Protein modifications in transcription elongation
ABSTRACT Posttranslational modifications (PTMs) of proteins play essential roles in regulating signaling, protein–protein modifications and subcellular localization. In this review, we focus on posttranslational modification of histones and RNA polymerase II (RNAPII) and their roles in gene transcription. A survey of the basic features of PTMs is provided followed by a more detailed account of how PTMs on histones and RNAPII regulate transcription in the model organism Saccharomyces cerevisiae. We emphasize the interconnections between histone and RNAPII PTMs and speculate upon the larger role PTMs have in regulating protein function in the cell.
- SourceAvailable from: Matthew Torres[show abstract] [hide abstract]
ABSTRACT: Post-translational modifications (PTMs) occur on nearly all proteins. Many domains within proteins are modified on multiple amino acid sidechains by diverse enzymes to create a myriad of possible protein species. How these combinations of PTMs lead to distinct biological outcomes is only beginning to be understood. This manuscript highlights several examples of combinatorial PTMs in proteins, and describes recent technological developments, which are driving our ability to understand how PTM patterns may "code" for biological outcomes.FEBS letters 02/2013; · 3.54 Impact Factor
Article: Emerging Views on the CTD Code.[show abstract] [hide abstract]
ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the "CTD code" hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a "code." Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.Genetics research international. 01/2012; 2012:347214.
- [show abstract] [hide abstract]
ABSTRACT: Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.PLoS Genetics 08/2012; 8(8):e1002822. · 8.52 Impact Factor
Protein modifications in transcription elongation
Stephen M. Fuchsa, R. Nicholas Laribeeb, Brian D. Strahla,c,⁎
aLineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA
bDepartment of Pathology and Laboratory Medicine, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA
cDepartment of Biochemistry and Biophysics, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA
a b s t r a c t a r t i c l e i n f o
Received 2 June 2008
Received in revised form 17 July 2008
Accepted 18 July 2008
Available online 30 July 2008
RNA polymerase II
Posttranslational modifications (PTMs) of proteins play essential roles in regulating signaling, protein–
protein modifications and subcellular localization. In this review, we focus on posttranslational modification
of histones and RNA polymerase II (RNAPII) and their roles in gene transcription. A survey of the basic
features of PTMs is provided followed by a more detailed account of how PTMs on histones and RNAPII
regulate transcription in the model organism Saccharomyces cerevisiae. We emphasize the interconnections
between histone and RNAPII PTMs and speculate upon the larger role PTMs have in regulating protein
function in the cell.
© 2008 Elsevier B.V. All rights reserved.
1.1. Protein modifications in biology
With the completed sequencing of the human genome nearly a
decade ago,it is now well established that a relativelysmall numberof
genes (b30,000) give rise to a substantially larger number of protein
products (∼3×106–3×107) [1,2]. This great protein diversity is
manifested at the level of mRNA by alternative splicing or utilization
of different transcription and translation start and stop sites. Once the
mRNA is translated into a protein product, further protein diversifica-
tion can be achieved by covalent modification of amino acid side
chains, hence the name “posttranslational modification” (PTM).
Protein modifications are very diverse in nature (totaling greater
than 200) and can be found on 15 of the 20 proteinogenic amino acids
[3,4]. Some modifications are familiar (e.g. phosphorylation) while
others are found on onlya fewsubstrates (e.g. diphthamide) . (For a
detailed examination of posttranslational modifications and asso-
ciated mechanisms see  and references therein). Regardless, many
PTMs, and their associated biochemical pathways, are evolutionarily
conserved between prokaryotes and eukaryotes, suggesting they play
indispensable roles in biology.
What are the functions of PTMs? Many modifications have evolved
as a way of incorporating more diverse functional groups into proteins
such as phosphate or sulfate . Others serve varied biological roles,
from controlling protein conformational stability  to controlling
localization of proteins within the cell . Perhaps the most widely
studied role of modifications is their ability to mediate protein–
protein interactions. A number of protein domains have evolved to
bind PTMs in specific protein contexts, such as SH3 and WW domains
that bind to proline-rich domains  or SH2 domains that recognize
phosphotyrosine . PTMs also appear to play key roles in epigenetic
processes as some of these modifications, such as methylation on
histones, can be maintained through cell divisions .
1.2. Scope of this review
Studies of PTMs are rooted in some of the earliest biochemical
research [3,11–13]. While the types of modifications and associated
enzyme mechanisms are well established, in many cases little is
known about how these modifications function. This review will
highlight some of the modifications for which biological roles have
been described and will try to put into a greater context how diverse
protein modifications modulate functions within the cell.
We will focus on two biologically connected processes, notably
posttranslational modification of histones and transcription by RNA
polymerase II (RNAPII). We chose these processes not only for their
wonderfully diverse modifications but also because recent research
has clearly shown that the two processes are inextricably connected
[14–17]. While there is an abundance of literature on these topics, it is
also evident that we have only scratched the surface of understanding
the full role of modifications in both these processes. Therefore, where
appropriate, we will attempt to speculate on what the future might
hold for the role of PTMs in these processes.
Biochimica et Biophysica Acta 1789 (2009) 26–36
⁎ Corresponding author. Department of Biochemistry and Biophysics, University of
North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA. Tel.: +1 919 843 3896; fax: +1
919 966 2582.
E-mail address: email@example.com (B.D. Strahl).
1874-9399/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagrm
Since there have been several comprehensive reviews on histone
modifications in higher organisms in the recent literature [16,18,19],
we will focus this review on histone PTMs in the model organism
Saccharomyces cerevisiae, as many of the modifications have been
extensively studied in this organism. Furthermore, yeast is an
attractive model for studies of PTMs as the number of proteins is
comparatively small and several whole-proteome studies of PTMs and
protein–protein interactions in yeast have already been completed
[20,21]. Much of what we have learned in yeast has proven to be
similar in higher organisms; therefore, many of the processes we
describe have parallels in mammalian cells as well.
We will review the historical links between histone modification
and transcription and briefly describe the common modifications
found on RNAPII, focusing primarily on the modification-rich C-
terminal domain (CTD), and the core histones (comprehensive
reviews of histone modifications can be found elsewhere [18,22]).
We will then put these modifications into greater biological context
highlighting several recent findings that demonstrate how modifica-
tions of histones dictate changes in chromatin structure leading to
changes in gene expression. Additionally we will show how
modification of RNAPII dictates changes to histones modifications
The goals of this review are twofold. One is to cover many of the
major recent findings linking histone modifications with transcrip-
tion. The second is to convey the enormous role of non-histone PTMs
in modulatingtranscriptionand chromatin remodelingaswell as their
likely role in many other biological processes.
1.3. History of the link between chromatin modifications and transcription
Histones have been known to associate with nucleic acids since
their discovery in the 1880s by Albrecht Kossel . Their biochemical
properties were heavily studied over the next half-century and in
1951, Stedman and Stedman first hypothesized that histones could
inhibit nucleic acid-based processes including replication and RNA
synthesis . In much of the early histone literature it was noted that
they purified in multiple, closely related forms. These forms were
found to differ primarily by modifications on the histone side chains
including phosphorylation, acetylation, and methylation. A biological
role forhistone modifications was first proposed by Allfreyand Mirsky
in 1964 when they observed that histone acetylation correlated with
increased rates of RNA synthesis [25,26].
Over the next thirty years, the functions of histone modifications
would take a backseat to the volumes of historic work detailing how
genetic information is stored and copied. During this time, the
enzymes responsible for replication and transcription were exten-
sively studied. Further, the functional association between DNA and
histones, the nucleosome, was reported [27,28]. Understanding this
genomic architecture would turn out to be essential to our under-
standing of the role histone modifications play in regulating not only
transcription but other DNA-based processes.
In 1996, the connection between histone modifications and
transcription again came to the forefront. Allis and coworkers showed
that a well-studied transcriptional activator, Gcn5, was in fact an
acetyltransferase enzyme that modified histones . This provided
biologicalcontext totheobservationsof AllfreyandMirskyof the 1960s.
In similar work Schreiber and coworkers linked transcriptional repres-
could modulate geneexpressionwas born.This ideawasmade manifest
a few years later with the “histone code” hypothesis [31–33].
The basic premise of the histone code hypothesis is that
modifications on histone tails are recognized by additional protein
factors. In turn, these factors alter chromatin structure and conse-
quently, transcriptional output giving rise to complex patterns of gene
expression. Some modifications are stable between generations and
presumably act as epigenetic marks as well.
The past ten years of research has resulted in the discovery of
numerous modifications on histones and the enzymes responsible for
their deposition, many of which we will discuss here. Further, many
proteins and protein domains that recognize posttranslational
modifications have been described. Whether a true “histone code”
exists is often a matter of debate, but the data are clear — histones are
modified to have complicated patterns of posttranslational marks
along their tails and these tails work both independently and in
conjunction with one another to recruit other factors to DNA. In the
case of transcription, the situation is further complicated as modifica-
tions on RNAPII can dictate which chromatin-modifying enzymes are
able to gain access to histones in a transcription-dependent manner.
2. Histone modifications associated with transcription
(Note: A new nomenclature for many of the histone-modifying
enzymes has been recentlyadopted. We will refer to proteins according
to the new nomenclature  and have the more established names in
parenthesis to facilitate searching of the published literature).
2.1. The architecture of the nucleosome
The nucleosome is the basic unit of chromatin and is comprised of a
protein core made up of histone proteins (2 each of H2A, H2B, H3, and
is wound. The histone proteins have a well-folded core domain and
unstructured tail domains on both the N- and C-termini . While
modifications occur within the core domains, the tail regions are rich
with protein modifications as depicted in Fig. 1A. Modifications on the
architecture bymodulatingthestabilityof nucleosomalparticles as well
as regulating what proteins are recruited to chromatin.
2.2. Acetylation and deacetylation of histones
Histone acetylation, the most prevalent modification on histones,
is catalyzed by a number of enzymes that transfer the acetyl moiety
from Acetyl-CoA to the ɛ-amino group of lysine residues. In yeast,
there are 9 reported histone acetyltransferases (HATs) that act on the
four core histones. These HAT complexes have different substrate
specificities, some recognizing only a few sites while others acting on
many lysine residues on multiple tails. HATs are generally found as
members of larger protein complexes, thus substrate specificity is
most likely regulated through associations with other complex
subunits . The intricacies of the individual marks are beyond the
scope of this discussion and are described in detail elsewhere [37,38].
Histone acetylation is generally associated with transcription
activation. Acetylation of lysine alters the chemical properties of the
ɛ-N-acetyllysine. In the context of chromatin, studies have shown that
acetylation not only reduces the interaction of histones with DNA but
also associations between nucleosomes [39,40]. Consequently, acety-
lated nucleosomes are destabilized, promoting both nucleosomal
rearrangement by ATP-dependent chromatin remodeling complexes
and binding of a diverse set of DNA-binding factors involved in
these remodeling factors are actually recruited to chromatin through
direct binding to the acetyl mark by bromodomains [43–46].
While histone acetylation generally promotes transcription, there
is a competing process of histone deacetylation that is generally
thought to act in a repressive manner [47,48]. In yeast, there are nine
reported histone deacetylase enzymes (HDACs) belonging to one of
two classes . Intuitively, HDAC function is generally associated
with nucleosome stabilization and repression of remodeling activities.
It is, however, now clear that while HDACs act in opposition to HATs,
HDACs also function as activators of gene expression [50–52].
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
2.3. Histone methylation
Methylation is a considerably more complex modification than
acetylation. Several amino acids can be methylated including lysine
and arginine on histones . Up to three methyl groups can be added
to one lysine residue while arginine can accept up to two methyl
groups, with dimethylarginine being either symmetric or asymmetric
. Unlike acetylation, the charge of the amino acid side chain of
lysine or arginine is unchanged by methylation. Similar to acetylation,
methylated residues serve as binding partners for a number of protein
domains including chromodomains  and PHD domains [55–57]
which bind methyllysine, and Tudor domains which can recognize
both methyllysine and methylarginine [58–61].
Lysine methylation of histones in budding yeast has only been
identified at a few locations: histone H3 Lys4 (H3K4) , Lys36
(H3K36)  and Lys79 (H3K79) [64–66]. Fission yeast and higher
eukaryotes have several additional sites of methylation, including
Lysine methylation differs from acetylation in that it appears that
methylation of different lysine residues is catalyzed by different
enzymes rather than one enzyme modifying multiple lysine targets.
For example, while Kmt2(Set1), Kmt3(Set2) and Kmt4(Dot1) modify
histoneH3 lysine residuesat K4, K36andK79respectively,Gcn5(Kat2)
is known to have a broad lysine substrate recognition on histone H3
that includes lysine residues K9, K14, K18, K27 and K36 [72,73]. One
reason some histone acetylases likely have such a broad substrate
preference is that, in addition to recruiting bromodomain-containing
factors, acetylation changes the overall charge of the histone tails,
which in turn can regulate nucleosome–nucleosome and histone tail–
DNA interactions that drive chromatin decompaction. As lysine
Histone methylation has many diverse roles in transcription.
Methylation at H3K4, H3K36, and H3K79, for example, is found
associated with active transcription [74,75]. In contrast, H3K9me in
gene silencing and heterochromatin formation [67,76,77]. Even though
several sites of methylation on H3 are all associated with active
transcription, they have very different roles in regulating transcriptional
events. For example H3K4 methylation is known to promote transcrip-
[78,79]. H3K36 methylation, however, is associated with recruiting
We will describe the roles of H3K36me in transcription in greater detail
later in this review. To date, the role of H3K79me in transcription is not
well understood although the mark can be found associated with
promoters and coding regions of most actively transcribed genes and is
connected to the process of DNA repair [59,66,75,83,84].
Argininemethylationis a modification most studied in RNA-binding
predominant arginine methyltransferase in yeast, Hmt1. Silver and
colleagues have reported a role for H4R3 methylation in gene silencing
silencing, but through a mechanism that involves antagonizing the
establishment of H3K4 methylation at gene promoters .
Methylation was believed to be an irreversible mark for many
years. Only recently have demethylase enzymes been described that
can act on both methyllysine and methylarginine [87,88]. In yeast, it
appears that like the methyltransferases, these enzymes have high
specificity and act on particular target residues and even on specific
methylation states. For example, the demethylase Kdm5(Jhd2)
removes di- and trimethylation from H3K4 [89,90], while H3K36me
is removed by both Kdm2(Jhd1) and Kdm4(Rph1) [91,92]. While these
enzymes clearly act on histones, their roles in chromatin remodeling
and transcription are largely unexplored inyeast although much more
is known in higher eukaryotes .
Phosphorylation of proteins is probably the most recognized
protein modification; however, phosphorylation is a relatively rare
eventonhistones. Like in otherprocesses,phosphorylationof histones
appears to correspond to changes in the extracellular environment.
Phosphorylation of Ser10 on H3 (H3S10) promotes transcription by
influencing acetylation at H3K14 in response to changes in carbon
source . Phosphorylation of H2B Ser10 (H2BS10) by Ste20 is a
signal for apoptosis in response to oxidative stress [94–96]. A
significant body of work has been established that shows phosphor-
ylation of the C-terminus of histone H2A is linked to DNA damage
repair . Similarly, phosphorylation of histone H4 at Ser1 (H4S1)
also accompanies DNA damage . While it is clear that H2A
phosphorylation leads to downstream changes in transcription,
definitive links between this phosphorylation and the transcription
machinery have not been reported. H4S1 phosphorylation, however,
has been clearlyshown torecruit the NuA4 HATcomplex as well as the
Swi/Snf chromatin remodeling complex to genes resulting in histone
acetylation and recruitment of RNAPII [99,100].
Fig.1. Posttranslational modifications (PTMs) found on histones and RNA polymerase II (RNAPII). (A) Most common PTMs found on the N- and C-terminal tails of the four canonical
histones inyeast. (B) Sites of phosphorylationwithin the 26-repeat C-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII. Phosphorylation of Ser5 is primarily catalyzed by
Kin28 and removed by Ssu72. Phosphorylation of Ser2 is primarily catalyzed by Ctk1 and removed by Fcp1. Ess1 is thought to facilitate prolyl isomerization within the CTD.
Differential phosphorylation of the CTD leads to recruitmentof distinct phospho-CTD associating proteins (PCAPs) in a spatiallyand temporallycontrolled manner toregulate specific
transcription-associated processes (e.g. chromatin remodeling and mRNA processing).
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
Ubiquitin is a 76 amino acid protein that is attached to protein
lysines through a complex system of protein ligases . These
modifications have a variety of functions, including acting as signaling
marks, as mediators of protein–protein interaction, and in regulating
protein stability. Inyeast, ubiquitin has to date only been found on one
histone, histone H2B at Lys123 . While the study of H2B
ubiquitylation has been restricted to monoubiquitylation (H2Bub1),
very recently, polyubiquitylation of H2B has been reported . In
higher eukaryotes, H2A is also ubiquitylated on Lys119 .
The role of ubiquitin in transcription is very complex. H2Bub1 is
catalyzed by the Rad6/Bre1 ubiquitin-ligase complex and functions to
promote transcriptional initiation and elongation [102,105–107]. For
example, H2Bub1 facilitates H3K4 methylation and works with the
FACT chromatin remodeling complex to promote transcription
elongation (Fig. 2) [108,109]. We will further discuss the interplay
between H2Bub1, H3K4 and RNAPII modifications later in this review.
Many roles of ubiquitination in transcription have recently been
reviewed elsewhere [110,111].
In addition to ubiquitin, there are several other ubiquitin-like
proteins that can be conjugated to proteins in higher eukaryotes .
Yeast has two ubiquitin-like proteins, Smt3 and Rub1. Smt3, SUMO
with transcription. In fact, it has been reported that all four histones can
be modified by SUMO . While the authors were unable to precisely
on at least 5 locations in the N-terminus H2B sumoylation is thought to
occur at Lys6/7 and Lys16/17. On H2A, SUMO is attached to Lys126.
The role of sumoylation in transcription is largely unknown. Berger
and coworkers have shown that sumoylation is in general a repressive
mark, and in fact antagonizes histone acetylation . However, the
exact mechanisms through which SUMO represses transcription are
still poorly understood.
2.7. Prolyl isomerization
Proline isomerization is the only noncovalent posttranslational
modification found to occur on histones to date. The ring structure of
theprolineaminoacid allows it to interconvert between two conforma-
propagate a significant change in protein structure, some of which may
serve functions within the cell . For example, the N-terminal tail of
histone H3 has several prolines within its sequence. Kouzarides and
coworkers have shown that mutation of Pro38 affects the ability of the
Kmt3(Set2) enzyme to methylate the nearby H3K36 . They also
described the corresponding enzyme, the prolyl isomerase Fpr4, which
antagonizes H3K36 methylation by Kmt3(Set2).
2.8. PMTs on histone variants
In addition to the canonical histones (H2A, H2B, H3, and H4), there
also exist variants of these histones that act in place of one of the four
main histones and perform alternate functions. Inyeast, there are two, a
variant of H2A called H2A.z (Htz1) and an H3 variant called Cse4 (also
[116,117] and while it hasbeen tied to chromatin remodeling complexes,
no role for Cse4 in transcription has been described [118,119]. Htz1 is
found to replace H2A in the promoter regions of genes. Htz1-containing
nucleosomes are thought to be less stable than H2A-containing
nucleosomes and thus probably play some role in marking gene
promoters. Htz1 is modified by the NuA4 acetylase complex [120,121]
kinases in vitro . While Htz1 acetylation has been correlated with
active transcription, it is not known if Htz1 phosphorylation occurs in
vivo and/or functions to regulate nucleosome dynamics in transcription.
2.9. Other modifications
It is important to note that most of the histone modifications
described above have been identified in the last decade. It is likely that
there are other modifications that have yet to be identified. For
example, the modifications described occur almost exclusively on the
histone tails. Modifications in the core regions are likely to occur and
in fact several new sites of acetylation in the core domains of H3 have
been identified recently [122–124].
3. Modifications on Pol II associated with transcription
3.1. The C-terminal domain (CTD) of RNA Polymerase II
The largest subunit of the RNA Polymerase II (RNAPII) holoenzyme
comprises not only the catalytic core of RNAPII but also a very long,
Fig. 2. H3K4 methylation and transcriptional activation. The COMPASS methyltransferase is recruited tothe promoterregions of genes through interactions with the PAF complex and
serine 5 phosphorylation, as well as by making contacts with H2Bub1 through Cps35 (see text). Recruitment of COMPASS leads to the restricted establishment of H3K4me3 near the
transcriptional start site This modification may serve to stabilize PIC formation and recruit chromatin remodeling and histone-modifying activities that facilitate the transcription
process (see text for details). Also associated with COMPASS and the elongating RNAPII complex is the histone chaperone complex, FACT, which disassembles and reassembles
nucleosomes to promote transcription. FACT function and COMPASS activity are both functionally regulated, at least in part, through H2Bub1 .
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
unstructured C-terminal region that is essential for function. This C-
terminal domain (CTD) is made up of multiple repeats (26 inyeast and
52 in humans) of a seven-amino acid consensus sequence YSTPSPS
 (see Fig. 1B). The CTD acts as a scaffold making significant
protein–protein interactions that link transcriptionwith PhosphoCTD-
associated proteins (PCAPs) that are involved in many biological
processes including chromatin remodeling, cell-cycle regulation, and
mRNA processing (see Fig. 1B) . These protein–protein interac-
tions are dictated by a complex set of posttranslational modifications
that occur on the CTD [125,127]. Here we will try to summarize CTD
modifications and their importance in transcription.
3.2. Ser2 and Ser5 phosphorylation
Early analysis of the largest subunit of RNAPII (Rpb1) showed that
the protein took on many forms as judged by SDS-PAGE electrophor-
esis . It was later determined that these different gel mobilities
signified varying levels of protein phosphorylation on the CTD,
specifically at Ser2 and Ser5 [129,130]. In yeast, Ser5 phosphorylation
of RNAPII is catalyzed by the cyclin-dependent kinase Kin28, which is
part of the transcription complex TFIIH [131–134]. There also exists
supporting data that a factor of the Mediator complex, Srb10, also
phosphorylates Ser5, but only when RNAPII is not associated with a
gene . Thus phosphorylation by Srb10 is thought to prohibit pre-
initiation complex (PIC) formation and thus is a negative regulator of
Ser2 phosphorylation is catalyzed in vivo by the cyclin-dependent
kinase Ctk1 . Phosphorylation of Ser2 serves as a mark that
denotes transcription elongation and requires many factors, including
Ser5 phosphorylation , removal of H2Bub1 , and association
of the PAF complex .
The phosphorylation state of RNAPII changes as the transcription
machinery moves along a gene . RNAPII bound at promoter
regions is largely unphosphorylated . Initiation of transcription
coincides with Ser5 phosphorylation by Kin28 . During the
elongation phase of transcription, Ctk1 phosphorylates Ser2 to give a
RNAPII that is modified at both Ser2 and Ser5 . As RNAPII travels
along a gene, phosphorylation on the CTD is removed. The
phosphatase Ssu72 has been shown to remove Ser5 phosphorylation
 while the phosphatase Fcp1 is responsible for removing Ser2
phosphorylation . It is thought that dephosphorylation of the
CTD is required for recycling of RNAPII on genes and may play a role in
gene looping [143–145].
3.3. Prolyl isomerization by Ess1
As described earlier, prolyl isomerases catalyze the interconver-
sion of the two conformations of proline residues. As is evident from
the protein sequence of the RNAPII CTD, there are two prolines in
every repeat. Hanes and coworkers found that the prolyl isomerase
Ess1 is essential for growth in yeast and binds to the RNAPII CTD
of the catalytic residues of Ess1 results in a temperature-sensitive
phenotype suggesting that at least one function of Ess1 may be to act
on the CTD.
Ubiquitylation is a common modification of lysine residues. Mono-
ubiquitylation often serves as a protein–protein interaction mark
while polyubiquitylation usually signals for degradation. The largest
subunit of RNAPII, Rpb1, associates with members of the ubiquitin-
polyubiquitylated substrates) [147,148]. WW domains within Rsp5
mediate binding to the elongating RNAPII CTD and Rsp5 then
ubiquitylates distant sites on Rpb1 in conjunction with an E2 protein
Ubc5, targeting Rpb1 for degradation in response to DNA damage
. This process is further regulated by other RNAPII subunits
including the nonessential subunit Rpb9 . A recent result from
Svejstrup andcolleagues shows that deubiquitylationof Rpb1 is also a
crucial process, as loss of the ubiquitin protease Ubp3 facilitates
removal of stalled RNAPII complexes in response to DNA damage
3.5. Other modifications
The richness of modifications on the CTD leaves one asking
whether there are still undiscovered modifications. In fact, late last
year, two groups reported that Ser7 of the CTD could be phosphory-
lated in mammalian cells [152,153]. Also, there are reports of
glycosylation of both Ser2 and Ser5 in higher eukaryotes as well
[154,155]. Tyrosine residues can also be modified in several ways
including phosphorylation, sulfation and nitrosylation . The
existence of these modifications in yeast has not been reported but
clearly these modifications are possible in this organism. Thus, the
future will reveal whether these additional modifications are
conserved and play important roles in regulating transcription-
associated processes or whether we have found evolutionary
differences in CTD modifications between yeast and metazoans.
We have chosen to restrict our discussion to modifications on the
CTD of the largest subunit of RNA polymerase. However, there are
eleven other subunits associated with the RNA polymerase holoen-
zyme in addition to Rpb1. Several of the other subunits have been
reported to be substrates of casein kinase II (CKII) indicating that
multiple members of the RNAPII holoenzyme are likely to be modified
. Future work will likely be aimed at understanding what role
PTMs of the RNAPII holoenzyme play in regulating transcription.
While the answers are still unknown, it is likely that these
modifications are important signals coordinating transcription with
other cellular processes.
4. The interplay between histone modifications and RNA
Hundreds of proteins are needed to properly carry out the process
of transcription. Hundreds more are required to properly integrate
transcription with other processes such as cell-cycle progression,
cellular metabolism, and RNA processing . As we have already
described, transcription is also tightly coupled to changes in
chromatin architecture. While researchers have determined most of
the chromatin and RNAPII modifications associated with transcrip-
tion, the mechanisms through which these modifications modulate
gene expression have only begun to be elucidated. In this section, we
will highlight a few stories for which we have considerable
information about how protein modifications and histone-modifying
enzymes interplay with RNAPII to dictate patterns of gene expression.
These exampleswill serve to emphasize oneessential rolethat protein
modifications play in cell physiology.
4.1. H3K4 methylation, H2B ubiquitination and CTD phosphorylation
H3K4 methylation in yeast is catalyzed by the enzyme Kmt2(Set1),
which is partof a larger proteincomplexcalled COMPASS [62,138,158].
It is well established that the presence of H3K4 methylation correlates
with nucleosomes residing within genes that are being actively
transcribed. As mentioned earlier, lysines can have one (me1), two
(me2) or three (me3) methyl groups attached to them. H3K4me3 is
most prevalent near promoter regions and then H3K4me2 and
H3K4me1 become more abundant in the coding regions of genes
. Several questions arise from these findings. For example, do
these different methylation states have different functions, and what
factors are required for establishing different methyl states?
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
As we described earlier, it appears that many protein domains are
able to recognize methyllysine. Though not confirmed, it is also likely
that these binding domains can also distinguish between different
methyl states (i.e. me1, me2, me3). Several different proteins have
been described that bind to H3K4me both in yeast and humans
[57,159–162]. These proteins have diverse functions including con-
trolling the extent of nucleosome remodeling, demethylation, acet-
ylation, and PIC formation. Therefore, it appears that the different
methyl states on H3K4 dictatewhat factors interact with chromatin. In
fact, H3K4 seems to be so important for regulating interactions of
binding proteins with nucleosomes that a motif has been recently
reported that recognizes unmodified H3K4 in mammalian cells .
There are numerous signals that control the methylation of H3K4.
H3K4me requires careful coordination of protein modifications not
only on histones (i.e. H2Bub) but also RNAPII, as well as recruitment of
other transcription factors. COMPASS recruitment to the CTD requires
phopshorylation at Ser5, catalyzed by the TFIIH member, Kin28. Kmt2
(Set1) does not appear to directly interact with the CTD tail, rather
COMPASS appears to be recruited to the CTD through interactions
with the PAF transcriptional complex, which is known to directly
interact with RNAPII . In addition to CTD phopshorylation, Kmt2
(Set1) also requires ubiquitylation of H2B (H2Bub1) for proper
methylation of substrates [164–166]. This finding was some of the
earliest support for the idea of the “histone code” because it showed
that protein modifications on one histone tail directly influenced
enzymatic modification of a different tail . This concept has
become known as a “trans-tail pathway” and since several other
similar mechanisms have been described .
Recently, Shilatifard and coworkers uncovered the basis for H2Bub1
regulation of H3K4me . They demonstrated that in the absence of
H2Bub1, theCps35 subunitof COMPASS does not associatewiththe rest
outlines a biological role for Cps35, the only essential subunit of
COMPASS. According to their model, H2B ubiquitylation directs H3K4
COMPASS, thus making the complex catalytically competent. This
finding is significant in that it biochemically defines the factors that
“translate” the “trans-tail” pathway. However, these results stimulate
many additional questions regarding the role of Cps35 in regulating
these processes. For example, as mentioned earlier, Cps35 is the only
essential member of COMPASS. It also happens to be a member of the
cleavage and polyadenylation factor complex (CPF). Is the essential role
for Cps35 in coordinating theactivity of COMPASS, in mRNA processing,
or perhaps in linking these two processes together? There is likely to be
an exciting future in unraveling the many functions of Cps35.
This story demonstrates how numerous modifications on several
proteins (including histones and RNAPII) regulate a histone mark that
has an important role in gene expression. Even with the considerable
questions still largely unanswered. For example, while H3K4me
correlates with the promoter and early coding regions of many genes,
it is not required for viability. What then is the actual role of H3K4me in
transcription? Reinberg and coworkers have uncovered a role for
H3K4me in RNA processing . Does something analogous to this
occur in yeast?
4.2. Kmt3(Set2) and CTD phosphorylation
The methyltransferase Kmt3(Set2) was described a few years ago
as an enzyme responsible for methylating Lys36 of histone H3
(H3K36) [63,170]. Investigators determined that Kmt3(Set2) methyla-
tion of H3K36 was occurring cotranscriptionally and that Kmt3(Set2)
associated directly with RNAPII. In a number of structural and
biochemical studies, a unique domain of Kmt3(Set2) was described
that bound specifically to the CTD of Rpb1 . In collaboration with
the Greenleaf laboratory, we showed that Kmt3(Set2) associates
preferentially with the CTD phosphorylated at both Ser2 and Ser5
. Deletion of the Ctk1 kinase responsible for Ser2 phosphoryla-
tion resulted in a loss of H3K36 methylation, suggesting that binding
of Kmt3(Set2) to the CTD is essential for its activity in vivo [172,173].
Subsequent work determined that H3K36me is most prominently
associated with nucleosomes positioned in the mid- to late coding
region of actively transcribed genes, consistent with Kmt3(Set2)
association to the actively elongating form of RNAPII [75,172–174].
Like H3K4me, H3K36me is recognized by methyllysine-binding
proteins. The Buratowski, Struhl and Workman labs showed that the
chromodomain of Eaf3 boundspecificallytoH3K36me [80–82]. Eaf3 is
a component of both the NuA4 HAT complex and the small Rpd3
deacetylase complex known as Rpd3S [81,82]. It was determined that
Rpd3S was the Eaf3-containingcomplex thatinteracts withH3K36me,
which also depends on an additional histone interaction mediated by
a PHD finger in the only Rpd3S-specific factor RcoI . It was also
clearly shown that methylation of H3K36 (Fig. 3) was capable of
recruiting Rpd3 to chromatin, which in turn, resulted in deacetylation
of nucleosomes [81,82]. More recently, the Strahl and Mellor labs have
reported that H3K36me2 is sufficient to recruit deacetylase activity to
genes . Thus methylation of H3K36me2 plays a repressive role in
transcriptional processes by recruiting enzymes to remove acetylation
from nucleosomes. Whether H3K36me3, which is preferentially
associated with actively transcribing genes, plays a distinct role in
Fig. 3. Role of H3K36 methylation in transcriptional dampening. Following CTD phosphorylation by Ctk1, the Kmt3(Set2) methyltransferase is recruited to the elongating form of
RNAPII via its SRI domain . Kmt3(Set2) methylates H3K36, leading to Rpd3(S) deacetylase recruitment and deacetylation of ORF specific nucleosomes. This event regulates the
removal of elongation-linked acetylation and thereby prevents inappropriate initiation events from occurring in gene bodies. Thus, H3K36me serves to maintain proper transcription
initiation at the promoter regions of genes.
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
gene transcription through the recruitment of a second H3K36me-
binding protein is unknown but remains an intriguing possibility.
The biological relevance of maintaining the coding regions of
RNAPII-regulated genes in a deacetylated state was demonstrated by
showing that when Kmt3(Set2) or several members of the Rpd3S
complex were deleted, nucleosomes in actively transcribed regions
were hyperacetylated and cryptic start-sites within genes were
inappropriately utilized . These results demonstrate how precise
modifications on histone tails can create changes in chromatin that
function to direct transcriptional initiation events to the appropriate
places within the genome.
how modifications on the CTD recruit chromatin-modifying activities,
which in turn dictate the expression patterns of certain genes. Thus
there exists a complicated regulatory mechanism where at least four
different modifications (phosphorylation, methylation, ubiquitylation
and acetylation/deacetylation) determine how a given gene is
expressed. This mechanism will only become more complex as we
learn more about the upstream signals that dictate CTD phosphoryla-
tion patterns. For example, our lab has recently found that binding to
RNAPII regulates the stability of Kmt3(Set2) protein in a proteasome-
specific manner (Fuchs, S.M. and Strahl, B.D., unpublished data). Thus
there appear to be additional mechanisms to tightly control Kmt3
(Set2) activity in response to changes in CTD phosphorylation.
4.3. H3K14, SAGA and RSC — chromatin remodeling and gene expression
The first two stories highlighted focus on the factors that regulate
regulation extends far beyond histone methylation. As an example, we
will highlight some work focusing on how other PTMs, specifically
acetylation and phosphorylation coordinate regulation of gene
Kat2(Gcn5), the catalytic subunit of the SAGA histone acetyltranf-
serase complex, is responsible for acetylation of a number of lysine
residues on both histones H3 and H4 . One of these marks,
acetylation at lys14 of histone H3 (H3K14ac) has receivedconsiderable
interest due to its role in recruiting the RSC chromatin remodeling
complex . In addition to modifying histones, Kat2(Gcn5) is also
known to acetylate non-histone proteins. In the case of RSC, in
particular, non-histone acetylation turns out to play a crucial role in
gene expression as well. In this section we will describe some of the
modifications that contribute to chromatin remodeling by RSC.
As previously stated, histone acetylation is an important factor in
loosening chromatin structure that acts to promote transcription.
H3K14 is only one of a number of residues that is acetylated by SAGA.
Interestingly, SAGA can be specifically recruited to H3K14 by signals
elsewhere on the H3 tail. Specifically, phosphorylation of Ser10 on
histone H3 (H3S10P) has been shown to increase H3K14ac on certain
genes leading to changes in gene expression . H3S10 is
phosphorylated by the kinase Snf1 in yeast in response to carbon
source to upregulate expression of genes involved in metabolism .
How does acetylation at H3K14 cause changes in transcription? At
least one answer is by recruiting the additional chromatin remodeling
activities of the RSC complex . RSC is a very abundant ATP-
roles in regulating gene expression during a number of processes
[118,180]. RSC functions to stimulate transcription elongation by
remodeling nucleosomes and recruiting HAT activities . RSC
contains at least 15 protein subunits several of which contain domains
(bromodomains and BAH domains) that interact with acetylated lysine
residues and other regions within histones. In particular, Cairns and
member of the RSC complex, specifically interact with H3K14ac .
RSC is also known to travel with RNAPII, but unlike Kmt3(Set2), it does
not appear to associate with the CTD. Instead, the Rsc4 subunit of RSC
interacts with the Rpb5 subunit of RNAPII holoenzyme and that this
interaction is critical to RSC function . Thus RSC appears to play an
important role in remodeling nucleosomes at sites of H3K14ac.
Regulation of RSC function by Kat2(Gcn5) goes beyond its ability to
acetylate H3K14. As a consequence of solving the crystal structure of
Rsc4, Cairns and coworkers noted the presence of an acetylated lysine
(Lys25) within Rsc4 itself . Acetylation of Rsc4 by Kat2(Gcn5) was
found to inhibit Rsc4 binding to H3K14ac. Thus it is appears as if Kat2
(Gcn5) acts to both stimulate and inhibit remodeling by RSC. This also
appears to be similar tothe role of H3S10P as phosphorylation recruits
Kat2(Gcn5) yet seems to impede Rsc4 binding. It is therefore likely
that H3S10P is a transient mark and an unknown phosphatase
removes this mark following H3K14 acetylation to allow Rsc4
recruitment (see Fig. 4).
Fig. 4. Acetylation both stimulates and inhibits RSC chromatin remodeling activities. Kat2(Gcn5) is recruited to acetylate H3K14 by Snf1-mediated phosphorylation of H3S10. During
transcription, RSC chromatin remodeling activities is recruited via Rsc4 which binds to H3K14ac via its tandem bromodomains and with RNAPII through interactions with Rpb5. Rsc4
can also be directly acetylated by Kat2(Gcn5) leading to inactivation of the complex.
S.M. Fuchs et al. / Biochimica et Biophysica Acta 1789 (2009) 26–36
own function raises many further questions about how protein
modifications regulate transcription. Are other transcription factors,
or regulators of transcription, modified by what are traditionally
thought of as “histone-modifying enzymes”? If so, how do these
modifications regulate their function? For example, in higher eukar-
yotes it is now well established that steroid hormone receptors are
highly regulated by phosphorylation . The oncogene p53 is
known to be phosphorylated as well as methylated, acetylated,
sumoylated and ubiquitinated . Therefore, it is clear that the
5. Concluding remarks
We began by noting that proteomics predictions estimate that
there are considerably more proteins in a cell than what are encoded
by the DNA. In this review we touched on a number of modifications
known to be important for regulating chromatin structure and
transcription. However, we limited our discussion to just a handful
of modifications on only a few proteins. With the rapid evolution of
high-throughput proteomics techniques, our understanding of the
extent of protein modification will undoubtedly increase in the next
few years. Deciphering the roles of newfound modifications will
become more difficult as the number of modified proteins increases.
Perhaps, understanding the complex networks of protein modifica-
tions is the key to understanding how transcription integrates with
other cell processes such as mRNA processing, cell-cycle regulation,
and signaling events.
Histone tails seem to be the richest sources of protein modifica-
tions in cells. This is likely due to their essential roles in maintaining
chromatin structure. Are there other modification-rich proteins that
have been largely overlooked? Preliminary evidence suggests there
are. RNA-binding proteins as well as ribosomal proteins are known to
be heavily methylated . What are the roles of methylation in
mRNA processing or translation? Structural proteins such as myelin
basic protein are decorated with a diverse set of modifications that
seem to play essential roles in neurodegenerative disorders .
How do modifications of these proteins regulate protein function?
Perhaps the most complex modifications are manifest outside the cell.
Researchers have spent decades studying how glycosylation patterns
on proteins dictate how a cell interacts with its environment .
Deciphering glycosylation is key to understanding a numberof today's
biomedical problems including cancer metastasis and cell differentia-
Protein modifications will likely continue to play a starring role in
future stories of chromatin structure and gene regulation. As more
modifications are identified, it is likely that these PTMs will be key
players in the regulation of gene expression as well as other biological
processes in the cell. In that respect, previous and current work on
histone modifications will serve as a roadmap for deciphering the
function of protein modifications in biology.
Work in the Strahl lab is funded by grants from the NIH (to B.D.S.
and S.M.F.) and the Pew charitable trusts. B.D.S. is a Pew Scholar in the
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