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Histone demethylase and it srole in cell biology review


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This document provides a scientific review of the histone demethylase enzymes; particularly the H3K4 demethlases (KDM5 family) focusing on their role in cell biology. This review was written in 2014

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Histone demethylase and it srole in cell biology review

  1. 1. Histone demethylation enzymes and dynamic cell biology Leanne Stalker and Christopher Wynder2 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.
  2. 2. 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).
  3. 3. 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
  4. 4. 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)
  5. 5. 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).
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. 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.
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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,
  15. 15. 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
  16. 16. 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).
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. 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.
  21. 21. 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
  22. 22. 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
  23. 23. 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,
  24. 24. 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
  25. 25. 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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