1. Heterochromatin Formation - It’s all about silencing!
Shhh!
As discussed in our prior lecture, eukaryotic genomes are very large, and most of the DNA does not code for proteins. The DNA is packaged up first into a nucleosome array, and then into ‘higher order’ structures. Heterochromatin (more condensed) and euchromatin (less condensed) can be observed visually, and appear to correlate with the activity state, with heterochromatin formation promoting silencing.
Sarah C. R. Elgin Bio 4342/434W Copyright 2017, Washington University in St. Louis

2. What determines phenotypes? It is not just your DNA….
Environment (diet)
(black bars = folate)
Phenotype
Epigenetics ?
Genotype
Slide: Nicole Riddle, Washington University in St Louis.
Source: RA Waterland, RL Jirtle (2003) Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 23: 5293 – 300.
Notes: Environment can have a big impact on phenotype. Data is shown here for the agouti mouse – the yellow coat color is due to an active transposable element (TE) driving a transcription factor in an incorrect tissue; if you feed a methyl donor (ie folate) to the mother mouse, you can shift the phenotype back to wildtype (brown), presumably due to silencing the TE..
Development
From N. Riddle
(Waterland and Jirtle 2003)

3. A tale of two mice – littermates (inbred line)
Yellow mouse
High risk of cancer, diabetes, obesity;
Reduced lifespan
Pseudo-agouti mouse
Low risk of cancer, diabetes, obesity;
Prolonged lifespan
Observation – sibs from the same litter can look very different, and have very different health outcomes. The proportion of the litter that shows the healthier Agouti phenotype can be increased by supplementing the mother’s diet with folate, providing the substrate for methylation of DNA and histones..
Maternal supplements with zinc, methionine, betaine, choline, folate, B12
Slide prepared by T Wang

4. Methylation and diet: impact on the agouti locus
Mothers fed BPA (soaks up methyl)  coats shift toward yellow (higher gene expression)
Mothers fed folate (methyl donor)  shift toward pseudoagouti
Trait stable at least to the following generation (your grandparents diet could effect your epigenetics!)
The agouti locus is the consequence of a transposable element insertion that drives inappropriate expression of a transcription factor. DNA and histone methylation promote silencing of this element, returning the gene to its normal expression pattern, producing a fairly normal mouse. Disruptors of methyl metabolism in the mother’s diet, such as BPA (which was used in plastic bottles), can result in a higher proportion of affected offspring.

5. What is epigenetics?
Study of gene expression states that are heritable, through mitosis and/or meiosis, but that are independent of DNA sequence changes.
How is this accomplished?
DNA modification – 5mC
Nuclear localization
Chromatin structure
Epigenomics is study of heritable states of gene expression on the level of the whole genome. A major determinant of epigenetics is chromatin structure. In this lecture we will look at the heterochromatic state, chromatin packaging that leads to stable inheritance of silencing.
(Brickner et al 2007)
from N. Riddle 5

6. Packaging large genomes:
First step - packaging in a nucleosome array
Second - differential packaging into heterochromatin & euchromatin
DNA
Chromatin
Histone protein core
DNA does not exist as a free molecule in the nucleus. It is packaged into chromatin which is defined as DNA plus its associated proteins. The basic subunit of chromatin is the nucleosome, which is 146 bp of DNA wrapped around a histone octamer core. Specific residues in the histones’ amino-terminal tails are enzymatically modified by acetylation, methylation, and phosphorylation. These modifications influence chromatin packaging and the level of gene activity in different regions of the genome.
Chromosome
(metaphase)
Lodish et.al., Molecular Cell Biology, 4th Edition
Felsenfeld et al. Nature 2003, 421: 448

7. DNA packaging domains Euchromatin Heterochromatin Less condensed
Chromosome arms
Unique sequences;
gene rich
Replicated throughout S
Recombination during meiosis
Heterochromatin
Highly condensed
Centromeres and telomeres
Repetitious sequences;
gene poor
Replicated in late S
No meiotic recombination
Slide: Gabriella Farkas.
Over time, characterization of euchromatin / heterochromatin expanded to the collection of properties given on the slide. During the last 20 years we have begun to define heterochromatin in biochemical terms; we will discuss several of the key experiments here.
Transcriptional activators
Hyper-acetylated histone tail
Heterochromatin Protein 1 complex
Hypo-acetylated histone tail; methylated H3/K9

8. The histone N-terminal tails are unstructured and accessible for modification
The complete histone octamer in the absence of DNA.
The view is down the superhelix axis.
Color code:
H2A
H2B H3 H4
Posttranslational modification of the histones is a key determinant of the chromatin packaging state.. All of the core histones have an unstructured N-terminal tail of about 30 amino acids which is the site of extensive post-translational modification. These modifications influence the protein-DNA interaction, and serve as signals for docking other chromosomal proteins.
Rhodes, Nature (1997) 389,

9. The core histone tails are extensively modified
Modifications include phosphorylation of serine, threonine or tyrosine residues, acetylation or methylation of lysine, methylation of arginine, ubiquitylation of lysines, ADP- ribosylation of lysines, and many others. We observed that such modifications occurred in patterns that were cell-type specific long before we understood the significance of these changes. We now know that the impact depends on the exact amino acid modified.
All of these modifications are reversible – the modification can be added or removed. We have identified a large set of nuclear proteins responsible for these enzymatic activities.
Histone modifications are reversible-
Examples: HAT / HDAC
HMT / KDM
Allis et al 2007 Epigenetics

10. Role of acetylation of histone tails in yeast transcription control Gcn5 is a histone acetyltransferase!
David Allis
The break-through came in 1996, when David Allis purified a histone acetyl transferase (HAT), and showed that this enzyme is Gcn5, a known positive transcription factor. We find that gene activation is associated with high levels of histone acetylation; thus positive transcription factors are those that anchor HAT’s at specific loci. Conversely, histone deacetylases (HDAC’s) are repressors of gene expression. An example is Rpd3. Acetylation of the lysines in the histone tails neutralizes the positive charges, and presumably decreases binding to the DNA, facilitating nucleosome displacement to allow transcription.
Lodish et.al., Molecular Cell Biology, 4th Ed

11. Specific mutations in the core histones have specific impacts on gene regulation
Mike Grunstein
Silencing at HML, HMR & telo
Repression of basal transcription
It is difficult to generate mutations in the histones, because in most eukaryotes these genes are present in multiple copies. However, it can be done in yeast (S.c.), and Michael Grunstein and colleagues demonstrated that different mutations had different impacts on gene regulation – some domains are needed to repress basal transcription, others to establish heterochromatin domains, with concomitant silencing. Specific point mutations (for example, substituting an arginine for a lysine at H3K9) also disrupted regulation – we now know this disrupted critical patterns of histone modification. These experiments proved that nucleosomes are not just “cellophane wrapping”, but play an active role in gene regulation.
Inter-nucleosomal contact
Silencing at HML, HMR & telo
Durrin et al 1991 Cell 65:1023
Repression of basal transctiption

12. Histone modification patterns can be inherited
Ragunathan et al 2015 Science 348: 90.
Patterns of histone modification are generally maintained through somatic cell division unless specific signals are introduced to alter the modifications at a specific site. Here silencing of a reporter gene is induced by tethering Clr4, resulting in H3K9 methylation; this state is maintained after the Clr4 is released. Maintenance of silencing is lost in ~10 generations in wild type yeast (S pombe), but is maintained >50 generations if the putative demethylase Epi1 is mutated (Ragunathan, Jih, & Moazed, 2015 Science 348:90).

13. Model system – fruit flies!
Inexpensive and easy to culture, short life cycle, easily visible phenotypes: good genetic approaches
Biochemical approaches
Polytene chromosomes:
excellent cytology
Simple genome, good reference sequence
PEV – reporter for gene silencing, heterochromatin formation
Metazoan useful for behavioral, developmental. and
human disease research
Mary Lou Pardue, MIT
Slide: Polytene chromosome showing in situ hybridization from Mary Lou Pardue, MIT.
Notes: The fruit fly is a great model organism for epigenetic studies: It’s short life span and simple genome (4 chromosomes) facilitates genetic studies; phenotypes easy to score; biochemical approaches are possible; the salivary gland polytene chromosomes provide a pretty high resolution visualization of the chromosomes, allowing studies of the distribution of DNA sequences (in situ hybridization) and proteins (immunofluorescent staining). We can use a reporter gene (white) to monitor the chromatin environment; when this gene is in its normal euchromatic environment, it is fully expressed giving a red eye, but when moved to heterochromatin (by rearrangement or transposition) it is silenced in some of the cells in which it should be active, resulting in variegation. Many of the deleterious mutations that cause health problems in humans can be modeled in the fruit fly, including Fragile X. (Flies are relevant!)
euchromatin heterochromatin
expressed silenced
GEP 2012 SCRE

14. Distribution of Heterochromatin Protein 1 Identified by screening monoclonal antibodies against tight-binding nuclear proteins
James et al, 1989, Europ J Cell Bio 50: 170
The D. melanogaster karyotype is shown; about 1/3 is heterochromatic (black domains), including all of the Y chromosome and all of the 4th chromosome. Polytene chromosomes form in the large cells of the larvae, which grow without division; endoreduplication of the euchromatic arms occurs (10 times in the salivary gland), but no mitosis – the chromatin strands remain juxtaposed in good order. The pericentric heterochromatin is not duplicated, and the chromosome arms fuse in a common chromocenter, giving the 5-arm pattern.
To look for proteins preferentially associated with heterochromatin, we prepared monoclonal antibodies against proteins that bind tightly in the nucleus, and screened by using the antibodies for immunofluorescent staining of the polytene chromosomes. Heterochromatin Protein I (HP1) is preferentially associated with the chromocenter, small fourth chromosome, telomeres, and a few sites in the chromosome arms. This suggests a role in heterochromatin formation. C C
Phase
HP1

15. Trans-acting modifiers of PEV:
Position Effect Variegation in Drosophila provides a functional assay for putative chromatin proteins
white
Wild Type
Inversion
PEV is the variegated expression pattern that results when a gene is moved by rearrangement or insertion in or near heterochromatin,as demonstrated by this white inversion giving rise to a mottled white eye. The gene is being silenced in some of the cells in which it is normally active. Given a variegating phenotype, one can screen for second site mutations that suppress or enhance that phenotype. Such studies have lead to identification of proteins involved in chromatin structure formation.
Trans-acting modifiers of PEV:
Su(var):
E(var):
Elgin lab

16. Heterochromatin-associated gene silencing is dependent on HP1
Mutations in
Source: Eissenberg et al (1990) Proc Natl Acad Sci USA 87: 9923 – 9927.
Notes: This experiment provides key genetic evidence that HP1 plays a role in the mechanisms of silencing; loss of HP1 (either a truncation mutant or a mutation in the coding domain) has this dominant phenotype, a loss of silencing in a reporter showing a variegating phenotype. The original mutation was recovered by T Grigliatti in a screen for mutations with this phenotype – suppression of variegation. Eissenberg et al then sequenced the DNA from these flies, proving that the mutation is in the gene coding for HP1a. The homozygous mutation is lethal, showing that HP1 is an essential protein.
gene for HP1a
Mutations recovered by T Grigliatti as suppressors of PEV.
Dosage dependent response.
Eissenberg et al, 1990, PNAS 87: 9923

17. Using a white transgene to sample chromatin environments
inject
transposon
P[white+]
white67c23
In flies we can use a P transposable element to put fragments of DNA back into the genome. A P element carrying the white gene can restore eye color; if we mobilize the P element, it will transpose at random. In most cases a red-eyed fly is recovered, indicating full expression of the gene, but in 1% of the cases we recovered flies with a variegating eye phenotype, suggesting insertion into heterochromatin.
Next slide summarizes what is known about the gene for HP1 and the HP1 gene
mobilize P element
by crossing to stock
with transposase
insertion into
euchromatin
(99%)
heterochromatin
( 1%)
Wallrath & Elgin, 1995

18. Transposition of a P element reporter allows sampling of heterochromatin domainsX 2L 3L 2R 3R 4
Silenced 1%
Source: L Wallrath & SCR Elgin (1995) Genes Dev 9: 1263 – 1277.
Notes: The P element construct used is diagrammed above. The hsp70-driven white gene gives us the eye phenotypic marker; the hsp26 gene marked with plant DNA has been used for the chromatin structure studies that follow. In situ hybridization showed that the variegating P element inserts all map to the pericentric heterochromatin, telomeres, Y, or fourth chromosome, all previously known heterochromatic domains (not to scale).
Active
99%
And the Y chromosome
Wallrath and Elgin, 1995

19. Assessing chromatin structure- same gene, different environments Analysis: MNase cuts between nucleosomes; DNase I and restriction enzymes cut hypersensitive sites
Slide: Gabriella Farkas
Now we can ask what happens to a gene that is normally in euchromatin when it is placed in a heterochromatic environment. It is being silenced inappropriately – is this due to a change in chromatin packaging at the nucleosome level, or the consequence of some higher order packaging? We can carry out chromatin mapping studies using nucleases, as we did earlier to examine the chromatin structure of the native gene.
What happens to the same gene in heterochromatin?
Elgin lab

20. The nucleosome array associated with the silenced transgene in heterochromatin shows more regular spacing and sharper MNase cleavage sites
In line 39C-X, the transgene is in euchromatin (full red eye); in line HS-2, the transgene is in heterochromatin (severe PEV – only a few facets of red). In the latter case we can score up to a 9- to 10-mer on MNase digestion, in contrast to 39C-X, where we can score only a 5- to 6-mer. This indicates that the nucleosome array in heterochromatin has much more regular spacing than that found in euchromatin..
Sun et al, 2001, Mol Cell Biol 21: 2867

21. Loss of accessibility in the heterochromatic hsp26 transgene is reversed in an HP1 mutant background
Cryderman et al 1999, Nucleic Acids Res 27: 3364
Digesting the chromatin with Xba!, we see loss of accessibility at the regulatory region (loss of the HS site; see loss of band at the proximal XbaI site); this is partially restored by crossing in a mutation in HP1a.

22. Assessing chromatin structure- same gene, different environments Analysis based on nuclease digestion of chromatin
Slide: Gabriella Farkas
Source: Based on experiments in FL Sun, M Cuaycong, SCR Elgin (2001) Mol Cell Biol 21: 2867 – 2879, and D Cryderman, H Tang, C Bell, DS Gilmour, LL Wallrath (1999) Nucleic Acids Res 27:
Notes: Now we can sum up what happens to a gene that is normally in euchromatin when it is placed in a heterochromatic environment. It is being silenced inappropriately – is this due to a change in chromatin packaging? We can carry out chromatin mapping studies using nucleases to examine the chromatin structure of the native gene. Digesting with micrococcal nuclease will reveal the nucleosome pattern, while digesting with DNase I will show where DNase hypersensitive sites – nucleosome free regions – are present. We find that when a gene is moved from a euchromatic to a heterochromatic domain, the nucleosome array becomes much more regular, with the loss of DH sites. The absence of DH sites most likely contributes to the observed silencing.
The euchromatic hsp26 transgene: - DH sites: accessibility at the TSS, upstream regulatory region irregular nucleosome array
The heterochromatic hsp26 transgene:
- loss of DH sites
- regular nucleosome array

23. HP1 sequence from Drosophila, mouse, human and mealy bug identifies conserved chromo & chromo shadow domains
Clark and Elgin, 1992 Nucleic Acids Res. 20:6067
Heterochromatin protein 1 is highly conserved – it is found in many fungi and in most animals checked to date. There are two conserved domains, the N terminal chromo domain (variations also found in several other chromosomal proteins) and the C-terminal chromo shadow domain. In the above comparison, gold residues are identical and orange are conserved.
HP1 from mammals can rescue mutations in flies and yeast!

24. HP1 interacts with both the modified histone H3K9me2/3 and the modifying enzyme
Chromo
Shadow
SU(VAR)3-9
HMT
Sources: Tschiersch et al (1994) EMBO J 13: 3822 – 3831; Lachner et al (2001) Nature 410: 116 – 120; Bannister et al (2001) Nature 410: 120 – 124.
Notes: How might heterochromatin be assembled? Work in mammalian cells showed that the chromo domain binds H3 iff it is methylated at K9 (Jenuwein, Kouzarides). The shadow domain generates HP1 dimers, forming a platform that binds several other chromosomal proteins. Among these is SUV3-9; Jenuwein showed that the human homolg has an enzymatic activity that methylates histone tails at lysine 9. Thus HP1 can both recognize a histone modification (H3K9 methylation), and promote that modification by interacting with the enzyme. This suggests a mechanism of recruit/methyl/recruit that could be used both in maintenance and propagation of heterochromatin. Note that because of its dimerization through the CSD, HP1 could work as a bifunctional crosslinker.
Histone 3
methyl-Lys9
H3 K9
methylation
[(SU(VAR)3-9 identified in screen by Reuter;
H3 interaction first shown from work in mammals – Jenuwein, Kouzarides;
demonstrated in flies by Imhof.]

25. Model for spreading of heterochromatin (and epigenetic inheritance): create a system that can recognize a histone modification mark and can generate that same mark.
Slide: Gabriella Farkas.
Animation of spreading of heterochromatin: HP1 recognizes H3K9me3 and binds; it then recruits SU(VAR)3-9, which can modify the next nucleosome accordingly.
Elgin summary

26. PEV transition: loss of euchromatin marks
Heterochromatin
Of course it is not that simple! Over 100 Su(var) mutations have been reported – over 100 proteins needed to generate / maintain silent chromatin! Some of these are needed to remove the “active marks” – for example, dLSD1, the enzyme that demethylates H3K4.
Reuter & Elgin, 2006, Epigenetics
-and probably others!

27. Establishing silencing: gain of heterochromatin marks
Euchromatin
Heterochromatin
And some are required to add the “silencing marks”, as well as being a structural component of the heterochromatin. Thus a cascade of events is required to shift a locus from a euchromatic, activatable status to a heterochromatic, silenced status. Remember – these processes are reversible – but seem to be fairly stable changes during differentiation, as a specific cell type acquires a limited set of active genes.
Reuter & Elgin, 2006, Epigenetics
And no doubt, additional proteins are required!

28. Three different H3K9 methyltransferases
Three different H3K9 methyltransferases! Mutations in these have different impacts on different heterochromatin compartments
It gets even more complicated! There are at least 3 different H3K9 HMT’s, and they seem to impact different heterochromatic domains differently. SETDB histone methyltransferase is a strong suppressor of variegation (pericentric chromatin and fourth chromosome). Haplo-insufficiency for SU(VAR)3-9 methyltransferase suppresses pericentric silencing but has a weak enhancement on the fourth chromosome. Evidence suggests that SU(VAR)3-9 is only active in the pericentric heterochromatin; thus when it fails, there is excess HP1 available which can bind to the fourth chromosome H3K9me3 generated by SETDB.
39C-12
118E-10
B Brower-Toland et al, 2008

29. Feedback loops to maintain silencing
There is variation among the eukaryotes. Yeast S cereviseae uses the Sir proteins to form a silencing complex. Most fungi and all animals checked to date use the HP1 – H3K9me2/3 system; many also use 5’mC as a mark for silencing. Plants use 5’mC, but their HP1 is rather different, and probably does not function in the same way. But all eukaryotes have a mechanism for silencing repetitious DNA in their genomes.
The various components required for silencing can stimulate each other – for example the H3K9me2/3 mark can bind proteins involved in DNA methylation. These self- and cross-reinforcing loops may be essential to maintain heterochromatin – and suppress expression of transposable elements and the like.
Richards & Elgin, 2002

30. Chromatin Immuno-precipitation – ChIP 
mapping chromatin structure
1. Crosslink proteins to DNA
2. Isolate chromatin and sonicate
qPCR
3. Incubate with antibody
ChIP-chip
Slide from N Riddle.
More recently we have looked at genome-wide patterns of histone modification using ChIP (chromatin immunoprecipitation) experiments. The chromatin is cross-linked (generally using formaldehyde), and then sheared into small fragments of a few hundred base pairs. The fragments are incubated with an antibody specific for a particular histone modification (for example, H3K9me3) or chromosomal protein; all of the chromatin fragments that bind the antibody are then collected. The cross-linking is reversed, and the selected population of DNA identified, either by qPCR (to look at a particular locus), or by using an oligonucleotide array to look at the whole unique genome, or by re-sequencing the DNA, and the identified population of DNA fragments is mapped back onto the sequenced genome. This map shows the distribution of the particular histone modification or chromosomal protein.
4. Isolate AB/chromatin complexes
ChIP-seq
5. Isolate DNA from complexes
N Riddle

31. ChIP-chip mapping of chromosomal proteins shows distinct differences between heterochromatin and euchromatin
Centromere
S2 cells
Euchromatin
Heterochromatin
HP1
Su(var)3-9
H3K9me2
H3K9me3
genes
Coming back to Drosophila, we can see a consistent transition at the base of the euchromatic chromatin arms, as we move into the pericentric heterochromatin. The solid pink bars indicate a 95% probability of enrichment of that mark. Note the concomitant shift in HP1 and H3K9me2/3 density, along with Su(var)3-9, a histone methyl transferase (HMT).
Euchromatin / heterochromatin transition point from Flybase
Riddle et al, 2011, Genome Research 21:

32. Define chromatin states by K-means clustering (using enrichment values for 1 kb chromatin fragments)
H3K4me3 
H3K9me3 
Slide: Nicole Riddle, Washington University in St Louis
Notes: The average value of enrichment for a histone modification mark for each 1 Kb fragment of chromatin is obtained, allowing that fragment to be plotted accordingly; in this example two modifications are used. A reiterative process is used to optimize the clustering of the data points. This approach, along with more sophisticated techniques, was used to derive the 9-state chromatin model described by Kharchenko et al (2011).
Assign each data point to closest mean.
The centroid of each cluster is the new mean.
Repeat the last two steps.
Select k means as starting points.
Slide by N Riddle

33. Karchenko et al, 2011, Nature 471: 480-5.
Clustering chromatin fragments according to histone modification marks suggests nine major chromatin states
H3K9me2
H3K9me3
TSS
We have repeated the ChIP experiment with 18 histone modification marks, and find that we can sort the chromatin fragments into 9 types. State 1 is associated with Transcription Start Sites, 2 with transcript elongation, 3-4 with enhancers and regulatory regions; 5 is specific to the male X chromosome, required for dosage compensation; 6 is the Polycomb state, maintaining silencing at certain developmentally regulated genes; 7 is constitutive heterochromatin, and 8 may be facultative heterochromatin; and 9 has no distinguishing features. Note green marks are associated with activation, red with silencing.
X chr
Polycomb
Hetero-chromatin
Similar to hetero-chromatin
S2 and BG3 cells

34. Histones modifications are critical for setting levels of gene expression.
H3/H4 acetylation associated with the active state
HAT’s are activators; HDAC’s are silencers
H4K16 acetylation associated with dosage compensation
H3K9 di- and tri-methylation are found in constitutive silent domains (centromeres, telomeres)
H3K27 tri-methylation is associated with developmentally regulated silencing (Pc complex)
H3K4 di- and tri-methylation occurs at the 5’ end of active genes, and H3K36me3 over the gene body
Chromatin modification states switch to achieve gene regulation, controlling frequency of transcription.
While we do not have time to review all of the experimental evidence, these conclusions hold in most cases.
SCR Elgin

35. Chromatin states identify large-scale genomic domains
1Mb
Mapping the nine chromatin states onto the Drosophila melanogaster genome. Note the heterochromatic regions around the centromeres (right) and entire fourth (state 7); heterochromatin-like state 8 is distributed within the chromosome arms in a cell-type specific pattern. State 6 is almost entirely on the male X chromosome, and is associated with dosage compensation. States 1-4 are associated with active genes.
Mapping the nine chromatin states onto the Drosophila genome (Bg3 cells).
Karchenko et al 2011 Nature 471: 480

36. Summing up….. Much of the genome is packaged in heterochromatin.
Heterochromatic structure differs-
- in a loss of accessibility, loss of 5’ HS sites;
- in having increased nucleosome stability;
- in having a more ordered nucleosome array.
Heterochromatin is marked by-
- hypoacetylated H4, H3K9me2/3, (methylated DNA)
Epigenetic inheritance and spreading are achieved by
- a protein (HP1) or protein complex that interacts with a modified histone and with the modifying enzyme.
The genome can be subdivided into different states based on histone modifications –
- specific patterns associated with transcription start sites, enhancers, transcript elongation;
- two types of silencing domains: H3K27me3 (Polycomb)
and H3K9me2/3 (HP1a)
- and more!
SCR Elgin