1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 13
Chromatin Structure and Its Effects on Transcription
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Chromatin Structure
Eukaryotic genes do not exist naturally as naked DNA, or even as DNA molecules bound only to transcription factors
They are complexed with an equal mass of other proteins to form chromatin
Chromatin is variable and the variations play an enormous role in chromatin structure and in the control of gene expression

3. 13.1 Histones Eukaryotic cells contain 5 kinds of histones
H2A
H2B H3 H4
Histone proteins are not homogenous due to:
Gene reiteration
Posttranslational modification

4. Properties of Histones
Abundant proteins whose mass in nuclei nearly equals that of DNA
Pronounced positive charge at neutral pH
Most are well-conserved from one species to another
Not single copy genes, repeated many times
Some copies are identical
Others are quite different
H4 has only had 2 variants ever reported

5. 13.2 Nucleosomes
Chromosomes are long, thin molecules that will tangle if not carefully folded
Folding occurs in several ways
First order of folding is the nucleosome, which have a core of histones, around which DNA winds
X-ray diffraction has shown strong repeats of structure at 100Å intervals
This spacing approximates the nucleosome spaced at 110Å intervals

6. Histones in the Nucleosome
Chemical cross-linking in solution:
H3 to H4
H2A to H2B
H3 and H4 exist as a tetramer (H3-H4)2
Chromatin is composed of roughly equal masses of DNA and histones
Corresponds to 1 histone octamer per 200 bp of DNA
Octamer composed of:
2 each H2A, H2B, H3, H4
1 each H1

7. H1 and Chromatin
Treatment of chromatin with trypsin or high salt buffer removes histone H1
This treatment leaves chromatin looking like “beads-on-a-string”
The beads named nucleosomes
Core histones form a ball with DNA wrapped around the outside
DNA on outside minimizes amount of DNA bending
H1 also lies on the outside of the nucleosome

8. Nucleosome Structure Central (H3-H4)2 core attached to H2A-H2B dimers
Grooves on surface define a left-hand helical ramp – a path for DNA winding
DNA winds almost twice around the histone core condensing DNA length by 6- to 7-X
Core histones contain a histone fold:
3 a-helices linked by 2 loops
Extended tail of abut 28% of core histone mass
Tails are unstructured

9. Crystal Structure of a Nucleosomal Core Particle

10. The 30-nm Fiber
Second order of chromatin folding produces a fiber 30 nm in diameter
The string of nucleosomes condenses to form the 30-nm fiber in a solution of increasing ionic strength
This condensation results in another six- to seven-fold condensation of the nucleosome itself
Four nucleosomes condensing into the 30-nm fiber form a zig-zag structure

11. Models for the 30-nm Fiber
The solenoid and the two-start double helix model each have experimental support
A technique called single-molecule force spectroscopy was employed to answer the question, ‘which model is correct?’
Results suggested that most of the chromatin in a cell (presumably inactive) adopts a solenoid shape while a minor fraction (potentially active) forms a 30-nm fiber according to the two-start double helix

12. Higher Order Chromatin Folding
30-nm fibers account for most of chromatin in a typical interphase nucleus
Further folding is required in structures such as the mitotic chromosomes
Model favored for such higher order folding is a series of radial loops
Source: Adapted from Marsden, M.P.F. and U.K. Laemmli, Metaphase chromosome structure: Evidence of a radial loop model. Cell 17:856, 1979.

13. Relaxing Supercoiling in Chromatin Loops
When histones are removed, 30-nm fibers and nucleosomes disappear
Leaves supercoiled DNA duplex
Helical turns are superhelices, not ordinary double helix
DNA is nicked to relax

14. 13.3 Chromatin Structure and Gene Activity
Histones, especially H1, have a repressive effect on gene activity in vitro
Histones play a predominant role as regulators of genetic activity and are not just purely structural
The regulatory functions of histones have recently been elucidated

15. Effects of Histones on Transcription of Class II Genes
Core histones assemble nucleosome cores on naked DNA
Transcription of reconstituted chromatin with an average of 1 nucleosome / 200 bp DNA exhibits 75% repression relative to naked DNA
Remaining 25% is due to promoter sites not covered by nucleosome cores

16. Histone H1 and Transcription
Histone H1 causes further repression of template activity, in addition to that of core histones
H1 repression can be counteracted by transcription factors
Sp1 and GAL4 act as both:
Antirepressors preventing histone repressions
Transcription activators
GAGA factor:
Binds to GA-rich sequences in the Krüppel promoter
An antirepressor – preventing repression by histones

17. A Model of Transcriptional Activation

18. Nucleosome Positioning
Model of activation and antirepression asserts that transcription factors can cause antirepression by:
Removing nucleosomes that obscure the promoter
Preventing initial nucleosome binding to the promoter
Both actions are forms of nucleosome positioning – activators force nucleosomes to take up positions around, not within, promoters

19. Nucleosome-Free Zones
Nucleosome positioning would result in nucleosome-free zones in the control regions of active genes
Assessment in SV40 DNA, a circular minichromosome, was performed to determine the existence of nucleosome-free zones - with the use of restriction sites it was found that the late control region is nucleosome free

20. Detecting DNase-Hypersensitive Regions
Active genes tend to have DNase-hypersensitive control regions
Part of this hypersensitivity is due to absence of nucleosomes

21. Histone Acetylation
Histone acetylation occurs in both cytoplasm and nucleus
Cytoplasmic acetylation carried out by HAT B (histone acetyltransferase, HAT)
Prepares histones for incorporation into nucleosomes
Acetyl groups later removed in nucleus
Nuclear acetylation of core histone N-terminal tails
Catalyzed by HAT A
Correlates with transcription activation
Coactivators of HAT A found which may allow loosening of association between nucleosomes and gene’s control region
Attracts bromodomain proteins, essential for transcription

22. Histone Deacetylation
Transcription repressors bind to DNA sites and interact with corepressors which in turn bind to histone deacetylases
Repressors
Mad-Max
Corepressors
NCoR/SMRT
SIN3
Histone deacetylases - HDAC1 and 2

23. Model for participation of HDAC in transcription repression
Assembly of complex brings histone deacetylases close to nucleosomes
Deacetylation of core histones allows
Histone basic tails to bind strongly to DNA, histones in neighboring nucleosomes
This inhibits transcription

24. Model for Activation and Repression

25. Chromatin Remodeling
Activation of many eukaryotic genes requires chromatin remodeling
Several protein complexes carry this out
All have ATPase harvesting energy from ATP hydrolysis for use in remodeling
Remodeling complexes are distinguished by ATPase component

26. Remodeling Complexes SWI/SNF ISWI In mammals, has BRG1 as ATPase
9-12 BRG1-associated factors (BAFs)
A highly conserved BAF is called BAF 155 or 170
Has a SANT domain responsible for histone binding
This helps SWI/SNF bind nucleosomes
ISWI
Have a SANT domain
Also have SLIDE domain involved in DNA binding

27. Models for SWI/SNF Chromatin Remodeling

28. Mechanism of Chromatin Remodeling
Mechanism of chromatin remodeling involves:
Mobilization of nucleosomes
Loosening of association between DNA and core histones
Catalyzed remodeling of nucleosomes involves formation of distinct conformations of nucleosomal DNA/core histones when contrasted with:
Uncatalyzed DNA exposure in nucleosomes
Simple nucleosome sliding along a DNA stretch

29. Remodeling in Yeast HO Gene Activation
Chromatin immunoprecipitation (ChIP) can reveal the order of binding of factors to a gene during activation
As HO gene is activated:
First factor to bind is Swi5
Followed by SWI/SNF and SAGA containing HAT Gcn5p
Next general transcription factors and other proteins bind
Chromatin remodeling is among the first steps in activation of this gene
Order could be different in other genes

30. Remodeling in the Human IFN-b Gene: The Histone Code
The combination of histone modifications on a given nucleosome near a gene’s control region affects efficiency of that gene’s transcription
This code is epigenetic, not affecting the base sequence of DNA itself
Activators in the IFN-b enhanceosome can recruit a HAT (GCN5)
HAT acetylates some Lys on H3 and H4 in a nucleosome at the promoter
Protein kinase phosphorylates Ser on H3
This permits acetylation of another Lys on H3

31. Remodeling in the Human IFN-b Gene: TF Binding
Remodeling allows TFIID to bind 2 acetylated lysines in the nucleosome through the dual bromodomain in TAF1
TFIID binding
Bends the DNA
Moves remodeled nucleosome aside
Paves the way for transcription to begin

32. Heterochromatin
Euchromatin: relatively extended and open chromatin that is potentially active
Heterochromatin: very condensed with its DNA inaccessible
Microscopically appears as clumps in higher eukaryotes
Repressive character able to silence genes as much as 3 kb away

33. Heterochromatin and Silencing
Formation at the tips of yeast chromosomes (telomeres) with silencing of the genes is the telomere position effect (TPE)
Depends on binding of proteins
RAP1 to telomeric DNA
Recruitment of proteins in this order:
SIR3
SIR4
SIR2

34. SIR Proteins
Heterochromatin at other locations in chromosome also depends on the SIR proteins
SIR3 and SIR4 interact directly with histones H3 and H4 in nucleosomes
Acetylation of Lys 16 on H4 in nucleosomes prevents interaction with SIR3
Blocks heterochromatin formation
Histone acetylation also works in this way to promote gene activity

35. Histone Methylation
Methylation of Lys 9 in N-terminal tail of H3 attracts HP1
This recruits a histone methyltransferase
Methylates Lys 9 on a neighboring nucleosome
Propagates the repressed, heterochromatic state
Methylation of Lys and Arg side chains in core histones can have either repressive or activating effects

36. Histone Methylation
Methylation of Lys 4 in N-terminal tail of H3 is generally tri-methylated (H3K4Me3) and is usually associated with the 5’-end of an active gene
This modification appears to be a sign of transcription initiation
Genome-wide ChIP analysis suggests that this may also play a role in controlling gene expression by controlling the re-starting of paused RNA polymerases

37. Summary
Histone modifications can affect gene activity by two mechanisms:
1. By altering the way histone tails interact with DNA and with histone tails in neighboring nucleosomes, and thereby altering nucleosome cross-linking
2. By attracting proteins that can affect chromatin structure and activity

38. Modification Combinations
Methylations occur in a given nucleosome in combination with other histone modifications:
Acetylations
Phosphorylations
Ubiquitylations
Each particular combination can send a different message to the cell about activation or repression of transcription
One histone modification can also influence other, nearby modifications

39. Nucleosomes and Transcription Elongation
An important transcription elongation facilitator is FACT (facilitates chromatin transcription)
Composed of 2 subunits:
Spt16
Binds to H2A-H2B dimers
Has acid-rich C-terminus essential for these nucleosome remodeling activities
SSRP1 binds to H3-H4 tetramers

40. Nucleosomes and Transcription Elongation
FACT facilitates transcription through a nucleosome by promoting loss of at least one H2A-H2B dimer from the nucleosome
Also acts as a histone chaperone promoting re-addition of H2A-H2B dimer to a nucleosome that has lost such a dimer