This document discusses transcriptional gene regulation in eukaryotes. It describes how gene expression is controlled through the regulation of transcription, which involves basal transcription factors, proximal promoter elements, distal enhancer and silencer elements, and the binding of transcription factors to cis-regulatory modules. Chromatin structure also influences transcription through modifications that can activate or repress gene expression. Multiple levels of gene regulation allow for fine-tuned and combinatorial control of transcription in eukaryotic cells.

1. Transcriptional Gene
Regulation in Eukaryotes
Sreeremya.s

2. Overview
 Gene expression
 Transcription
 Regulation of eukaryotic transcription
 Influence of chromatin structure
 Oncogenes
 Techniques

3. Orphanides, Cell 2002

4. Control of gene expression at any stage:
Activation of
gene structure
Initiation of
transcription

5. What is a gene?
“The entire nucleic acid sequence that is necessary for
the synthesis of a functional polypeptide or RNA
molecule”

6. Overview
 Gene expression
 Transcription
 Regulation of eukaryotic transcription
 Influence of chromatin structure
 Oncogenes
 Techniques

7. Transcription
Initiation, elongation, termination
Catalyzed by RNA polymerase
“Transcription bubble”: DNA transiently separated into
single strands
One strand is used as a template
Unwinding point & rewinding point
Rate ≈ 40 nucleotides/second at 37° for bacteria
RNA polymerase
Many subunits: catalytic site, CTD with (YSPTSPS)n
pol I, pol II, pol III

10. Bacteria
ω

11. Molecular details of
gene expression
control in bacteria: lac
operon E. coli (Jacob &
Monod 1960s)

12. Glutamine synthase

13. Eukaryotes

14. Basal transcription apparatus (general factors &
RNA polymerase)
Proximal cis-regulatory module
Distal cis-regulatory modules
Modules = discrete DNA elements that contain specific
sequence motifs with which DNA binding proteins
interact and transmit molecular signals to genes
Promoter
Enhancer

15. BTA
General factors: TFIIx
Mechanics of initiating RNA
synthesis at all promoters
Determines location of
transcription startpoint
Complex with RNA polymerase
TATA
 ~ 25bp upstream
 8bp consensus of A•T pairs
 Tends to be surrounded by
G•C rich regions
 TBP, 11 TAFs : TFIID (~800kD)

16. TATA-less promoters:
 Inr Py2CAPy5 (-3 to +5)
Promoter-prediction: TATA-box, C-G enrichment
50%
TATA Inr
Inr
TATA
6 4
132
23
3
12 19
Eponine
CpG islands
Promoter
Inspector

17. Promoter-proximal region
Efficiency and specificity of transcription depend
on binding of transcription factors

18. Promoter
recognition
Function = to be recognized by proteins; so
differs from exon, …
Any essential nucleotide sequence should be
conserved
Some variation is permitted
When is it sufficiently conserved?
Idealized sequence with base most often present:
consensus sequence by aligning all known examples
Only conservation of very short sequences; 60 bp
associated with RNA pol lack conservation

19. Variety of elements can contribute, none is essential
for all promoters (mix & match principle)
CAAT box ~ -80bp GGCCAATCT
increases promoter strength
Bound by CTF/NF1 family, CP1 & CP2, C/EBP, ACF
GC box GGGCGG
SP1
Octamer (8bp) ATTTGCAT
Bound by Oct1 (ubiquitous): activates histon H2B
Bound by Oct2 (lymphoid cells): Ig kappa light chain
context is important

20. Modular nature of the promoter:
Equivalent regions can be exchanged
Main purpose = to bring the factors they bind into
the vicinity of the initiation complex
Protein-protein interactions determine the
efficiency of the initiation reaction
Sequence elements influence the frequency of
initiation
Repression of transcription:
Generally by influencing chromatin structure
By repressors, e.g. Dr1/DRAP1 binds to TBP and
CAAT displacement protein (CDP)

22. Modules
Enhancers, silencers
5’ region, distal

23. Modules
50 bp to 1.5 kbp in size
4-8 TFs (often multiple sites); higher density of
regulatory elements than in the promoter
Many elements are common elements in promoters,
e.g. AP1 and the octamer
Can stimulate any promoter placed in its vicinity
Can function anywhere (cfr β-globin: 200 fold in vivo) ;
Position relative to promoter can vary substantially;
can function in either orientation

25. Binding sites for activators that control transcription of the mouse transthyretin (TTR)
promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell
Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]

26. Example: muscle specific
modules
Creatine kinase, myosin light chain, skeletal actin, myosin heavy chain

27. Model for the control of the human β-globin gene. Some of the gene regulatory proteins shown,
such as CP1, are found in many types of cells, while others, such as GATA-1, are present in only
a few types of cells including red blood cells and therefore are thought to contribute to the cell-
type specificity of β -globin gene expression. (Adapted from B. Emerson, In Gene Expression:
General and Cell-Type-Specific [M. Karin, ed.], pp. 116-161. Boston: Birkhauser, 1993.)

30. Current view:
same sort of interaction with basal apparatus as the
proximal promoter module
Increase the concentration of transcription factors
in the vicinity of the promoter
Intervening DNA: extruded as a large “loop”
Generality: not yet clear (what proportion of
promoters require an enhancer?)

31. Four activators enriched in
hepatocytes plus the ubiquitous
AP1 factor bind to sites in the
hepatocytespecific enhancer and
promoter-proximal region of the
TTR gene.
The activation domains of the
bound activators interact
extensively with co-activators,
TAF subunits of TFIID,
Srb/Mediator proteins, and
general transcription factors,
resulting in looping of the DNA
and formation of a stable
activated initiation complex.
Cooperative
assembly

32. Limited
knowledge
Experimentally verified binding sites
Experimentally verified “composite elements” or
CE’s
GR site + AP-1 in proliferin promoter
Synergistic: result in non-additively high level
Antagonistic: overlapping sites, masking an activation
domain,…
Direct or through coactivator
Few modules characterized that have multiple
elements, some in developmental biology

33. Side-track: Transcription
factors
5% of our proteins
Activities controlled in regulatory pathways
Independent domains responsible for activities:
Recognition of specific target sequences
Binding to other components
of the transcription apparatus
E.g. yeast GAL4

34. Protein-DNA interactions
Proteins with high affinity for a specific sequence
also possess a low affinity for any (random) DNA
sequence
E.g. Lac repressor E. coli: Free:bound = 10-4
High-affinity site competes with the large number
of low-affinity sites; repressor binds ≈107
times better
to operator DNA (bound 96% of time for 10
molecules/cell)

35. How the different base
pairs in DNA can be
recognized from their
edges without the need to
open the double helix.

36. The binding of a gene regulatory protein to the major
groove of DNA.
Typically, a protein-DNA interface
consists of 10 to 20 such contacts,
involving different amino acids, each
contributing to the binding energy of
the protein-DNA interaction.

37. Zinc finger motif
Common motif in DNA binding, e.g. SP1 has 3
(A) The structure of a fragment of a mouse gene regulatory protein bound to a specific DNA site. This protein
recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats. (B) The three
fingers have similar amino acid sequences and contact the DNA in similar ways. In both (A) and (B) the zinc
atom in each finger is represented by a small sphere. (Adapted from N. Pavletich and C. Pabo, Science252:810-
817, 1991. © 1991 the AAAS.)

38. All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red
cylinder) are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the
helix-turn-helix motif is colored blue. The lambda repressor and cro proteins control
bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite
activator protein (CAP) control the expression of sets of E. coli genes.
Helix-Turn-Helix

39. Homeodomains
Related to helix-turn-helix bacterial repressors
Homeobox = 60 AA residues
E.g. en, eve, Hox, Oct-1, Oct-2 (Oct also have Pou
domain next to homeodomain)
The homeodomain is folded into three alfa helices, which are packed tightly together by hydrophobic interactions (A). The part
containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts
with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor
groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein, but it is
nearly identical to two homeodomains from Drosophila, which interact with DNA in a similar fashion. (Adapted from C.

40. Helix-loop-helix (HLH)
DNA binding (helix) & dimerization
Class A: ubiquitouslyh expressed proteins, e.g.
E12/E47
Class B: tissue-specific expression, e.g. MyoD,
myogenin, Myf-5
Myc proteins (separate class)
Leucine zippers fig 21.15
Dimerization motif
E.g. Jun+Fos = AP1
Gcn4 ->

41. Steroid receptors
Independent domains: DNA binding, hormone
binding, and dimerization
Cortisol - glucocorticoid receptor (GR).
Retinoic acid - retinoic acid A receptor (RAR).
Thyroxine - thyroid hormone receptor (TR).

42. Figure 1 Genome-wide comparison of transcriptional activator families in eukaryotes.
The relative sizes of transcriptional activator families among Homo sapiens,
D. melanogaster, C. elegans and S. cerevisiae are indicated, derived from an analysis of
eukaryotic proteomes using the INTERPRO database, which incorporates Pfam, PRINTS
and Prosite. The transcription factors families shown are the largest of their category out
of the 1,502 human protein families listed by the IPI.

44. Transcription
factories
cfr. replication factories
Active RNA polymerases are concentrated in discrete
'factories' where they work together on many different
templates
Complexes for transcription and RNA processing are
likely to be immobile structures within the gel-like
nucleoplasm (Burns et al, 2001; Kimura et al, 1999)
Transcriptional interference: phenomenon where
transcription of one gene prevents transcription of an
adjacent gene. Discovery: Cells were transfected with a
retroviral vector encoding resistance to neomycin and
azaguanine, and clones harboring a single copy of the
vector selected. Expression of the 3' gene was
suppressed when selection required expression of the 5'

45. Cook, 1999 (Science)• Enhancers
•dynamic equilibrium
•enhancing the probability of the key transcription cycle interactions
•Element 5’ or 3’ doesn’t matter!

46. Recap: evolution of understanding of
eukaryotic transcription
Lemon and Tjian, Genes Dev. 14: 2551-2569 (2000)

47. Termination
Bacteria

48. Overview
 Gene expression
 Eukaryotic transcription
 Regulation of eukaryotic
transcription
 Influence of chromatin structure
 Oncogenes
 Techniques

49. Activate/inactivate a
TF
Transport through nuclear pores from
cytoplasm to nucleus (e.g. masking NLS,
nuclear localization signal, can regulate this
transport)
Link to Ubiquitin protease system
Rapid turnover of promoter bound TF: resets
signaling pathway: cell can continuously monitor its
environment
Tissue-specific synthesis
Development, e.g. homeodomain proteins
Modification
Phosphorylation, acetylation, methylation
E.g., AP1 (= Jun+Fos) → active form by
phosphorylation

50. Ligand binding
E.g. Steroid receptors
Influence: localization or DNA-binding ability
Cleavage
Inhibitor release
E.g. NF-κB + I- κB (release in B lymphocytes)
Change of partner (active partner displaces
inactive partner)

51. Examples:
GATA-1 CAP NtrC Adenovirus E1A NF-KB/
+ CBP/p300 glucocorticoid receptor
Pathways…

52. 1
2

53. Level 1 = active/inactive factor
Level 2 = cooperation of multiple factors
within a module (all present and active, and all
repressors inactive or absent)
Level 3 = multiple autonomous modules per
gene
Each module can independently activate the gene
Each has a specific function (e.g. activation in
certain cell type or at particular stage in dvl)
different circuits of regulation, e.g. metallothionein
gene (MT): heavy metals and steroids, fig 21.1
Gene can respond to multiple signaling pathways
Facilitates fine-tuning of transcript levels

54. Combinatorial and context dependent regulation of
transcription
one factor can induce transcription of one gene while
repressing that of another

55. Experiment demonstrating the modular
construction of the eve gene regulatory region.
(A) A 480-nucleotide-pair piece of the eve
regulatory region was removed and inserted
upstream of a test promoter that directs the
synthesis of the enzyme β-galactosidase (the
product of the E. coli lacZ gene). (B) When this
artificial construct was reintroduced into the
genome of Drosophila embryos, the embryos
expressed β-galactosidase (detectable by histo-
chemical staining) precisely in the position of the
second of the seven eve stripes (C).
(Metamerization)
-
+

56. rho
• Dorsal (Dl)
• Twist (HLH)
• a HLH
• Snail (-)

57. Principles for
specification
1. cis-regulatory transformation of input patterns into
spatial domains of differential gene expression
2. Always assemblages of diverse target sites because
multiple inputs are required
3. Output=novel with respect to any one of the incident
inputs + more precise in space and time => “information
processing”
4. Every specific type of interaction that can be detected in
vitro is fundamentally significant (it is unlikely that highly
specific site clusters, which are of improbable random
occurrence would have no function)
5. Negative & positive inputs(Davidson, 2001)

58. Cis-regulatory logic device
endo16 of Strongylocentrotus (zee-egel)
Secreted embryonic gut protein
“hardwired biological computational device”

62. Overview
 Gene expression
 Initiation of transcription
 Regulation of transcription
 Influence of chromatin structure
 Oncogenes
 Techniques

63. Chromatin
Eukaryotic genomes are
packaged with chromatin
proteins
Heterochromatin (highly
condensed, untranscribed)
Euchromatin (more
accessible, transcribed)
Each cell: unique pattern of
heterochromatin and
euchromatin

64. Nucleosome
s
Workman and Kingston
Ann. Rev. Biochem. 67: 545 (1998)
•146 bp
• H2A, H2B, H3, H4

65. Chicken and egg
scenario
TF binding requires chromatin decompaction by
certain factors but the latter also need to interact
with DNA
Solution: probably some TFs can bind to their
recognition sequences even when they are
packaged (e.g. glucocorticoid receptor: only
contacts DNA on one side ⇔ NF1 surrounds
double helix)

66. Narlikar, 2002 (Cell)
1. ATP-dependent remodeling
Modify chromatin structure

67. 2. Histone-modifying complexes
 Phosphorylation, methylation, acetylation
 Histone acetyltransferase (HAT), histone deacetylase
(HDAC)
 How do they impact the structure of the template and
the ability of the transcription machinery to function?
 lowered positive charge on acetylated N termini, lowered
stability of interaction with DNA
 Disrupting internucleosomal interactions
 Recruiting additional TFs
 A lot of combinatorial possibilities: histon code?

68. Jenuwein T, Allis CD. Science 293:1074-80 (2001).

69. Model of the protein
interactions and functions
of the Myc/Max/Mad
transcription network.
Myc-Max and Mad-Max (along with Mnt-Max and Mga-Max) complexes bind to DNA to E-boxes. Binding can be
affected by the context, sequence, cooperativity, and location of the E-boxes. Myc-Max heterodimers activate
transcription by recruiting HAT's via TRRAP. This leads to the acetylation of histone tails and the opening of local
chromatin structure. Additionally, Myc-Max appears to repress transcription through Inr elements via an undefined
mechanism. As a result of these activities at target genes, Myc affects proliferation, cell cycle, growth, immortalization,
and apoptosis. When deregulated, Myc cooperates with other oncogenes to cause a variety of cancers.
Mad-Max and Mnt-Max heterodimers repress transcription by recruiting HDAC's via mSin3A. This leads to the
deacetylation of histone tails and the closing of local chromatin structure. As a result of target gene repression, Mad
causes an increased cell doubling time, growth arrest, and the maintenance of differentiation.
Grandori C, Cowley SM, James LP,
Eisenman RN.
Annu Rev Cell Dev Biol. 2000;16:653-
99

70. Cytosine methylation
mCG often in inactive vertebrate genes
After replication of methylated DNA, methyl groups
are added to daughter strands
CpG islands
imprinting

71. Imprinting
Imprinted genes are genes whose expression is determined by the
parent that contributed them.
Imprinted genes violate the usual rule of inheritance that both alleles
in a heterozygote are equally expressed.
Examples of the usual rule:
 If a child inherits the gene for blood group A from either parent and the
gene for group B from the other parent, the child's blood group will be AB.
 If a child inherits the gene encoding hemoglobin A from either parent and
the gene encoding hemoglobin S from the other parent, the child's red
blood cells will contain roughly equal amounts of the two types of
hemoglobin.
But there are a few exceptions to this rule. A small number of genes in
mammals (~50 of them at the most recent count) have been found to
be imprinted. Because most imprinted genes are repressed, either
 the maternal (inherited from the mother) allele is expressed exclusively
because the paternal (inherited from the father) allele is imprinted or
 vice-versa.

72. Link between DNA methylation and histone deacetylation

73. Chromatin compartmentalization
Heterochromatin: gene poor, inaccessible,
perifery, transcr. inactive
Euchromatin: gene rich, transc. active

74. Consistent correlation between gene silencing (e.g. in
B en T lymphocytes) and presence in
heterochromatin regions
LCR, enhancers, insulators: act by maintaining
endogenous loci in a chromatin compartment that is
either transcr. permissive or nonpermissive?

75. Position variegation
Position effects can be observed for the Drosophila white gene. Wild-type flies with a
normal white gene have red eyes. If the white gene is inactivated by mutation, the eyes
become white (hence the name of the gene). In flies with a chromosomal inversion that
moves the white gene near a heterochromatic region, the eyes are mottled, with red
and white patches. The white patches represent cells where the white gene is silenced
and red patches represent cells that express the white gene. (After L.L. Sandell and
V.A. Zakian, Trends Cell Biol. 2:10-14, 1992.)

76. Overview
 Gene expression
 Initiation of transcription
 Regulation of transcription
 Alteration of chromatin structure during
transcription
 Oncogenes
 Techniques

77. The development and metastasis of human
colorectal cancer and its genetic basis.
A mutation in the APC tumor-suppressor gene in
a single epithelial cell causes the cell to divide,
although surrounding cells do not, forming a
mass of localized benign tumor cells called a
polyp. Subsequent mutations leading to
expression of a constitutively active Ras protein
and loss of two tumor-suppressor genes, DCC
and p53, generates a malignant cell carrying all
four mutations; this cell continues to divide and
the progeny invade the basal lamina that
surrounds the tissue. Some tumor cells spread
into blood vessels that will distribute them to
other sites in the body. Additional mutations
cause exit of the tumor cells from the blood
vessels and growth at distant sites; a patient with
such a tumor is said to have cancer. [Adapted
from B. Vogelstein and K. Kinzler, 1993, Trends
Genet. 9:101.]

78. Overview
 Gene expression
 Initiation of transcription
 Regulation of transcription
 Alteration of chromatin structure during
transcription
 Oncogenes
 Techniques

79. Technique
s
Mutational analysis
EMSA electorphoretic mobility shift assay
Microarrays: level of mRNA
ChIP: Chromatin ImmunoPrecipitation
Yeast2Hybrid
Sequence analysis, phylogenetic footprinting

80. Free DNA probe
*
*
Protein-DNA complex

82. Sources
B Lewin, Genes VII
Lodish et al. Molecular Cell Biology
EH Davidson: Genomic Regulatory Systems
Alberts et al. Essential Cell Biology
EM Blackwood & JT Kadonaga: Going the distance: a
current view of enhancer action
Cell, February 22, 2002: 108 (4) "Reviews on Gene
Expression"