Epigenetics- Transcription regulation of gene expression
1.
2. • First Introduced;1942 (Conrad Waddington)
• “Above genetics”
• Any process that alters gene function without changing
the DNA sequence, and is heritable
Study of heritable changes which affect gene function without
modifying the DNA sequence
3. DNA (genome) -hardware of computer,
Epigenetics (epigenome) -“software”.
• Every cell in our body contains exactly the same genes, but inside the individual
cells some genes are activated while others are silenced.
• When genes are active they are capable of being translated into proteins. When
genes are silent, they are inaccessible for translation into proteins.
4. • Nucleosomes constitute the basic repeating
subunit of chromatin.
• Nucleosome = Core particle + linker DNA (10–60 bp) + linker histone (H1).
Core particle = Histone octamer (two copies each of four core histone
proteins) + 146 bp DNA
5. Small (11-15 kDa), very basic proteins
Large proportion of the positively charged (basic) amino acids.
DNA is negatively charged - phosphate groups on its backbone.
Strong attraction and high binding affinity between histones and DNA
Functions;
Compact DNA and regulate chromatin, therefore impacting gene regulation.
Eukaryotic cells commonly contain five abundant histones: H1, H2A, H2B,H3, and H4.
Histones H2A, H2B, H3, and H4 are the core histones, and two copies of each of
these histones form the protein core around which Nucleosomal DNA is wrapped.
Histone H1 binds to the linker DNA (The DNA between each nucleosome is called
linker DNA. ) - linker histone
6. Nucleosome is composed of a
core particle plus histone H1
and linker DNA.
The nucleosome core particle is
composed of a histone octamer
and about 146 bp of DNA.
7. Histones H2A, H2B, H3, and H4
are the core histones, and two
copies of each of these histones
form the octamer around which
Nucleosomal DNA is wrapped.
Histone H1 binds to the linker
DNA (The DNA between each
nucleosome is called linker DNA. )
- linker histone
8. Histone-fold domain, mediates assembly.
The core histones each have an amino-terminal extension, called a tail-
lacks a defined structure and is accessible within the intact nucleosome.
Sites of extensive posttranslational modifications -alter the function of
individual nucleosomes.
These modifications include phosphorylation, acetylation, and methylation
on serine, lysine, and arginine residues.
The histone fold forms an
extensive protein-protein
interface described as a
‘handshake’ interaction
that directs
heterodimerization of
histones H2A with H2B
and H3 with H4.
10. DNA Methylation
• C5 of the cytosine
• DNMTs.
Occurs exclusively at C that are followed immediately by a G-
~1% of the total genome;
Characteristic; G+C content >50% and minimum size; 200bp
Present at 10 times their average density in certain regions, called CpG
islands.
The human genome contains about 20,000 CpG islands and they usually
include promoters of genes.
• For example, 60% of human protein-coding genes have promoters in
CpG islands and these include all the promoters of the housekeeping
genes—those genes that code for the many proteins that are essential for
cell viability and are constitutively expressed in nearly all cells .
11. DNA methylation prevents transcription via several mechanisms, including
inhibition of transcription factor binding.
12. These regions are unmethylated.
• Methylation of cytosines at CpG dinucleotides recruits methyl-CpG binding
proteins (MeCP1, MeCP2, MBD1, MBD2, MBD3, MBD4)
• Have both a methyl-DNA binding domain (MBD) and a transcription-regulatory
domain (TRD).
• MBD proteins recognize these cytosine residues in the DNA, they bind, then
they go and recruit repressors or co-repressors.
• These co-repressors /suppressor complex usually contain either, HDACs or
HMTs and when these are recruited to the promoter -leads to transcription
repression.
• MeCP2 recruits a transcriptional corepressor complex containing Sin3 and
associated proteins; to methylated CpG islands and results in target gene
transcription repression
13.
14.
15.
16. NA methylation is one of the most important epigenetic modifications [1],
playing key roles in the regulation of gene expression, genomic imprinting, X
chromosome inactivation, and tumorigenesis [2, 3]. In mammals, DNMT1,
DNMT3A and DNMT3B, the generally recognized three types of DNA
methyltransferases (DNMTs), execute the genomic methylation process [4].
These proteins are highly conserved and have similar amino acid sequences.
The N-terminus contains a regulatory domain, which allows DNMTs to anchor
in the nucleus and recognize nucleic acids or nucleoproteins, and the C-
terminus possesses a catalytic domain, which is responsible for the enzymatic
activity [5]. DNMT1, DNMT3A and DNMT3B have different functions in the
methylation process. DNMT1 is required for the maintenance of all
methylation in the genome. During replication, DNMT1 restores the specific
methylation pattern on the daughter strand in accordance with that of the
parental DNA. DNMT3A and DNMT3B are referred to as de
novo methyltransferases, which are responsible for establishing DNA
methylation patterns during embryogenesis and setting up genomic imprints
during germ cell development
17. • Although they are highly expressed in early mammalian embryos, DNMT3A
and DNMT3B decrease in expression over the course of cell differentiation.
These two proteins have distinct functions throughout embryonic
development, showing both spatial and temporal differences. DNMT3A
primarily methylates a set of genes and sequences at the late stage of
embryonic development and especially after birth, whereas DNMT3B
modifies a broader region of genomic sequences in early embryos [2, 6]. Very
recently, one study identified a new de novo DNA methyltransferase
DNMT3C in murine germ cells. DNMT3C exhibits high identity with DNMT3B,
and is specialized at methylating the young retrotransposons [7]. Beside the
above-mentioned enzymes, which are essential for the methylation of
mammalian DNA, the DNMT family also includes two additional members,
DNMT2 and DNMT3L.
• There are two ways in which DNA methylation can control transcription by
inhibition. The first is interference with the interaction of transcription
factors and other DNA-binding proteins (5). The second method of control is
the inhibition of gene expression by recruiting methyl-CpG binding protein
(MBD) (6).
18. • The enzymes that are involved in DNA methylation are called DNA
methyltransferases (DNMTs). They catalyze the transfer of a methyl group from
an S-adenosyl-L-methionine to cytosine residues. The mammalian DNMT family
consists of five members: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3-
like (DNMT3L) (7).
• Only DNMT1, DNMT3A, and DNMT3B possess methyltransferase activity. The
catalytic members of the DNMT family are classified into either de novo DNMT
(DNMT3A and DNMT3B) or maintenance DNMT (DNMT1) groups. De novo
DNMTs are highly expressed in stem cells and are downregulated after the
process of differentiation. This modification consists of the addition of a methyl
group at the cytosine residues of the DNA template. DNMTs enzymes catalyze
either the de novo or maintenance methylation of hemimethylated.
• DNA following DNA replication. They transfer a methyl group from the methyl
donor S-adenosylmethionine (SAM), resulting in 5-methylcytosine (Figure 2). 5-
methylcytosine is recognized by the methyl-CpG binding domain (MBD) of
certain proteins. In mammals, there are five members: MeCP2, MBD1, MBD2,
MBD3, and MBD4. Most of the MBD proteins are located in highly methylated
chromatin regions, which influence silencing of imprinting genes and in
endoparasitic sequences, promoting transcriptional repression and genomic
stability (8).
21. Regulate various biological processes, including transcription, DNA
replication and DNA repair.
22. How PTMs influence chromatin structure???
1. CHROMATIN REMODELLING; Addition of any PTM on histone protein affects
inter/intra-nucleosomal interactions and their binding to DNA by steric hindrance.
Eg; histone acetylation -chromatin relaxation and transcription activation, H4K16ac
inhibits the formation of compact 30 nm fibers and higher order chromatin
structures
2. DOCKING SITES TO RECRUIT OTHER PROTEINS
• Example ; “histone readers,” -specifically bind histone modifications and affects a
defined nuclear process such as transcription, DNA repair and replication, etc.
• Evolutionary conserved chromodomain heterochromatin protein 1 recognize and
gets recruited to H3K9me3 and leads to the formation of compact chromatin which
in turn inhibits the access of the transcriptional machinery.
23. Acetylation
Acetylation at lysine residues- neutralizes positive charge of histones.
Modulate electrostatic histone tail interactions.
Causes unfolding of the chromatin fiber and thereby permits transcription
24. Histone acetyltransferases (HATs) catalyze the direct
transfer of an acetyl group from acetyl-CoA to the ε-
NH+ group of the lysine residues within a
histone.52,53
The bromodomain is an evolutionarily conserved
protein module that functions as an acetyl-lysine
(AcK)-binding domain.
By recognizing regions of acetylated histones and by
recruiting other proteins capable of remodeling
chromatin conformation, bromodomain-containing
proteins are able to reinforce the open conformation
of chromatin where higher levels of histone
acetylation are present.
Many proteins involved in genome regulation have
bromodomains as part of their structures.
25. Many proteins involved in transcriptional factors activation such as transcription
factors, or coactivators, or even the TAFs, possess HAT activity
How are histones Acetylated?
Bromodomains bind to acetylated histones and act as chromatin remodeller
26. HAT family of proteins
I. GNAT family -Gcn5 related N-
acetyltransferases, and
II. MYST family, which consists of
MOZ, Ybf2, Sas2, Tip 60,
III. CBP or p300.
IV. GTFs-TAFII250
V. Nuclear receptors like SRC1 and
ACTR
HDAC family of proteins
The Class I Rpd3-like proteins (HDAC1,
HDAC2, HDAC3, and HDAC8);
The Class II Hda1-like proteins (HDAC4,
HDAC5, HDAC6, HDAC7, HDAC9, and
HDAC10);
The Class III Sir2-like proteins (SIRT1,
SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and
SIRT7); and
The Class IV protein - HDAC11
Non-histone substrates of HDAC; E2F,
p53, Rb, Importin-α, β-catenin etc..
27. Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a multicomponent regulator
of acetylation.[1] It has been found that this complex is highly conserved between different
organisms, such humans, Drosophila, and yeast. This 15 subunit complex has been best
characterized for its histone acetyltransferase activity (HAT)
• The first and foremost observation that a transcription factor actually
possesses a HAT activity came from a transcriptional activator called Gcn5
protein- transcriptional activator in yeast cells.
• The human homolog of the Gcn5p also has HAT activity, -enzymatic activity is
conserved from yeast to humans.
• Yeast Gcn5 was found to exist in at least 2 distinct multiprotein complexes–
Ada and SAGA.
• Free Gcn5 protein, when not present as a multiprotein complex, -can acetylate
only free histones; cannot acetylate histones which are bound to DNA in the
form of chromatin,
• As a part of the SAGA complex, it could actually acetylate histones.
28. Recruitment of a HAT
leading to activation of
transcription
Only the phosphorylated
form of CREB can bind to
CRE and CBP/P300
29. Recruitment of HDAC, can result in a repression of
transcription.
Rpd3
HDAC
Rpd3
HDAC
T3
LBD interacts with a corepressor
complex, which contains a
HDAC .
T3 binding causes conformational change in
LBD ; which can no longer bind to the
corepressor complex.
30. HDAC inhibitors-Anticancer drugs
• They can induce cell cycle arrest; they can cause differentiation of
cells; they induce apoptosis in colon cancer cell lines
Synthetics Natural Products
31. • HDACs are actually over expressed in a number of cancer tissues. For example,
the HDAC 1 is actually over expressed in prostate cancers, gastric and colorectal
cancers. Now, this is always only correlation that tells me that in many of these
cancers, there is a very high HDAC activity. Similarly, the HDAC2 is over expressed
in a number of colorectal and gastric cancers; HDAC3 is over expressed in lung
cancer and several solid tumors, whereas HDAC 8, if you knock down, it inhibits
the cell growth in several human tumor cells. So, these studies clearly told that,
the HDAC, the histone deacetylases, when they are active, they can actually may
be involved in cell proliferation.
• So, what could be happening is that these HDACs may actually be deacetylating
the promoters of many tumor suppressor genes, and therefore, the expression of
these tumor suppressors may be blocked, and as the result, the cell has become
cancerous, and evidence actually comes from here. In the case of HDAC 8, which
is actually expressed in the particular tumor cell line, when we knocked out this
HDAC tumor 8, you can actually inhibit the cell growth of… that means, some of
this tumors promoter genes expressions is getting turned down, and therefore, it
is resulting in the succession of growth, indicating that these HDACs plays a very
important role
33. Three families of enzymes have been identified thus far that catalyze the addition of methyl
groups donated from S-adenosyl methionine4 to histones. The SET domain containing
proteins14 and Dot1 like proteins15have been shown to methylate lysines and members of
the PRMT family have been shown to methylate arginines
Does not alter the charge of the histone protein.
Lysine may be mono-, di- or tri-methylated,
Arginine may be mono-, symmetrically or asymmetrically di-methylated
The multiple methylation states of lysine can alter transcriptional response
Dimethylated state –H3K4me2 - readiness for gene transcription (called permissive
chromatin state), whereas active transcription of the gene always requires the presence of
trimethylated lysine at position 4 (H3K4me3).
So, in addition to lysine 4 and lysine 9, lysine 27 is also plays a very important role in the
regulation of gene expression and chromatin structure.
Methylation of the lysine 27 - transcription repression.
Methylation of lysine 9 or lysine 27 in histone H3 - heterochromatin formation and
transcription repression.
Lysine 27 methylation of H3 histone is catalyzed by a multi molecular complex, PRC.
34. Methylated histones are recognized by proteins, which contain specific domains
called as chromodomains.
Depending upon which kind of lysine is methylated, Chromodomain containing
proteins recruit protein complexes which can either result in transcription activation or
transcription repression.
Chromodomains have the highest affinity for tri-methyllysine, and the lowest for
monomethyllysine.
Eg of Chromodomain containing proteins;
Heterochromatin protein 1, HP1-recognize K9, and
Polycomb protein ,Pc- recognize K27
35. Specific HMT methylate the specific histone residues in histone H3.
H3 K residue Drosophila Humans
K4 Trx MLL
K9 Su(var)3-9 Suv39H1/2
K27 E(Z) EZH2
H3; A R T K Q TA R K S T G G K A P R K A R K S A
4
Me
9 27
MLL
Trx
Suv39H1/2
Su(var)3-9
EZH2
E(Z)
Me Me
Humans
Drosophila
The SET domain is a conserved
catalytic domain present in
HMT
36. Histone methylation is traditionally linked to repression, but it can also lead to
transcriptional activation
For example;
Lysine 4 methylation by a HMT called Set1p -activation of transcription, whereas
Lysine 9 methylation by SUV39H1 can result in repression of transcription.
Set1p is associated wit HAT complex
H3; A R T K Q T A R K S T G G K A P R K A R K S A
4
Me
9
Me Me
27
Set1p
SUV39H1
In heterochromatin, the K9 of H3 was found to
be methylated.
heterochromatinEuchromatin
Repression of transcription
Heterochromatin
Methylation of H3K4 residue of histone H3
impairs methylation at H3K9.
Activation of transcription
Euchromatin
38. TAFII250
Bromodomain
H3; A R T K Q T A R K S T G G K A P
9
Ac
14
Ac
Transcriptional
activation
HP1
Chromodomain
Me
Transcriptional silencing
Heterochromatinization
Histone modification dependent recruitment of proteins
40. •All core histones contain phospho-acceptor sites in their N-terminal domains.
H2A can be phosphorylated on serine 1, H2B on serine 14 and 32, H3 on
serines 10 and 28, and H4 on serine 1.
•Histone phosphorylation is that of H3S10 - involved in the initiation of the
chromosome condensation process that is essential to mitosis.
Phosphorylation of serine 10 on H3 is mediated by at least three kinases;
ribosomal S6 kinase-2 (Rsk2), which is downstream of extracellular signal-
regulated kinase (ERK);
mitogenand stress-activated kinase-1 (Msk1), which is downstream of both
ERK and p38 mitogenactivated kinases (MAPK); and
the aurora kinase family member Ipl1.
Phosphatases remove phosphate groups from histones.
Phosphatases PP1 and PP2A, regulate H3 dephosphorylation
41. Protein domains that recognize phosphorylated amino acids ; SH2, BRCT, WW,
FHA, WD40, 14-3-3 and LRR domains
42. Phosphorylation of tyrosine 41 of histone H3, -carried out by an enzyme
JAK2, and this has implications in hematopoiesis and leukemia.
Phosphorylation of H3 can actually result in the modulation of expression of
genes involved in hematopoiesis,
Decreased phosphorylation of Y41 of H3 results in the expression of an
oncogene called lmo2.
Over expression of lmo2, results in leukemia.
In fact, in some gene therapy trials, SCID is cured by retroviral gene transfer,
found out that this gene actually goes and integrates near lmo2 gene, and
results in activation of this lmo2 gene, and as a result, some of these patients,
which actually underwent gene therapy for severe combined
immunodeficiency syndrome, they develop leukemia
43. •Ubiquitinated through attachment of a ubiquitin to the ε-NH+ group of a lysine.
•Attached to histone lysines via the sequential action of three enzymes,
E1-activating; Ubiquitin is first activated by an ATP-dependent reaction involving a ubiquitin
activating enzyme (E1)
E2-conjugating; followed by its conjugation via a thioester bond to a cysteine residue in a
ubiquitin-conjugating enzyme (E2)
E3-ligating enzymes; In the final enzymatic step, ubiquitin is transferred from the E2
enzyme to a target lysine residue in a particular substrate protein by a ubiquitin-protein
isopeptide ligase.
The enzyme complexes (E1, E2, E3) determine both substrate specificity (i.e., which lysine is
targeted) as well as the degree of ubiquitylation (i.e., either mono- or poly-ubiquitylated)
Polyubiquitination targets proteins for degradation via the 26S proteasome,
monoubiquitination generally acts as a tag that marks the substrate protein to signal
for a particular function
44. •Ubiquitin is a 76 amino acid polypeptide that can be enzymatically coupled
•to all sorts of proteins. Its major function in most cells is undoubtedly the "tagging" of
worn-out or damaged proteins that are destined for degradation
•by the proteasome, but it also has uses in epigenetic genome control.
•The principal targets of ubiquitin are histones
•H2A (lysine 119) and
•H2B (lysine 120),
•although the exact positions of the target amino acids vary between species. The two
lysines indicated above are for mammals, but they will suffice for the time being to
demonstrate the importance of adding ubiquitin to these two histones. Many other
applications of ubiquitination in the cell rely on the addition of multiple ubiquitin units
(polyubiquitination), but histones only ever seem to be conjugated to a single ubiquitin
moiety (monoubiquitination); however, even this was sufficient for ubiquitinated H2A to
be originally considered a unique histone-like chromosomal protein, named A24
because the extra amino acids greatly alter the protein’s molecular weight.
•The functions of H2A and H2B ubiquitination are quite variable.
•The latter seems to be a prerequisite for the dimethylation and trimethylation of H3K4
and H3K79, although it does not seem to influence monomethylation.
45. •H2AK119ub1 is involved in gene silencing, whereas H2BK123ub1 plays an
important role in transcriptional initiation and elongation.
•H2B ubiquitination ; required for transcription by RNA polymerase II
•The process of transcriptional elongation of the nascent RNA molecule is
hindered by the presence of nucleosomes, because RNA polymerase II needs
to translocate along the DNA.
•H2B ubiquitination might assist the histone chaperones (FACT) in stimulating
the passage of RNA polymerase II through a nucleosomal template. FACT can
displace an H2A–H2B dimer from a nucleosome core, enhancing transcription
elongation on the chromatin template.
•The modification is removed via the action of isopeptidases called de-ubiquitin
enzyme and this activity is important for both gene activity and silencing
46.
47. Is a modification related to ubiquitylation
The small ubiquitin-related modifier protein (SUMO) shares 18% sequence
identity with ubiquitin and adopts a similar 3-D structure, unlike ubiquitin it
has no apparent role in targeting proteins for Proteasomal degradation.
Involves the covalent attachment of SUMO molecules to histone lysines via
the action of E1, E2 and E3 enzymes.
SUMOylation; function by antagonizing acetylation and ubiquitylation that
occurs on the same lysine side chain .
Addition of this group to lysines in the histone N-terminal polypepetide is
facilitated by E3-SUMO ligases in a manner somewhat analogous to
ubiquitination
H4 can be modified by SUMO and this modification leads to the repression of
transcriptional activity through the recruitment of HDACs and HP1 proteins
48. E1 activates SUMO (SU) in an ATP-dependent manner.
E2 attaches SUMO to the lysine in the target protein, supported by
E3-LIGASE.
SUMO protease removes SUMO from the protein, which is now free
to be reused in another cycle.
Epigenetics is underpinned by
molecular mechanisms that regulate how, when and where genes are expressed
Genome function (gene expression) is regulated by the interaction between DNA sequence, histones and non-histone proteins (eg.transcription factors).
Chromatin and the nucleosome are key players in epigenetic processes
Most cytosine methylation occurs in the sequence context 5'CG 3’
CpG, "—C—phosphate—G—" nucleotides on one DNA strand (left), and complementary C-G base-paring on two DNA strands (right)
The CpG sites or CG sites are regions of DNA where a cytosinenucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG is shorthand for 5'—C—phosphate—G—3' , that is, cytosine and guanine separated by only one phosphategroup; phosphate links any two nucleosides together in DNA. The CpGnotation is used to distinguish this single-stranded linear sequence from the CG base-pairing of cytosine and guanine for double-stranded sequences. The CpG notation is therefore to be interpreted as the cytosine being 5 prime to the guanine base. CpG should not be confused with GpC, the latter meaning that a guanine is followed by a cytosine in the 5' → 3' direction of a single-stranded sequence.
Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosine. In mammals, methylating the cytosine within a gene can change its expression, a mechanism that is part of a larger field of science studying gene regulation that is called epigenetics. Enzymes that add a methyl group are called DNA methyltransferases.
In mammals, 70% to 80% of CpG cytosines are methylated
The majority of DNA methylation occurs on cytosines that precede a guanine nucleotide or CpG sites. Although the brain contains some of the highest levels of DNA methylation of any tissue in the body, 5mC only accounts for ∼1% of nucleic acids in the human genome (Ehrlich et al, 1982). The majority of DNA methylation occurs on cytosines that precede a guanine nucleotide or CpG sites. Overall, mammalian genomes are depleted of CpG sites that may result from the mutagenic potential of 5mC that can deaminate to thymine (Coulondre et al, 1978; Bird, 1980). The remaining CpG sites are spread out across the genome where they are heavily methylated with the exception of CpG islands
4. DNA Methylation
In mammals, DNA methylation is a common epigenetic change in DNA. After DNA synthesis, cytosines within the dinucleotide CpGs are methylated at their carbon 5 by DNA methyltransferase (DNMT) (Figure 1). CpGs which undergo methylation could be found either in singular situation or in clusters so-called CpG islands [5]. But if the methylation happens in the promoter region of the genes, it would likely lead to gene silencing [6]. Normally, long-term silencing of genes occurs only in X-linked, imprinted, and germ-cell specific genes. CpG islands of DNA sequence that contain plenty of C and G nucleotides are commonly hypermethylated in tumor cells which could result in silencing of tumor suppressor genes
CpG islands in promoters[edit]
In humans, about 70% of promoters located near the transcription start site of a gene (proximal promoters) contain a CpG island
HDAC1 and HDAC2 , RbAp46, RbAp48, SAP30, SAP18, and SDS3
Moreover, the fact that there are different variants of each histone protein differing from few to many amino acids adds another level of complexity in functional aspects of histone PTMs. Such complicated and multilayered regulatory mechanisms of cellular processes through histone modifications have led to the hypothesis of “histone code” where a set of histone variants and modifications together perform a specific function[29]. However, due to its complexity histone code is still not fully understood[30]. Further, the status of one histone modification also regulates that of another by cross-talk and affects chromatin remodeling and gene expression. Cross-talk between H3S10ph and H3K14ac, H2Bub and H3K4me and H3K4ac and H3K4me3 and H3K14ac are few prominent examples regulating gene expression[31]. For example, acetylation of H3K18 and H3K23 by CBP (CREB binding protein) can promote the methylation of H3R17 by Coactivator-Associated Arginine Methyltransferase 1 (CARM1), resulting in activation of estrogene-responsive genes
already entered clinical trials. This particular example, called SAHA, which is a very potent inhibitor of HDACs, is actually now commercially available as Zolinza as a trade name, and Zolinza was actually approved on October sixth 2006 by United States Food and Drug Administration. So, for the treatment of a specific type of a skin cancer called cutaneous T cell lymphoma, and Sezary’s disease.
Methylation of lysine can come in three forms; monomethyl, dimethyl,
and trimethyl adducts are known. Of these, only the trimethyl form, being
a quaternary amine, preserves the positive charge on the nitrogen atom
of the -amino group. The other two are secondary and tertiary amines,
respectively, and are uncharged at physiological pH (Figure 6.5).
Because of the multiple methylation states that are possible for a single
lysine, the methylation of histone N-terminal tails can include a great
deal more information than is possible with acetylation. An example
of this is the difference between dimethylation and trimethylation of
lysine 4 on histone H3, in which the dimethylated state is often associated
with readiness for gene transcription (a so-called permissive chromatin
state), whereas active transcription of the gene almost always
requires the presence of trimethylated lysine at position 4 (H3K4me3).
The effect is also determined by the position of the methylated lysines.
H3K4 may be an excellent way to promote gene activity, but if lysine 9 on
H3 becomes methylated (often by replacing acetyl groups), gene expression
is decreased.
Because of its recruitment of heterochromatin protein 1 (HP1), methylation
of lysine 9 on histone H3 is perhaps the best characterized example
demonstrating the different effects of histone methylation. HP1 was first
identified as a cytological component of Drosophila heterochromatin that
Cross-talk between histone marks Cross-talk between different histone marks can also have a profound effect on enzyme activity [1]. For instance, ubiquitylation of H2B K123 by the E2 ubiquitin conjugating enzyme Rad6 is required for subsequent di-methylation of H3 K4 by Set1p or H3 K79 by Dot1p [38]. Prior histone marks can also inhibit subsequent modifications
ribosomal S6 kinase-2 (Rsk2), which is downstream of extracellular signal-regulated kinase (ERK);
mitogenand stress-activated kinase-1 (Msk1), which is downstream of both ERK and p38 mitogenactivated kinases (MAPK); and
the aurora kinase family member Ipl1.
Evidence also implicates aurora kinases in the phosphorylation of serine 28 in histone H3.81 Phosphatases remove phosphate groups from histones.82 To date, the phosphatases PP1 and PP2A, and the PP1 inhibitor DARPP32, have been shown to regulate H3 phosphorylation in the CNS.78,83,84 In most cases, phosphorylation of histones is associated with gene activation, although much further work is needed to define the prec
Rsk2, which is downstream of ERK;
Msk1, which is downstream of both ERK and MAPK; and
The aurora kinase family member Ipl1.
Previously described histone modifications result in relatively small molecular changes to amino-acid side chains.
Ubiquitylation - much larger covalent modification.
occur only
although
the interaction is reversible and specific isopeptidases release the SUMO
moiety. SUMOylation can compete with other lysine modifications such
as acetylation. For the most part, it seems to lead to the inhibition of transcription,
often through the addition of one or more SUMO peptides to all
four core histones; however, the extent of SUMOylation does not seem
to be equal for all histones. The primary targets seem to be histones H2A
and H2B (lysine 126 in H2A, and lysines 6, 7, and possibly 16 in H2B), but
there is also evidence to suggest the involvement of all five lysines on the
N-terminal tail of H4.
Analyzing the functions of this modification by using chromatin immunoprecipitation