Histone demethylation enzymes and dynamic cell biology

Leanne Stalker1 and Christopher Wynder2,3

1-Department of Biomedical Science,,University of Guelph, 2-Department of Biochemistry, University of Western Ontario, 3PTM Discoveries, London Ontario

Introduction

In order to maintain structure and organization within the nucleus of a

eukaryotic cell, the large DNA macromolecule is structured in to

chromosomes. To provide an additional layer of organization, these

chromosomes are wrapped around protein complexes containing proteins

known as histones to form the basic unit of chromatin, the nucleosome

(Kornberg, 1974; Kornberg & Lorch, 1999). Each nucleosome is comprised

of an octameric core containing two each of Histone H2A, H2B, H3 and H4

around which 146bp of DNA is wound. This DNA is then secured to the

core by an additional histone, histone H1(Kornberg & Lorch, 1999;

Kouzarides, 2007; Sims et al, 2003; Volkel & Angrand, 2007). This

DNA/protein complex provides a mechanism by which to conform the large

DNA molecule to the confined space of the nucleus, allows protection from

DNA damage during cell division, and plays a pertinent role in

transcriptional regulation(Kooistra & Helin, 2012; Kouzarides, 2007). Each

individual histone protein contains two highly conserved protein domains

including a large globular core and an amino terminal tail that protrudes

from both the histone individually and the nucleosomal structure as a

whole(Luger et al, 1997). From a gene regulation perspective, these N-

terminal tails represent an infinite ability for the nucleosomal structure to

become modified.

Histone tail modifications

Due to their availability outside of the core nucleosome, many amino

acid residues on histone tails are targets of extensive post transcriptional

modifications. These occur on specific amino acid residues and include

acetylations, phosphorylations, SUMOylations, ubiquitinations and

methylations. The result of the addition of these molecular groups is

varied and depends highly on both the specific amino acid modified and

the modification itself (Kouzarides, 2007). The addition of these various

groups tends to result in one of two possible consequences. First, it may

change the interaction between DNA and the histone directly leading to an

alteration of the chromatin structure as a whole. This activity is observed

mostly when a posttranscriptional modification, such as an acetylation,

alters the charge of an amino acid on the histone tail. Acetylation of a

lysine (K) residue acts to neutralize its basic charge. This loosens the

interaction between the histone and DNA, increasing the accessibility of

the DNA and generally resulting in transcriptional activation(Shogren-

Knaak et al, 2006; Workman & Kingston, 1998). Acetylation is the most

extensively studied of the post-transcriptional modifications and occurs

most frequently on residues K9, K14, K18 and K56 of Histone H3. The

enzymes responsible for both the addition of the acetyl group, Histone

Aceytl Transferases (HATs) and the enzymes responsible for the removal

of the acetyl group, Histone Deacetylases (HDACs) have been increasingly

popular targets for drug discovery(Khan & Khan, 2010; Kuo & Allis, 1998).

The second consequence of histone modification is the alteration of

non-histone protein recruitment to histone tails. For example, histone

phosphorylating enzymes MSK1/2 and RSK2 tend to target serine residues

at H3S10. Phosphorylation of this residue is found to attract the phospho-

binding protein 14-3-3, which is thought to activate NFB-regulated

genes(Banerjee & Chakravarti, 2011; Kouzarides, 2007). Greater

understanding of the role of histone phosphorylation is yet to be

determined. Ubiquitylation and SUMOylation differ from the

aforementioned mechanisms because they require the addition of large

moieties(Berger, 2007). The function of ubiquitylation remains unclear but

its mechanism of action is believed to either act to recruit supplementary

proteins to histone tails or physically “wedge” chromatin open due to its

size. Functional effects of ubiquitylation appear to vary depending on the

residue to which the moiety is added. For example, ubiquitylation of

H2BK123 is associated with the activation of transcription while

ubiquitylation of H2AK119 by NSPc1 has been found to cooperate with

DNA methylation correlate with the transcriptional silencing of Hox genes.

(Wright et al, 2011; Wu et al, 2008). Conversely, the result of sumoylation is

believed to be mainly transcriptionally repressive(Nathan et al, 2006).

Histone Methylation

Recently, much interest has been placed on the regulation of histone

tail methylation. Unlike the previously mentioned modifications,

methylation can occur on both lysine and arginine (R) residues on amino

terminal histone tails(Shilatifard, 2006; Sims et al, 2003). This modification

has also been found to be processive, suggesting that unlike acetylation,

which is either present or absent, methylation potentially allows for an

increased ability to fine tune regulation. An arginine can become mono or

dimethylated, the latter of which can be either symmetrical or

asymmetrical. Whereas a lysine can be modified in a mono- or di- and tri-

methylated form, each of which has been found to have a differing effect

(Cloos et al, 2008; Santos-Rosa et al, 2002). Methylation does not alter the

charge of the histone tail. Therefore, this modification is not thought to

play a direct role in DNA/ histone interactions. Rather, methylation can

result in a modulation of chromatin structure, altering the accessibility to

chromatin to effector proteins, or may act as a recruitment signal for

regulatory factors(Cloos et al, 2008). This results in transcriptional

alterations due to changes in the chromatin landscape as a whole

(Bannister et al, 2002; Lachner et al, 2001). Histone methylations have been

found to be associated with both transcriptional activation and repression

with methylation of K4 and K36 of H3 being generally ascribed to gene

activation, whereas association with K9 and K27 of the same histone are

generally thought to be involved with transcriptional repression(Berger,

2007). The enzymes responsible, known as lysine methyltransferases

(HMTs) are unique in the sense that they are residue specific. For instance,

the Set1/COMPASS or MLL class of histone methyltransferases are specific

for the methylation (mono-, di-, and tri-) of H3K4, while the Su(var)3-9 family

is restricted to methylation of H3K9 (Kouzarides, 2007)

Demethylation of the histone tail

Historically, methylation was considered to be a mark of

permanence. Without the discovery of an enzyme class capable of the

removal of methylation, it was thought that these marks were static. The

discovery of the enzyme KDM1a (also known as LSD1, BHC110) in 2004,

changed this notion. KDM1a was found to have the ability to catalyze the

demethylation of histone residues by a flavin adenine dinucleotide (FAD)-

dependent amine oxidase reaction. However, the enzymology of this

demethylase requires a protonated methyl -ammonium in its substrate.

This is absent in the trimethylated version of methylation, resulting in the

conclusion that this enzyme was restricted to mono and dimethylated

modifications(Shi et al, 2004). Since then a more novel, larger protein

group named the Jumonji (JMJC) domain family has been discovered.

These enzymes catalyze the removal of methylation marks utilizing a

hydroxylation reaction through their JMJC domain. This reaction no longer

requires a protonated methyl -ammonium, allowing for the demethylation

of all three methyl states. In several cases, the trimethylated version is

actually the preferred substrate(Christensen et al, 2007; Fodor et al, 2006;

Klose et al, 2006; Tsukada et al, 2006; Whetstine et al, 2006). Historically, F-

Box and Leu-rich repeat protein 11 (FBXL11) was the first enzyme

discovered in this class; it has demethylase activity towards both the mono

and dimethylated versions of H3K36 (Tsukada et al, 2006).

To date, JMJC enzymes of this class have been found to be active on

H3K4(Iwase et al, 2007; Klose et al, 2007; Lee et al, 2007; Secombe &

Eisenman, 2007; Seward et al, 2007; Tahiliani et al, 2007; Yamane et al,

2007) ; H3K9(Yamane et al, 2006), H3K27(Agger et al, 2007; De Santa et al,

2007; Lan et al, 2007), H3K36(Fodor et al, 2006) and H4K20(Liu et al, 2010).

This has led to the current understanding that methylation represents an

extremely flexible and dynamic modification state resulting in the active

modulation of transcription. Though the JMJC class of demethylases as a

whole is an expansive protein family (the human genome encodes 30

different JMJC containing proteins, 18 of which have been proven to show

demethylase acitivity on both arginine and lysine residues(Kooistra &

Helin, 2012) phylogeny has suggested that within this family there are

several clusters of proteins which appear to group together in both

structure and function. The KDM5 family of demethylases, known to target

all three methylation states of H3K4, represents one such cluster(Cloos et

al, 2008)

Specific function of the KDM5 family of HDM enzymes

The KDM5 family of JMJC demethylases includes four known

members: KDM5a, KDM5b, KDM5c and KDM5d (previously known as

Jarid1a, Jarid1b, Jarid1c and Jarid1d respectively). As seen in Figure 1;

these demethylases are highly conserved structurally and are

characterized by the presence of five protein domains:JmjN and JmjC

domains required for demethylation activity a BRIGHT/ARID domain for A/T

DNA binding, and both a C5HC2-Zinc finger domain and several PHD (plant

homeobox domains) involved in the enzymes ability to recognize and bind

methylated residues and regulate protein-protein interactions(Cloos et al,

2008). This review will concentrate on the known roles of KDM5 proteins in

transcriptional regulation, development and disease. For a recent review

encompassing all histone demethylases, please see Kooistra et al.

(Kooistra & Helin, 2012)

H3K4 methylation: a fine balancing act

The KDM5 family of histone demethylases act specifically on H3K4

methylation marks, with a preference for trimethylated H3K4 (H3K4me3).

Studies of H3K4 methylation and its biological roles have been vast and the

majority of studies report the presence of methylated H3K4 as a sign of

transcriptional activation(Barski et al, 2007; Pokholok et al, 2005; Schubeler

et al, 2004);. H3K4me3 localized to gene promoters allows for

transcriptional activation by binding a subunit of TFIID, which then leads to

the formation of the initiation complex(Sims et al, 2003; Vermeulen et al,

2007). Though both mono- and di-methylated versions of H3K4 span

further into the transcribed protein and have even been found at enhancer

elements(Heintzman et al, 2009; Robertson et al, 2008) H3K4me3 remains

strongly conserved to the transcriptional start site (TSS)(Cloos et al, 2008;

Kooistra & Helin, 2012; Santos-Rosa et al, 2002). As expected due to their

conserved enzymatic targets, KDM5 demethylases have been suggested as

potent transcriptional repressors through their known ability to remove this

activating mark. Recent genome studies have suggested however, that the

presence of H3K4me3 at the transcriptional start site is not sufficient to

assume active transcription (Guenther et al, 2007). Within embryonic stem

cells (ESC) for example, a very high proportion of transcriptional start sites

possess marks of both transcriptionally active, and transcriptionally silent

chromatin. These sites are said to be bivalent and represent the ability of a

non-committed cell to be poised for commitment and development(Azuara

et al, 2006; Bernstein et al, 2006). This phenomenon has also been

observed lower on the evolutionary scale, with C. elegans showing

H3K4me3 and H3K27me3 co-occupying promoters early in development

(Wang et al, 2011).This suggests that modification of this methyl mark may

represent an ability of the cell to tweak transcription in one direction or the

other, without requiring an absolute condition of “On” or “Off”. Studies of

both KDM5a and KDM5b have suggested that these demethylases actually

co-localize with their substrate, with target genes showing expression of

both the enzyme and H3K4me3(Lopez-Bigas et al, 2008; Schmitz et al,

2011). Though expression of H3K4me3 was generally found to be lower at

sites of demethylase recruitment, the methylation mark was not completely

absent, suggesting that these enzymes function to maintain low levels of

H3K4me3 but not to abolish the mark completely. This also suggests that

recruitment of additional factors may be required for full demethylase

activity of the enzyme, or that the context of the protein complex in which

the KDM5 demethylase is present may alter its enzymology.

Roles for KDM5 outside of the transcriptional start site

Additional groups have suggested a role for KDM5 family members

in intragenic regions of the genome. Liefe et al. suggest that KDM5a plays

a role in Notch-mediated silencing and that demethylation at specific

regulator elements rather than entire promoter TSS regions, is sufficient to

result in gene silencing(Liefke et al, 2010) where Xie et al. have also

recently suggested that KDM5b may play a role in intragenic transcription

and elongation of KDM5b target genes, though these results are currently

under debate (Schmitz et al, 2011; Xie et al, 2011). This adds an additional

layer of regulation, suggesting that the accuracy of these enzymes for

transcriptional regulation is most likely extremely pertinent to sensitive

biological functions within the cell, with potentially significant impact on

processes including development and differentiation, and that even the

smallest of perturbations could wholly or in part give rise to disease or

transformation.

H3K4me3 and Cellular Identity

Previous studies in D. melanogaster have shown that the KDM5

homologue Little Imaginal Disc (LID) is required for normal development to

proceed through the regulation of homeotic genes (Gildea et al, 2000)

Additionally, the homologue of KDM5 in C.elegans, rbr-2, has been found to

be both an active demethylase and to play a role in the normal

development of the nematode, dependent upon this enzymology

(Christensen et al, 2007). Knock down of rbr-2 was found to result in an

increase in H3K4me3 expression and resulted in a disruption to normal

vulval development. Most recently, rbr-2 has also been implicated in

regulation over C.elegans lifespan (Greer et al, 2010). As both flies and

worms only possess one copy of the KDM5 homologue, there is no chance

for functional redundancy. Within higher eukaryotes however, the role of

these proteins in development becomes increasingly complex.

Roles for KDM5 in higher order organisms

Though higher order organisms possess four KDM5 family members,

their roles appear, in many cases, to be functionally distinct. Knock out

studies of KDM5c in a zebrafish model leads to impaired neuronal

development. Similar phenomena are observed in rats where dendritic

development becomes impaired(Iwase et al, 2007). This suggests that any

functional redundancy exhibited by KDM5 family members does not

include the role of KDM5c in neural development. This is of interest

considering how similar KDM5c and KDM5d, in specific, are, and reiterates

the importance of target specificity and expression profile differences

between the four family members.

Knock out studies in mice continue to support functionally distinct

roles for these enzymes. Though viable and possessing only mild

behavioural abnormalities, KDM5a -/- mice have been found to have altered

transcription of several cytokine genes known to be KDM5a targets. This

has been shown to lead to aberrant hematology, altered cell cycle and a

resistance to apoptosis of hematopoietic cancers(Wang et al, 2009b).

Knockout of KDM5b in mice however, in contrast to family member KDM5a,

has been reported to be embryonic lethal around E4.5(Catchpole et al,

2011). This suggests that KDM5b is required in early embryonic

development and that this role cannot be taken over by another KDM5

family member. This early functional importance of KDM5b is somewhat to

be expected due to differences in KDM5 family member expression

profiles. Where KDM5a appears to be widely expressed through all tissues

showing high expression in the haematopoetic system(Christensen et al,

2007; Cloos et al, 2008; Klose et al, 2007; Lopez-Bigas et al, 2008) KDM5c,

an X linked gene which escapes X linked inactivation(Wu et al, 1994a; Wu

et al, 1994b) appears to have more limited expression, showing neuronal

expression patterns and playing a role in neuronal development (Iwase et

al, 2007). KDM5b shows a completely different profile, widely expressed in

ESCs and undifferentiated progenitors(Dey et al, 2008), but limited in adult

tissues: restricted to the testis and differentiating mammary gland(Barrett

et al, 2002; Lu et al, 1999). Of interest however, KDM5b is highly expressed

in several forms of cancer(Barrett et al, 2002; Barrett et al, 2007; Madsen et

al, 2003; Roesch et al, 2006; Roesch et al, 2010; Xiang et al, 2007).

Catchpole et al. additionally report the creation of a KDM5b mouse strain

containing a mutation in which the ARID domain is removed. This mutation

has previously been documented to completely obliterate the demethylase

activity of KDM5b(Tan et al, 2003; Yamane et al, 2007) though Catchpole et

al. suggest that some residual activity is a possibility (Catchpole et al,

2011). Interestingly, though these mice display what is referred to as a

“mammary phenotype” they are both viable and fertile suggesting that the

role of KDM5b in embryonic development may not hinge completely on its

enzymology (Catchpole et al, 2011). To increase the complexity of the

KDM5b knockout story, Schmitz et al. have recently suggested that they

were successful in creating a KDM5b knock out mouse that is both viable

and fertile and suggest that compensation by other family members may

rescue the knockout phenotype previously described (Schmitz et al, 2011)

KDM5; master regulators of differentiation and development

Though KDM5 family knockout mice may remain viable, distinct and

numerous defects in differentiation and development are frequently noted.

This is suggestive of a protein family involved in the regulation of

differentiation control. In 2005, Benevolenskaya et al. found the first

evidence of pRB-KDM5a complexes in cells and determined that KDM5a

was a key regulator of differentiation control by demonstrating that pRB

must displace KDM5a from key promoters in order to promote

differentiation (Benevolenskaya et al, 2005). This work was completed

previous to the knowledge of KDM5a enzymology. Further study in ESC

suggests that during differentation the removal of KDM5a from Hox genes

correlates with increased levels of H3K4me3 (Christensen et al, 2007),

consistent with its role in cellular differentation and development. Previous

work on KDM5b has found that this family member can also repress

several target genes important to differentiation including HOXA5(Yamane

et al, 2007), Brain Factor-1 (BF-1) and Pax9 (Tan et al, 2003).

Recently, work in our laboratory has suggested that KDM5b plays a

role in mouse embryonic stem cells (mESC) to maintain a population of

uncommitted progenitors. Overexpression of KDM5b in mESC was

additionally found to impair specification, and delay or destroy neural

differentiation (Dey et al, 2008). More recent studies have supported this

work, suggesting that KDM5b is required for neural differentiation, most

specifically, the generation of neural progenitors (NPC) from ESC (Schmitz

et al, 2011). KDM5b was found to occupy developmental regulator genes in

ESC, and as seen previously (Dey et al, 2008) plays a pertinent role in gene

regulation in this cell type. Their findings however, suggest that KDM5b is

dispensable for the self-renewal capacity of ESC, but absolutely required

for differentiation. Of interest, the modulation of KDM protein expression

in most cell types results in no change in global H3K4me3 levels, including

neural stem cells (NSC) (Schmitz et al, 2011) and MCF7 (Yamane et al, 2007)

after the knockdown of KDM5b; and MEFs after the knockdown of KDM5a

(Klose et al, 2007). This is however different in ESC where alteration to

KDM5b levels appears to have a direct effect on global H3K4me3 levels

(Dey et al, 2008; Schmitz et al, 2011). Genome wide chromatin studies have

suggested that the global levels of H3K4me3 decrease from the ESC stage

over the course of differentiation (Ang et al, 2011) with bivalency being

removed through demethylation of H3K4me3 positive promoters (Bernstein

et al, 2006). H3K27me3 expression however, appears to remain present.

This suggests that the presence of H3K4me3 may be required for early

development, although its removal may also represent a required

checkpoint for certain stages of differentiation. This selective removal of

H3K4me3 seems to be required for appropriate cell fate determination to

occur.

Studies in C. elegans demonstrate that the appearance of H3K4me3 is both

regulated according to cell lineage and that the deposit of this tri-

methylation is extremely dynamic (Wang et al, 2011) lending credence to

the theory that both the presence and absence of this mark may represent

significant methods of gene regulation during development. Interestingly,

recent studies categorizing the role of H3K4 methylation in fully

differentiated cells such as the cardiomyocyte adds to this work,

suggesting that maintenance of H3K4me3 is required to maintain cellular

integrity even in a non dividing, fully committed cell type (Stein et al, 2011).

This also supports an ideal where though the expression of H3K4me3 may

be required to be reduced at certain developmental check-points, that re-

expression of this mark does occur at later stages of development. All

these data together paint a picture where a fine balance between

methylation and demethylation must be maintained in both a lineage and

commitment dependent manner. Slight alterations to the expression level

or localization of, enzymes required to maintain this balance may result in

changes in levels of H3K4me3 in either a global, or gene specific manner

which, in turn, could easily result in disease or abnormal cellular

phenotypes.

Demethylation and disease; a fine balance disrupted

Known for their potent roles in development, it is of no surprise that

misregulation of several KDM5 family members has been found to play role

in several developmental diseases. Mostly targeted to the neurological

system, where several KDM5 family members have been studied as

developmental regulators, KDM5 family member involvement in diseases

other than cancer has been a target of recent study.

Past studies of KDM5c have resulted in the striking conclusion that

KDM5c regulation is pertinent to appropriate neural development. Though

it is known as an H3K4me3 demethylase, KDM5c has also been found to

recognize Histone 3 Lysine 9 trimethylation (H3K9me3) (Iwase et al, 2007),

and to play a role in RE1 silencing transcription factor (REST) mediated

repression, as it has been found to co occupy several REST target genes

(Ballas & Mandel, 2005). Loss of KDM5c causes de-repression and

increases in H3K43me at key REST targets leading to an impairment of

neuronal gene regulation (Tahiliani et al, 2007). Strikingly, KDM5c has been

found to be involved in several diseases of neurodevelopment including X

linked mental retardation/X linked Intellectual Disability (XLMR/XLID),

epilepsy, and autism spectrum disorders (ASD). Many mutations, currently

a total of more than 21, to KDM5c have been found and continue to be

found associated only with cases of XLMR (Abidi et al, 2008; Jensen et al,

2010; Santos-Reboucas et al, 2011; Tzschach et al, 2006) several of these

mutations resulting in a decrease in the ability KDM5c to recognize

H3K9me3, or to demethylate H3K4me3; suggesting that the enzyomology

of KDM5c may be linked to pathology. One novel mutation was found to

alter the start site of KDM5C, presumably resulting in a complete lack of

translation (Ounap et al, 2012). Additionally, mutations to KDM5c have been

connected to distinct symptomology within XLMR such as memory loss

(Simensen et al, 2012). This suggests that specific areas of the brain may

be targeted by KDM5c misregulation. In 2008, KDM5c was connected to

another neurocognitive phenotype when a missense mutation in exon 16

was found connected to ASD. Though several KDM5c target genes such as

BDNF and SCN2A had previously been known to show altered expression

in patients presenting with ASD, KDM5c itself had never been implicated

(Adegbola et al, 2008). KDM5c is not the only family member with a

neurodevelopmental phenotype.

KDM5b, another KDM5 family member which is a known regulator of

neurological development (Schmitz et al, 2011) has also recently been

implicated as a possible player in a congenital variant of Rett Syndrome*, a

severe neurodevelopmental disease. Molecular causes of Rett syndrome

include the persistent expression of early developmental genes (Urdinguio

et al, 2008). Although Rett syndrome is normally classified by a mutation

in the X-linked methyl-CpG-binding protein MeCP2 (Kramer & van

Bokhoven, 2009), a congenital variant showing FOXG1 truncation has

recently been discovered (Ariani et al, 2008; Bahi-Buisson et al, 2010;

*

Mencarelli et al, 2009; Papa et al, 2008) Further analysis of the FOXG1

truncation shows that in both (of the two) observed truncation events, the

domain known as the JBD or the KDM5b binding domain, is missing,

suggesting that the interaction between FOXG1 and KDM5b is pertinent to

the regulation of this disease. A reduction in KDM5b binding would result

in a series of downstream effects, causing a reduction in the ability of

FOXG1 to repress transcription. This transcriptional change would, in turn,

result in a reduction of MeCP2 binding due to a delay in neural

differentiation. This may mimic what occurs when MeCP2 itself is mutated,

resulting in a similar disease phenotype.

Taken together, this data supports the conclusion that alterations to

KDM5 proteins result not only in impaired development at the embryonic

level, but that these alterations and mutations may translate into long term

disabilities- either through functional deficits in the demethylase itself, or

through downstream effects on interacting proteins. This also provides

additional evidence that each KDM5 family member plays a unique role in

the regional and temporal control of chromatin structure, and that

compensation by additional family members may not be sufficient to result

in phenotypic rescue. (Figure 2)

Though examples of KDM5 demethylases in disease appear limited

to diseases of a neuro-developmental decree, an increased understanding

of these enzymes and how they are regulated will undoubtedly uncover a

wide range of diseases in which they contribute to pathogenesis. Research

efforts have, until recently, concentrated on understanding the roles of

these enzymes in various types of cancer, as detailed below. In many

cases KDM5s appear to play a role in turning on correct genes at an

incorrect time. This leads us to question whether these enzymes may also

play a role in degeneration in disorders such as Alzheimers and

Huntington’s disease, by encouraging incorrect signaling, leading to

alterations in neural regulatory networks later in life.

Demethylation and Cancer; a fine balance turned back on incorrectly?

Though they are currently know as transcriptional repressors

through their demethylase activity, several KDM5 family members first

garnered the attention of researchers long before their enzymology was

discovered. KDM5a, for example, was originally identified as an interaction

partner for retinoblastoma protein (pRB). As such, it was originally named

Retinoblastoma Binding Protein 2 (RBP2) (Benevolenskaya et al, 2005).

Further work on KDM5a showed that it binds to genes known to be

involved in pluripotency and is active in CD34+ and CD105+ cell

populations (known to be markers of HSCs and mesenchymal stem cells

(MSCs) respectively (Wang et al, 2009a). KDM5a target gene activation and

repression may therefore play a key role in the determination of

differentiation profiles in HSCs vs MSCs. Paired with the information

gathered from KDM5a null mice, mentioned earlier, this presents a strong

case that KDM5a may play a key role in the regulation of the haemotopoetic

system including the modulation of haematopoietic cell resistance to

apoptosis, a hallmark of several blood cancers. Multitudinous KDM5a

target genes are preferentially expressed in leukemia and lymphoma and

interestingly, KDM5a has recently been found to be a gene partner involved

in Acute Myeloid Leukemia (Wang et al, 2009a). This suggests that its

involvement in cancer may not be limited to retinoblastoma and that the

pRB/KDM5a axis may be a pertinent player in leukemia pathogenesis as

well as a regulator of differentiation and development. Additional studies

have supported the role of KDM5a in a tumour suppressor role, including a

recent study by Liefke et al. Here they suggest that the switch that

regulates Notch target genes includes KDM5a and that through this target

specific role, KDM5a may act as a potent tumour suppressor in Notch

mediated carcinogenesis (Liefke et al, 2010).

KDM5b was additionally recognized prior to its enzymology becoming

apparent. Originally known as Plu-1, this protein was first discovered as a

target up-regulated in response to Her2/c-ErbB2 in breast cancer cell lines

and primary breast cancers (Lu et al, 1999). Of limited expression in most

adult tissues, KDM5b shows consistent up regulation in breast and

prostate cancers in both human and mice, and has been suggested as a

possible oncogene in multiple cancer types. (Barrett et al, 2002; Hayami et

al, 2010; Lu et al, 1999; Roesch et al, 2010; Xiang et al, 2007; Yamane et al,

2007) Hayami et al. draw on previous work completed in breast (Yamane)

and Prostate (Xiang) cancers and demonstrate that KDM5b is directly

involved in the proliferative rate and ability of both lung and bladder cancer

cells to escape apoptosis. In concordance with other groups, they

demonstrate that reduction of KDM5b level results in alterations to the cell

cycle of tumour cells, and a reduction in oncogenic potential (Hayami et al,

2010). Delineating the exact role that KDM5b exerts in cancer has become

complex and more and more evidence points towards the theory that

cancer should be categorized as a group of diseases, rather than a single

dysfunction. KDM5b is known to be a regulator of both oncogenes and

tumour suppressors through direct interaction with their promoters, such

as BRCA1 in breast cancer (Yamane et al, 2007). It has also been

associated with cell cycle control in both an accelerating (breast cancer)

(Yamane et al, 2007) and decelerating (Melanoma) (Roesch et al, 2010)

fashion and has been found to increase the invasive potential of non

invasive cell types through repression of the tumour suppressor KAT5

(Yoshida et al, 2011). Recently, KDM5b has also been demonstrated to

promote cell cycle progression in breast cancer cells by the epigenetic

modulation of the expression of micro RNA let7e suggesting an additional,

indirect method to regulate of gene expression (Mitra et al, 2011).

In the recent years, another role of KDM5b in tumor survival

has surfaced, suggesting that KDM5b may be required for the adaptation of

cells to hypoxia. Solid tumors are considered to be highly hypoxic

compared to surrounding tissue, and adaptation to this state is pertinent

for tumor survival (Semenza, 2003). Adaptation to hypoxia is driven

through Hypoxia inducible factor-1 (HIF-1) and is largely mediated through

transcriptional repression. Recent screens for proteins that facilitate this

adaptation noted several Jumonji family demethylases, including KDM5b.

KDM5b was found to be a direct HIF target and shows increased

expression under hypoxic conditions (Xia et al, 2009). Previous work has

shown that reduced H3K4 methylation is linked to poor prognosis in cancer

patients (Seligson et al, 2005), suggesting that an ability to demethylate

H3K4 is important for tumor survival. Due to the requirement of

dioxygenases such as KDM5b, for molecular oxygen, Xia et al. propose that

the increased expression level of these enzymes may represent a

compensatory mechanism in response to decreasing oxygen availability

(Xia et al, 2009). Without this compensatory mechanism, H3K4me3 levels

would be expected to increase as tumors increase in size and oxygen

levels decrease, leading to the death of the hypoxic tumor cells. An

increase in the expression level of demethylases such as KDM5b may

provide a mechanism for the tumor to maintain low H3K4me3 levels even in

situations where decreased oxygen levels are present. This novel

mechanism may allow tumors to literally skirt death and continue to

proliferate.

KDM5c, a demethylase more commonly thought to exert

control over neuronal identity, is over expressed in both prostate tumors

and seminomas, and has been shown to act as a co-repressor to Smad3.

Binding of KDM5c to Smad3 blocks its transactivation ability, thus

reducing its ability to act as an effector of the TGF-B pathway. Blockage of

this pathway is apparent in several cancer types suggesting that KDM5c

may possess oncogenic potential through its ability to block Smad3. Most

interestingly, this appears to be independent of its demethylase activity

(Kim et al, 2008). Recently, Niu et al. explored the role of KDM5c in clear

cell renal cell carcinoma (ccRCC). A high proportion of ccRCCs show

inactivation of the tumour suppressor von Hippel-Lindau (VHL).

Additionally, VHL-/- tumours show decreased levels of H3K4me3 compared

to their VHL +/+ counterparts. Interestingly, this was also shown to be

Hypoxia inducible factor- (HIF1-) dependent. Previous work, demonstrating

gene alterations in patient samples of ccRCC, had provided evidence that

mutations to KDM5c were higher than would be expected by chance in

ccRCC patients (Dalgliesh et al, 2010), suggesting a connection between

KDM5c alterations and aberrant levels of H3K4me3. Niu et al. have shown

that KDM5c is responsible for suppressing HIF response genes by removal

of H3K4me3, and that mutations to KDM5c are promote tumour growth.

This tumour suppressor role of KDM5c is specific to this family member as

loss of KDM5c (but not KDM5a or KDM5b) abolished the difference between

VHL-/- and +/+ tumors (Niu et al, 2012).

Given their role in stem cell biology and development, we

are left to question whether KDM5s simply do the “right” job at the “wrong”

time in cancers; exerting control similar to non-pathogenic contexts during

differentiation and development, but with aberrant results within a fully

developed tissue. The roles of KDM5s during carcinogenesis appear to

focus on helping tumour cells to survive in contexts when appropriate

cellular signaling would lead to cell death; survival of hypoxia, escaping

apoptosis, increasing potential for invasion, and alterations to cell cycle

leading to over proliferation and the development of inappropriate cell

types. However, information on the roles of these proteins are often

contradictory, with several being classified as proteins with both

oncogenic and tumour suppressor abilities depending on cellular context.

Though, as previously mentioned, reduced H3K4 methylation levels appear

to be linked to poor prognosis in cancer patients (Seligson et al, 2005), in

the case presented above, increased H3K4 in the context of HIF response

genes in ccRCC appears to be tumour-promoting. This again draws

attention to the fine balance of H3K4me3 expression and the regulation of

the enzymes that control this methylation, both are highly dependent upon

cellular context.

KDM5s in tumour sub populations

Several groups have now suggested that KDM5 family

members exert control in specific subsets of a tumour population to

maintain or promote growth. Sharma et al. noted a population of

“reversibly drug tolerant” cells within several human cancers which

maintain viability through an altered chromatin state requiring KDM5a.

These cells appear absolutely required to protect tumors from eradication

(Sharma et al, 2010). Roesch et al. show another angle of the KDM5 cancer

story, using the expression of KDM5b as a biomarker to flag a small

population of slow cycling cells within the heterogeneous population of a

melanoma (Roesch et al, 2010). These “slow” cells appear to be required

for tumour maintenance, giving rise to progeny which express low levels of

KDM5b, and knock down of KDM5b results in an exhaustion of tumour

growth. Interestingly the same group has also proposed that KDM5b has a

tumour suppressor role (Roesch et al, 2006; Roesch et al, 2008). It has

been suggested that the acceleration of cell cycle in these melanocytes

after KDM5b expression decrease may be due to a derepression of E2F-

target genes, thus accelerating cell cycle. Both KDM5b and KDM5a have

been shown to be members of the Rb repression complex, required for the

repression of E2F target genes during senescence (Chicas et al, 2012;

Nijwening et al, 2011). Though repression of E2F targets would generally be

considered a tumour suppressive function, mutations to Rb are common in

cancer progression, allowing pro-proliferative effects to override normal

suppression and could lead to increased oncogenic potential. Following in

this theory, loss of KDM5a in a pRb defective tumour context promotes

senescence and differentiation, suggestive of an oncogenic role in the

absence of Rb (Lin et al, 2011). As noted by Chicas et al., this highlights

the context- dependent role of these demethylases (Chicas et al, 2012).

These results together suggest that though they are involved in

oncogenesis, KDM5s appear to exert their “tumourogenic potential” in

different ways, depending on cellular context and may respond differently

depending on which upstream cellular cues become activated (Figure 3).

These aspects of KDM5 demethylases, though complex,

make them potentially lucrative targets for pharmaceutical intervention.

Enzymes are known to provide excellent drug targets and KDM5b in

particular, due to its low expression level in most adult human tissues, may

provide a potentially safe target for pharmaceuticals. Immunotherapy

approaches against KDM5b have been investigated recently with results

suggesting that KDM5b may represent a tumour associated antigen (TAA)

for breast cancer (Coleman et al, 2010).

The major question that remains for future clinical use of

KDM5 targeting therapeutics is: How can we utilize this knowledge of

KDM5 biology to combat cancer and disease? Histone deacetylase

inhibitors have long been the “king” of the epigenetic pharmaceutical

industry, with drugs such as Valproic acid, Entinostat and Romadepsin

showing large potential in the clinic and earning FDA approval (Song et al,

2011). However, little has been done targeting demethylase enzymes as

possible treatment options. Recent studies have demonstrated the release

of therapeutic agents against KDM1 and studies of agents against JMJD2

demethylases (Hamada et al, 2010), and novel assays are being developed

to screen and identify novel candidates against these targets (Yu et al,

2012). The KDM5 family is not special in this contextual activity. The

importance of context and the flexibility that KDMs in general bring to

transcriptional control is the key to a variety of processes. Understanding

how and when the KDMs interact with both each other and the basal

transcriptional machinery will likely provide clues into a myriad of

diseases.

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