
RNP complexes with mRNA's. If the
match is exact, the mRNA is
destroyed, similar to siRNA
mechanisms. If the match is less-
than-exact, then binding (usually of
several miRNA's) inhibit
translation. Genes for miRNA's
make up 0.5-1.0% of the total
number of genes in multicellular
organisms, i.e. 200-250 miRNA
genes in humans.

51.  Several changes can still take place that determine
whether or not the protein will be active.
 In the ER, the protein becomes further processed (e.g.
formation of disulfide bonds, glycosylation,
phosphorylation). The folded proteins are carried in
vesicles to the Golgi apparatus where further processing
of the carbohydrates occurs, and then to exocytotic
vesicles where they are released.

52. Proteins can be marked for
destruction by the addition of
ubiquitin. 1) A protein targeted for
degradation is bound at its N-
terminus by a ubiquitinating enzyme
complex. 2) In an ATP-dependent
series of reactions, ubiquitin
molecules are sequentially attached
to the protein's lysine residues. The
ubiquitinating enzyme complex then
detaches. 3) A proteasome
degrades the ubiquitinated protein
into short peptides. The ubiquitin is
released and can be recycled.

53.  Some newly synthesized proteins are inactive
without the cleaving away of certain inhibitory
sections. (e.g., insulin is processed from its larger
protein precursor).
 Some proteins must be "addressed" to their
specific intracellular destinations in order to
function.
 Proteins are often sequestered in certain regions,
such as membranes, lysosomes, nuclei, or
mitochondria.
 Some proteins need to assemble with other
proteins to form a functional unit.
 Some proteins are not active unless they bind an
ion such as calcium, or are modified by the
covalent addition of a phosphate or acetate group.

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