DNA, CHROMATIN &

CHROMOSOMES
Sebastian Schmeier

Learning objectives
DNA, building blocks and DNA structure (recap)
Composition of chromatin
Histones and DNA packaging
Nucleosome structure
Nucleosome sliding

Levels of
chromatin
packaging

HIGHER-ORDER CHROMATIN
STRUCTURES

Learning objectives
Chromosome structure
Centromeres
Kinetochores
Telomeres and telomerases
The formation and existence of different forms of

chromatin

CHROMATIN
REMODELING

Learning objectives
Different forms of histone modifications
Understand how enzymes regulate gene

expression by modulating and responding to the

epigenetic code

What controls the condensation of the

chromatin?

Where are the n-termini of the core

histones?

Luger, Mader, Richmond, Sargent &

Richmond Nature 389, 251-260 (1997)

Question 1: What is the function of histone N-termini ?

Question 2: Are all N-termini functionally equivalent ?
Molecular Cell Biology, Lodish, 7th edition

10

The histone code

Specific combinations of the histone modifications

in different chromatin regions have been

suggested to constitute a histone code
The number of possible distinct markings on
individual nucleosomes is enormous
This code is thought to influence chromatin
function by creating or removing binding sites
of chromatin-associated proteins

11

Model for the formation

of heterochromatin
Higher eukaryotes express a

number of proteins containing a so

called chromodomain that binds
to histone tails when they are
methylated at specific lysines, e.g.
heterochromatin protein (HP1)
HP1 binds to histone H3 N-terminal
tails tri-methylated at lysine 9
HP1 has a second domain
(chromoshadow) which binds
other chromoshadow domains !
results in a condensed chromatin
structure
Molecular Cell Biology, Lodish, 7th edition

12

Model for the formation

of heterochromatin (2)

Heterochromatin condensation can spread along a chromosome

because HP1 binds a histone methyltransferase (HMT) that methylates

lysine 9 of histone H3.
This creates a binding site for HP1 on the neighboring nucleosome.
The spreading process continues until a "boundary element" is
encountered.
Boundary elements so far characterized are generally regions in
chromatin where several non-histone proteins bind to DNA, possibly
blocking histone methylation on the other side of the boundary
Molecular Cell Biology, Lodish, 7th edition

2384

H. Santos-Rosa, C. Caldas / European Journal of Cancer 41 (

13

Establishment of transcriptionally active

chromatin (1)
Establishment of transcriptionally active
chromatin by lysine 4 H3 methylation
1. Set1p methylates lysine 4 H3.
2. Methylated lysine 4 recruits the
chromatin remodelling factor Chd1p
in physical association with histone
acetyltransferases.
3. Acetylation of lysine residues
prevents repressive modifications to
occur and recruits transcription
activators.

Chromatin modifier enzymes, the histone code and cancer. Santos-Rosa et al. 2005:41(16) European Journal of Cancer

2384

H. Santos-Rosa, C. Caldas / European Journal of Cancer 41 (2005) 23812402

14

Establishment of transcriptionally active

chromatin (2)
Establishment of transcriptionally active
chromatin by lysine acetylation:
1. GCN5 acetylates several residues
within histones H3 and H4
2. Acetylated lysines recruit the
chromatin remodelling complex SWI/
SNF.
3. SWI/SNF, via its ATPase activity,
displaces and twists nucleosomes
exposing DNA areas for interaction
with the transcription machinery.

Chromatin
modifier enzymes, the
histone code
and cancer.
et al. 2005:41(16)
Journal ofofCancer
Fig. 3. Recruitment of bromoand chromo-domain
containing
proteins
by Santos-Rosa
histone modifications.
(a) European
Establishment
silent

TRANSCRIPTION,
RNA-POLYMERASES &
REGULATION
Sebastian Schmeier

16

Learning objectives
Recap of the transcriptional process
Similarities and differences between RNA

Polymerase I, II, and III

Gain an understanding of the structure of the
eukaryotic RNA Polymerase II and its function in
transcription
Similarities and differences between eukaryotic
and prokaryotic polymerases

17

Transcription

Transcription is catalyzed by DNAdependent RNA polymerases,

which produce an intermediate called precursors

Similar to DNA polymerases but they incorporate ribonucleotides
instead of deoxyribonucleotides into the newly synthesized strand
They do not require a primer

18

RNA Polymerases
Eukaryotic cells contain at least three different types of

RNA polymerases
RNA polymerase I synthesizes an RNA with a
sedimentation coefficient of 45S, which serves as
precursor for three ribosomal RNAs
RNA polymerase II produces hnRNAs (heterogeneous
nuclear RNA), from which mRNAs later develop, as well
as precursors for snRNAs (small nuclear RNA)
RNA polymerase III transcribes genes that code for
tRNAs (transfer RNA), 5S rRNA, and certain snRNAs

19

Eukaryotic vs. prokaryotic RNA Pol

Molecular Cell Biology, Lodish, 7th edition

20

Carboxy-terminal repeat domain (CTD)

CTD is an extension

appended to the C terminus

of RPB1
Proteins bind the CTD in
order to activate RNA Pol II
activity
Reversibly phosphorylated
Not present in prokaryotes
CTD is essential for life.
Cells containing only
RNAPII with none or only
up to one-third of its
repeats are inviable
Cramer, 2004

TRANSCRIPTIONAL
REGULATION (1)

22

Learning objectives
Transcriptional regulation in bacteria
The lac operon
Differences eukaryotic and bacterial

transcriptional regulation
Promoter structure in eukaryotes
General transcription factors in eukaryotic
transcription initiation

23

Modes of gene/protein level control in

eukaryotes

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 415

24

Transcription initiation in bacteria

Chain elongation continues
(at a speed of approximately 50

nucleotides/sec for bacterial RNA

polymerases)

until the enzyme the terminator in

the DNA, where the polymerase

halts
Here, it releases both the newly
made RNA chain and the DNA
template

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 337

25

Transcription in eukaryotes
1. Pre-initiation complex (PIC) assembly
2. PIC activation
3. Initiation
4. Promoter clearance
5. Elongation
6. Termination

26

Model of eukaryotic genes

Molecular Cell Biology, Lodish, 7th edition, Page 304

27

Overview eukaryotic transcriptional

regulation
Highly complex machinery as

opposed to bacterial
transcriptional regulation
Initiation requires core
promoter, general
transcription factors and
enhancer elements
Mediator also specific to
eukaryotic cells

Molecular Cell Biology, Lodish, 7th edition, Page 281

28

Initiation of transcription
Pre-initiation complex

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 341

TRANSCRIPTIONAL
REGULATION (2)

30

Learning objectives
Regulatory sequences in protein coding genes and the

proteins through which they function

Promoter-proximal elements
Distant Enhancers/regulatory regions
Mediator protein complex
Transcription factors (TFs): activators and repressors

31

Different promoter classes

Lenhard B et al., Metazoan promoters: emerging characteristics and insights into transcriptional regulation, Nature Reviews Genetics 2012

32

Chromatin
immunoprecipitation
(ChIP)
CpG promoter can have

bidirectional transcription
initiation
Antibody to RNA Pol II
Extract sequence bound by
RNA Pol II
Sequence DNA
Map sequence to genome

Molecular Cell Biology, Lodish, 7th edition, Page 298

33

Mediator protein complex

A central component of

gene regulation in
eukaryotes is the
Mediator, a 24-subunit
protein complex
Mediator, allows activator
proteins to communicate
properly with the
polymerase II and with the
general transcription
factor
Mediator provides an
extended contact area
for gene regulatory
proteins
Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 441

34

Activator proteins
Promote the assembly of RNA

polymerase and the general

transcription factors
Activators bind enhancers up to
several 10000bp distant to the
TSS
The simplest activators have a
modular design consisting of two
distinct domains
DNA-binding domain containing

structural motif for recognition of

specific DNA sequences (next lecture)
Activation domain for interaction with
other proteins (e.g. general TFs)

Molecular Cell Biology, Lodish, 7th edition, Page 308

35

Modular design of activator proteins

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 442

36

Modes of action of repressor proteins

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 446

37

Combinatorial control
In some heterodimeric TFs,

each monomer recognizes the

same DNA sequence, creating
different activation domains
with different partners (a)
TF monomers recognising
different TFBSs, the
combination of TFBSs will
determine the activation
domain (b)
Inhibitory factors also play a
role in repressing certain TFs
(c)
Molecular Cell Biology, Lodish, 7th edition, Page 313

TRANSCRIPTIONAL
REGULATION (3)

39

Learning objectives
DNA-binding domains of transcription factors (TFs)
How to experimentally determine DNA-binding proteins
Transcription factor binding sites (TFBS) and how to

experimentally and computationally determine them

Regulation of the regulators

40

How to experimentally detect DNA-binding proteins?

Gel-mobility shift assay
based on the effect of a bound protein

on the migration of DNA molecules in

an electric field
A DNA molecule is highly negatively
charged
the electrophoretic mobility of a DNA
fragment is reduced when a protein is
bound, causing a shift in the location of
the fragment band
The method does not identify the
specific proteins
Once a sequence-specific DNA protein
has been purified, the gel-mobility shift
assay can be used to study the
strength and specificity of its
interactions with different DNA
sequences, the lifetime of DNA-protein
complexes
Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 428

41

How to experimentally detect DNA-binding proteins? (2)

DNA affinity chromatography
protein-purification method
Through purification, one can

isolate enough protein to obtain

its AS sequence through mass
spectrometry

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 429

42

How to experimentally determine TFBSs?

DNase I footprinting
DNA gets randomly digested
This leads to fragments of different

sizes
Because initial sequence is known,
one can count the positions on the
bands and thus identify the exact
sequence that is covered by the
protein

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 430

43

How to experimentally determine

TFBSs? (2)
Chromatin immunoprecipitation

(ChIP)
Use antibody against specific protein of
interest to fish out all protein-sequence
pairs
Remove protein
Identify sequences by either PCR
(classical ChIP) or by sequencing (ChIPseq)

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 432

44

How to determine TFBSs in silico?

Phylogenetic footprinting

Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 431

RNA PROCESSING AND

REGULATION
Sebastian Schmeier

46

Learning objectives
Understand that transcription and mRNA processing affect

one another
Pre-mRNA processing
5 capping
Cleavage
3 polyadenylation

Mechanisms of Termination

47

mRNA processing in eukaryotes

Molecular Cell Biology, Lodish, 7th edition, Page 348

48

Learning objectives
Pre-mRNA processing
Polyadenylation (revisited)
Splicing

49

Alternative polyadenylation
About half of all protein-coding genes have more than one

polyadenylation site a gene can code for several

mRNAs that differ in their 3' end
Alternative polyadenylation can
shorten the coding region
making the mRNA code for a
different protein
Alternative polyadenylation
changes the length of the 3'
untranslated region change
which binding sites for
microRNAs the 3' untranslated
region contains
Sven Danckwardt, Matthias W Hentze, and Andreas E Kulozik.
3 end mRNA processing: molecular mechanisms and implications for health and disease.
EMBO J. 2008 February 6; 27(3): 482498.

74:3171; courtesy of P. A. Sharp.]

50
\

mRNA

Splicing signals
An intron has to have three parts to be able to get spliced:

center portion of introns can be removed without affecting

net result of these two reactions is that two exons are ligated
splicing; generally only 30-40 nucleotides at each end of an
and the intervening intron is released as a branched lariat
structure.
1ntron are necessary for splicing to occur at normal rates.
Analysis of the intermediates formed during splicing of
pre-mRNAs in vitro led to the discovery that splicing of exons
The
splicetransesterification
donor site includes
an almost invariant sequence GU at the
proceeds via two
sequential
reactions (FigDuring Splicing, snRNAs Base-Pair
ure 8-8). lntrons
removed
as aintron,
lariat structure
in which
the
5' are
end
of the
within
a larger,
less highly conserved region
with Pre-mRNA
5' G of the intron is joined in an unusual2' ,5 '-phosphodiester
Splicing requires the presence of small n uclear RNAs
bond to an adenosine ncar the 3' end of the intron. This A
(snRNAs), important for base pairing with the pre-mRNA,
residue is called the branch point A because it forms an RNA
and
170 associated
proteins.point,
Five U-rich
snRNAs, desigbranch in the
lariat structure.from
In eachthe
transestcrification
re Upstream
polypyrimidine
rich- tract
is a branch
which
nated U1, U2, U4, US, and U6, participate in pre-mRNA
action, one phosphoester bond is exchanged for another.
includes
an
adenine
nucleotide
splicing. Ranging in length from 107-210 nucleotides, these
Since the number of phosphoester bonds in the molecule is
snRNAs are associated with 6- 10 proteins each in the many
not changed in either reaction, no energy is consumed. The

1. A donor site (5' end of the intron)

2. A branch site (near the 3' end of the intron)

3. An acceptor site (3' end of the intron)

The splice acceptor site at the 3' end of the intron terminates the intron

with an almost invariant AG sequence.

5' splice site

Branch point

3' splice site

Pre-mRNA
Frequency of
occurrence (%)

70

60

80

100 100

95

70 80 45

80

90

80

100

80

80

100 100 60

1+---------- 20- 50 b

FIGURE 8 -7 Consensus se quences around splice sites in

adenosine, also invariant, usually is 20-50 bases from the 3' splice site.

and the 3 ' AG of the intron (blue), although the flanking bases

50 kilo bases in length, generally is unnecessary for splicing to occur.

exon/GUintronAG/exon
vertebrate pre-mRNAs. The only nearly invariant bases
are the 5' GU
The central region of the intron, which may range from 40 bases to

Molecular Cell Biology, Lodish, 7th edition, Page 352

51

Exon definition
The information for defining the splice sites that

demarcate exons is encoded within the exon sequence

A family of RNA-binding proteins, the SR proteins,
interact with the sequences called exonic splicing
enhancers
The complex of SR proteins, snRNPs, and other splicing
factors (e.g., U2AF) that assemble across an exon, which
has been called a cross-exon recognition complex,
permits precise specification of exons in long pre-mRNAs

52

Learning objectives
Alternative splicing
Cytoplasmic mechanisms of post-transcriptional control
microRNA and RNAi

53

Alternative splicing
Widely distributed general mechanism for regulating gene

expression
Alternative splicing generates genetic diversity without the
need for additional genes
Single gene transcribed and processed in different ways
to yield different protein products

http://compbio.berkeley.edu/people/ed/rust/

54

Advantages of alternative splicing

Split genes allow multiple protein isoforms to be

expressed from a single sequence by alternative splicing

events.
Molecular analyses during the last decade demonstrate
that alternative splicing determines the binding properties,
intracellular localization, enzymatic activity, protein
stability and posttranslational modifications of a large
number of proteins
Gene regulation through alternative splicing is more
versatile than regulation through promoter activity

55

Nonsense-mediated decay (NMD)

It has long been known that mRNAs

carrying a premature termination codon

are highly unstable
NMD recognizes these mRNAs and
degrades them
During mRNA processing, a complex is
deposited near sites of intron removal
These exon-junction complexes are important

both for facilitating export from the nucleus and

for remembering gene structure
They mark the sites where the introns were
spliced out
This relative positioning appears to be checked
during the pioneering round of translation

http://compbio.berkeley.edu/people/ed/rust/

56

Regulated unproductive splicing and translation (RUST)

There are cases in which alternative splicing does not affect the coding region:
It only affects whether the isoform will be down regulated by NMD
Regulation of this kind, termed RUST, is mediated by the splice environment,

the set of splicing factors present and active at a given time and place
Under certain conditions, one set of

splice sites could be used that generate

an isoform whose stop codon is on the
last exon translation
Under different conditions or in a
different cell, alternative splice sites
could be used that introduce a premature
termination codon, generating an
unproductive isoform
This can be done by splicing in an
alternative exon, causing a frameshift, or
splicing out an intron downstream of the
normal termination codon. NMD

http://compbio.berkeley.edu/people/ed/rust/

57

miRNA action

494

Chapter7:Controlof GeneExpression

The mature miRNA is part of an

active RNA-induced silencing

complex (RISC) containing Dicer
and many associated proteins
lf the RNA:RNA match is
extensive as is commonly seen
in plants, Argonaute cleaves the
target mRNA causing its rapid
degradation
In animals the miRNA-mRNA
match does not extend beyond a
short 7-nucleotid seed region
near the 5' end of the miRNA
This less extensive base-pairing
leads to inhibition of translation,
mRNA destabilization and,
transfer of the mRNA to Pbodies for eventual degradation

CLEAVAGE
V

lllililillililt

"CROPPING"

ilililt1

NU C L E U S

I
A r g o n a u t ea n d
other proteins 3,
e x t e n s i v em a t c h

amRNA

rilrflrilrilil

AAAAA

CLEAVAGE
V
l nL
A

CYTOSOL
"DlClNG"

t
5'

RISC

l e s se x t e n s i v em a t c h

OmRNA

"sLrcrruc"

illillililrrililt

AAAAA

@*1 I
ADP <_{

l*

R I S Cr e l e a s e d

I
r a p i d m R N AD E G R A D A T I O N

T R A N S L A T I ORNE D U C E D
t r a n s f e ro f m R N Ai n t o
P-bodiea
s n d e v e n t u a ld e g r a d a t i o n

polymerase II and are capped and polyadenylated. They then undergo a special
type of processing,after which the miRNA is assembledwith a set of proteins to
form an RNA-indttced silencing complex or fiISC. Once formed, the RISC seeks
Alberts,
5th edition,
7-112
out its target mRNAs by searching for complementary
nucleotide
sequences

58

RNA interference through siRNA

496

Chapter7:Controlof GeneExpression

RNA interference (RNAi)

d o u b l e - s t r a n d eR
d NA

induces degradation of
precisely complementary
mRNAs
siRNAs have a well-defined
structure: a short (usually
21-bp) double-stranded
RNA (dsRNA) with
phosphorylated 5' ends and
hydroxylated 3' ends with
two overhanging
nucleotides
The Dicer enzyme
catalyzes production of
siRNAs from long dsRNAs
and small hairpin RNAs.

A r g o n a u t ea n d
o t h e r R I S Cp r o t e i n s

RISC

A r g o n a u t ea n d
other RITSproteins

q4]##'.

I
I

PATHWAYNOW FOLLOWS
ONEOF
T H O S ES H O W Nl N F i g u r e7 - 11 2

H I S T O NM
E ETHYLATION
DNAMETHYLATION
T R A N S C R I P T I O NRAELP R E S S I O N

response resembles certain aspects of animal immune systems; in both, a

invading organism elicits a customized response,and-through
Alberts, 5th edition,
7-115 o
amplification

59

Base pairing miRNA versus siRNA

(a) miRNA

L.LI

inhibition

I I I I I I I I I I I I

(b) siRNA

LLr.

I I

cleavage

II"'TI"T"Inii'"T'I'I"1111"'TI'T'l:-;

:"T"IT"""j

Target RNA

Target RNA

uc

C A
C A
5' -UCCCUGAGA
GUGUGA 3'
11111 1 11
I I II I
3' -UCCAGGGACUCAACCAACACUCAA- 5'

1111111111111 111111111
3' - CUUAUCCGUCAAAGUACAACAACCUUCU- 5'

lin-4 miRNA and lin-14 mRNA (C.elegans)

miR-196a and HOXBB mRNA (H. sapiens)

5' -UGUUAGCUGGAUGAAAACTT- 3'

11111111
111111111
3' -GCCACAAUCGAAACACUUUUGAAGGC- 5'

5' -UCGGACCAGGCUUCAUUCCCC_ 3'

1111111111111 1 11111
3' - UUAGGCCUGGUCCGAAGUAGGGUUAGU- 5'

CXCR4 m iRNA and target mRNA (H. sapiens)

miR-166 and PHAVOLUTA mRNA (A. thaliana)

URE 8-25 Base pairing with target RNAs distinguishes miRNA

siRNA. (a) miRNAs hybridize imperfectly with their target mRNAs,
essing translation of the mRNA. Nucleotides 2 to 7 of an miRNA
hlighted blue) are the most critical for targeting it to a specific

mRNA. (b) siRNA hybridizes perfectly with its target mRNA, causing
cleavage of the mANA at the position indicated by the red arrow,
triggering its rapid degradation. [Adapted from P. D. Zamore and B. Haley
2005, Science 3 09:151 9.]
Molecular Cell Biology, Lodish, 7th edition, Page 371