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histone methylation in hypoxia: a review of the set domain family and set family
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Histone methylation in hypoxia .
Histone methyltransferases and demethylases .
Histone methylation generally occurs at lysine and arginine residues. The well-known lysine methylation sites include H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20, and arginine methylation sites include H3R2, H3R8, H3R17, H3R26, and H4R3 15 . The transcriptional effects of histone methylation depend on the methylation site. For example, methylation of H3K4, H3K36, H3K79, and H3R17 is found in transcriptionally active regions, whereas methylation of H3K9, H3K27, and H4K20 is found in transcriptionally repressed regions.
The various histone methyltransferases responsible for these types of methylation are categorized into three families: the SET (Su[var]3–9, Enhancer of Zeste, Trithorax) domain family, the DOT1L (DOT1-like) family, and the PRMT (protein arginine N-methyltransferase) family 15 , 16 . The SET domain family is divided into four subfamilies, namely, SUV39, SET1, SET2, and RIZ, as well as others that remain unclassified. Specifically, the SUV39 subfamily includes SUV39H1/2 and ESET for H3K9me2/3 and G9a/GLP for H3K9me1/2. In addition, the SET1 family includes SETD1A, SETD1B, and MLL1–4 for H3K4me1/2/3 and EZH1/2 for H3K27me2/3. In addition, the representative members of the SET2 family are NSD1 and NSD2, which are responsible for H3K36me3 and H3K36me2/3, respectively. Moreover, the nonclassified members, such as the SMYD subfamily, SUV4–20 subfamily, and SET7/9, are related to H3K4me2/3, H4K20me2/3, and H3K4me1, respectively. Furthermore, DOT1L is the only member of the DOT1L family catalyzing H3K79 methylation 17 . Last, the PRMT family is divided into 3 types: types I, II, and III. The type I PRMTs are PRMT1, 2, 3, 4, 6, and 8, which facilitate monomethylation and asymmetric dimethylation, whereas the type II PRMTs are PRMT5 and PRMT9, which perform mono- and symmetric dimethylation. PRMT7, the only member of the type III PRMT subgroup, is responsible for arginine monomethylation 17 .
Histone demethylases or lysine demethylases (KDMs), which contribute to protein methylation homeostasis, can be classified into 2 families—lysine-specific demethylases (LSDs) and JmjC domain-containing histone demethylases (JMJC demethylases)—according to their catalytic mechanisms 16 , 18 . The LSD family, containing LSD1 and LSD2, exhibits flavin adenine dinucleotide-dependent amine oxidation activity for catalytic reactions. LSD1 demethylates H3K4me1/2 and H3K9me1/2 in concert with interacting proteins, whereas LSD2 demethylates only H3K4me1/2. In contrast, JMJC demethylases are members of the 2-oxoglutarate-dependent dioxygenase superfamily; they use Fe 2+ and oxygen to remove methyl groups through hydroxylation. KDM2–6 belong to the JMJC demethylase family. The KDM2 subfamily members, KDM2A and KDM2B, demethylate H3K36me2, while KDM2B demethylates H3K4me3. In addition, the KDM3 subfamily members, KDM3A and KDM3B, remove H3K9me1/2. Moreover, the KDM4 subfamily members, KDM4A-D, are involved in removing H3K9me2/3 and H3K36me2/3. Furthermore, the KDM5 subfamily members, KDM5A-D, are responsible for the demethylation of H3K4me2/3. Last, the KDM6 family members, KDM6A and KDM6B, are related to the removal of H3K27me2/3 18 .
Effects of histone methyltransferases under hypoxic conditions .
The SET domain family plays a major role in regulating gene expression in response to hypoxia (Tables 1 and 2 ) 19 . Among the SET domain family members, G9a/GLP has been extensively studied regarding its function under hypoxic conditions 20 . However, the role of G9a/GLP in transcription under hypoxic conditions remains controversial. G9a/GLP activates or represses hypoxia-inducible genes depending on the target of methylation. For example, Reptin and Pontin are chromatin remodeling factors methylated by G9a during hypoxia. Reptin methylated at Lys67 binds to the promoters of hypoxia-responsive genes, such as PGK1 and VEGF , and represses the transcription of these genes, resulting in negative regulation of hypoxic responses 21 . On the other hand, methylation of Pontin by G9a/GLP under hypoxic conditions increases the recruitment of p300 and HIF-1α to the promoters of HIF-1α target genes, including Est1 , thereby activating the expression of these target genes 22 . According to Bao et al., HIF-1α is methylated by G9a under hypoxic conditions; methylation at Lys674 of HIF-1α inhibits its transactivation domain activity, repressing the transcription of NDNF and SLC6A3 23 . Another study revealed that hypoxia increases G9a stability by reducing prolyl hydroxylation-mediated G9a degradation. Then, G9a suppresses transcription under hypoxic conditions by promoting H3K9 methylation in the promoter region of tumor suppressor genes, including HHEX , GATA2 , and ARNTL 24 .
Table 1 Histone methylation in hypoxia. Full size table
Table 2 Nonhistone methylation in hypoxia. Full size table
Several HMTs in the SET1 family are also associated with hypoxic responses. According to Heddleston et al., hypoxia-induced MLL1 increases the expression of HIF-2α, and inhibition of MLL1 decreases H3K4m3 levels while increasing H3K27m3 levels. These findings indicate that MLL1 regulates HIF-2α transcription via histone modification 25 . A recent study showed that SETD1B contributes to the activation of hypoxia-inducible genes. SETD1B associated with the HIF complex can be localized in the promoter region of hypoxia-related genes, such as CA9 , PHD3 , and VEGF , increasing the H3K4me3 levels at these loci. Therefore, in response to hypoxia, the HIF complex recruits the H3K4 methyltransferase SETD1B to facilitate the transcription of HIF target genes 26 .
In addition, EZH2 is involved in TWIST-induced epithelial-mesenchymal transition (EMT) under hypoxic conditions in pancreatic cancer cells. Hypoxia increases TWIST expression, which represses the transcription of E-cadherin and p16INK4A . TWIST overexpressed due to hypoxia interacts with EZH2 and Ring1B and binds to the promoters of E-cadherin and p16INK4A , increasing H3K27me3 and H2AK119ub1 in the promoter of E-cadherin 27 . In addition, under normoxic conditions, EZH2 modulates the expression of HIF-1α through H3K27 methylation in the promoter of HIF-1α. Furthermore, EZH2 is guided to the HIF-1α gene promoter via the lncRNA HITT , a hypoxia-responsive lncRNA whose expression decreases with increasing hypoxia. Thus, as HITT is downregulated under hypoxic conditions, the recruitment of EZH2 to the promoter of HIF-1α is reduced, increasing the expression of HIF-1α 28 .
SETD3 and SETD7, other members of the SET domain-containing methyltransferase family, are also known for their regulation of gene expression under hypoxic conditions 29 , 30 . Under normoxic conditions, SETD7 methylates HIF-1α at K32, blocking the transcriptional activity of HIF-1α and in turn repressing the expression of HIF-1α target genes, including LDHA , PDK , and VEGF 29 . Furthermore, methylation of HIF-1α at K32 by SET7/9 in the nucleus decreases the stability of HIF-1α 31 . Under hypoxic conditions, the SET7 protein level is reduced, which increases the stability and transactivity of HIF-1α, thereby inducing the expression of HIF-1α target genes. In addition, SETD3 is a negative regulator of VEGF expression during hypoxia 32 , 33 . Moreover, SETD3 interacts with and methylates FOXM1, which binds to the promoter of VEGF . Hypoxia decreases the SETD3 level, leading to the disassociation of SETD3 and FoxM1 from the VEGF promoter.
Taken together, these observations indicate that HMTs differentially regulate gene expression in hypoxia by methylating nonhistone proteins and histones. It is likely that methylation of nonhistone proteins typically affects their stability and/or interaction with other proteins (e.g., transcription factors). Moreover, hypoxia drives HMTs to cooperate with transcription factors or lncRNAs to control histone methylation. Eventually, these methylation events may modulate gene expression, leading to adaptation to cellular hypoxia.
Effects of histone demethylases under hypoxic conditions .
According to a growing body of evidence, hypoxia affects the gene expression and functions of histone demethylases (Tables 1 and 2 ) 2 . For example, LSD1 affects hypoxic responses by demethylating HIF-1α and histones. The demethylase activity of LSD1 toward HIF-1α facilitates HIF-1α stabilization by inhibiting VHL-induced HIF-1α degradation. Recent studies have shown that LSD1 demethylates HIF-1α at K32 and K391 in response to hypoxia-mimicking conditions 31 , 34 . In addition, pharmacological inhibition or siRNA-mediated silencing of LSD1 expression effectively reduces the HIF-1α protein level 35 . Furthermore, LSD1 increases the transcription of MTA1 via H3K9 demethylation in the promoter region of MTA1 , enhancing NuRD complex-mediated deacetylation of HIF-1 34 .
JMJC demethylases require oxygen to remove methyl groups. The results of many studies indicate that some JMJC demethylases are inactivated as oxygen availability decreases and that their expression is upregulated to compensate for the reduced enzymatic activity 1 , 36 . KDM3A and KDM4B are upregulated via HIF-1α in hypoxia 36 , 37 , 38 . Although the expression of KDM3A and KDM4B is induced by hypoxia, the levels of H3K9me2 and H3K9me3 are unchanged or even increased 37 , 39 . Chromatin immunoprecipitation assays in macrophages revealed increases in repressive marks H3K9me2 and H3K9me3 in the specific promoter regions of Ccl2 , Ccr1 , and Ccr5 that resulted in decreases in their expression under hypoxic conditions (1% O 2 ) 39 . These results suggest that hypoxia suppresses the demethylase activity of KDM3A and KDM4B while increasing their expression levels. In contrast, another study revealed that hypoxia in prostate cancer cells increased the expression of KDM3A and that its catalytic activity was maintained under severe hypoxic conditions (0.5% O 2 ) 40 . KDM3A occupies the PSA enhancer region, demethylating H3K9me1 and H3K9me2. This recruits p300 and MLL4, thereby resulting in the addition of active histone marks (i.e., H3K9ac and H3K4me3) and increased gene expression. Furthermore, Mimura et al. reported that HIF-1 and KDM3A upregulate glycolytic genes in response to hypoxia (1% O 2 ) independent of cell type 41 . In particular, KDM3A is recruited to the SLCA3 locus in a HIF-1-dependent manner and demethylates H3K9me2. In some cases, the KDM4 subfamily members, which also regulate HIF genes, exhibit increased expression levels and are functional in hypoxia. According to Dobrynin et al., KDM4A stimulates the expression of HIF-1α by removing a methyl group from H3K9me3 at the HIF-1α locus under mild hypoxic conditions (2% O 2 ). Loss of KDM4A decreases the HIF-1 α mRNA level and HIF-1α protein stability, thus reducing the HIF-1α level 42 . However, KDM4A demethylase activity is abolished under more severe hypoxic conditions (less than 0.1% O 2 ). Similarly, Hancock et al. showed that KDM4A enzymatic activity decreased gradually with oxygen depletion (0.1–5% O 2 ) 43 . These results suggest that KDM4A acts as an oxygen sensor. KDM4B expression is induced in a HIF-1α-dependent manner under hypoxic conditions (1% O 2 ); it upregulates the expression of a subset of hypoxia-inducible genes by decreasing H3K9me3 in their promoters 44 . KDM4C expression is also induced under hypoxic conditions (1% O 2 ). KDM4C selectively interacts with HIF-1α, which mediates the recruitment of KDM4C to the HREs in HIF-1 target genes, allowing KDM4C to decrease H3K9me3 and promote the binding of HIF-1 to the HREs, thereby activating the transcription of BNIP3 , LDHA , PDK1 , and SLC2A1 45 . These results suggest that KDM4 demethylase activity is maintained or decreased based on the hypoxia status.
Some histone demethylases, such as KDM5A, KDM6A, and KDM6B, act as direct oxygen sensors 46 . According to Batie et al., KDM5A inactivation under hypoxic conditions (1% O 2 ) is related to hypermethylation of H3K4 in cancer cells. In addition, hypoxia causes a rapid increase in global histone methylation independent of HIF. KDM5A, upon sensing low oxygen levels under hypoxic conditions, becomes enzymatically inactivated, thus inhibiting the removal of a methyl group from H3K4me3 in the promoters of hypoxia-inducible genes, such as BNIP3L and KLF10 47 . Consistent with this finding, KDM5A demethylase activity is decreased during hypoxia (1% O 2 ) in lung cancer cells, which increases the H3K4me3 levels in the promoters of the HMOX1 and DAF genes 48 . Chakraborty et al. also showed that KDM6A senses oxygen, determining cell fate. They found that hypoxia (2–5% O 2 ) induces HIF-independent hypermethylation at H3K27. Hypoxia blocks C2C12 cell myogenic differentiation, which is not due to HIF activation and 2-hydroxyglutarate. Similar to their effects on hypoxia, treatment with the KDM6 family inhibitor GSK-J4 and knockdown of KDM6A inhibited myogenic differentiation and increased the level of H3K27me3, a repressive mark. During muscle differentiation, the reduction in H3K27me3 at late myogenic genes, such as Actc , Myl1 , and Myog , is blunted by hypoxia. These results suggest that KDM6A inactivation by hypoxia increases H3K27me3 levels and inhibits transcriptional activation of genes involved in differentiation 49 . On the other hand, Li et al. reported that hypoxia (1% O 2 ) in cardiomyocytes significantly upregulates KDM6A expression, which increases the expression of Ncx , encoding the Na+/Ca 2 + exchanger, by reducing the H3K27me3 level in the Ncx promoter, thus decreasing intracellular calcium influx 50 . Liu et al. showed that hypoxia (1% O 2 ) induces KDM6B expression, which elevates VEGF gene expression and angiogenesis via removal of H3K27me3 51 . However, another study reported that severe hypoxia (0.1% O 2 ) increases genome-wide H3K27me3, suggesting that the changes in the chromatin state in response to hypoxia are due to the inactivation of KDM6B 52 .
Considering these findings, it is clear that hypoxia increases the expression of some JMJC demethylases. However, the demethylase activity of JMJC demethylases such as KDM3–6 under hypoxic conditions is still incompletely understood. Further studies will be needed to clarify this issue. .
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