**4. HIF regulation in epigenetic modification**

Wadding first proposed the concept of epigenetics in 1915 and believed that the phenotypes generated from certain genotype within the scope of epigenetics. Meanwhile, he explained the expression of the genetic materials in the entire life process for the first time by using the concept of "Whole View" [25]. Holiday summarized with a more comprehensive explanation that "epigenetics refers to the changes of the gene expression manner with no difference and/ or change of heritage DNA sequence" [26]. Epigenetics is involved in individual development and the gene expression regulation in the biological process, however, it could also lead to human diseases when it is interfered [27].

#### **4.1. Histone acetylation**

oxygen sensor called factor-inhibiting HIF-1 (FIH-1) involves hydroxylation of an asparagine residue (Asn803) in the C-TAD of HIF-1α, which also utilizes oxygen as a substrate [11]. The hydroxyl modification of Asn803 blocks the binding of the cofactor proteins) CREB-binding protein (CBP) and p300 thus inhibit HIF transcriptional activity. In hypoxia, HIF-α is regulated through "bicephalous" transcriptional nature in an FIH-dependent or FIH-independent manner [12]. In short, PHD has a lower affinity for oxygen than HIF and therefore is more rapidly inhibited. Consequently, genes require only the N-TAD to be induced. As oxygen decreases further, the inhibition of C-TAD is released and HIF-1α retains full transcription activity.

The metabolic intermediates are also the key regulators disrupting the hemostasis of HIF activates. As mentioned earlier, PHDs are 2-OG-dependent dioxygenases, catalyzing the conversion of a prolyl residue, molecular oxygen, 2-OG to hydroxyprolyl, carbon dioxide, and succinate using ferrous iron as cofactor. In addition, succinate also intermediates in the tricarboxylic acid (TCA) cycle catalyzed by succinate dehydrogenase (SDH) to fumarate in mitochondria. SDH dysfunction in cells raises the levels of succinate, which accumulates and leaks out to cytosol [13]. The increased level of succinate also inhibits PHDs activity due to accumulation succinate feedback, leading to the stabilization of HIF-α and activation of HIF complex. Similarly, deficiency of fumarate hydratase (FH) leads to accumulation of fumarate in the cytosol. Due to chemical similarity of fumarate to succinate, FH-deficient cells could also inhibit PHDs [14]. Other metabolic changes, such as diseases related to iron homeostasis, also crosstalk with HIF regulation. Hepcidin, a small polypeptide, plays a central role in regulating iron uptake. Iron demand in bone marrow increases when erythropoiesis is stimulated by hypoxia via increased erythropoietin (EPO) synthesis. Iron overload disease like hemochromatosis and iron decrease in anemia, feedback hepcidin production through VHL-HIF regulation [15].

HIF is the central master regulator of adaptation to decreased oxygen availability in both physiological and pathological conditions. It is evolutionary pressure to reestablish metabolic balance to allow normal tissue and/or even tumor to survive. Physiologically, in the woundhealing area, damaged tissue leads to hypoxia and facilitates vascular growth. However, pathologically, in the solid tumor region, oxygen demand is in continuous increase due to the uncontrollable growth of the cancer cell. Hypoxia also represents the unifying feature of the microenvironment of solid tumors. The adaptive changes of tumor survival pattern referred

HIF upregulation in tumors plays a central role in metabolic switch from aerobic metabolism to anaerobic metabolism. In turn, all the enzymes (e.g., aldolase A and C, enolase 1, hexokinase 1 and 2, pyruvate kinase M (PKM), phosphofructokinase) and glucose transporters (GLUT1, GLUT3) involved in glycolytic pathway are upregulated [16]. Moreover,

**2.3. Metabolic-dependent HIF regulation**

108 Myeloid Leukemia

**3. HIF regulation in mitochondria metabolic change**

to as "hypoxia tumor phenotype" are greatly noticed.

**3.1. HIF regulation of metabolic change**

Histone acetylation, a process closely related to transcriptional activation is one of major epigenetic modifications. Histone acetylation occurs in the lysine residue located at the end of the histone N-terminal. Histone acetylation induces relaxed and/or higher-order structure of chromatins through transcriptional regulation [28]. In addition, the acetylated histone produces a signal that binds to the protein, producing intrinsic activities or capped-chromatin remodeling complexes, thereby promoting the transcriptional induction. Histone acetylation is mainly controlled by the inhibitory activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), and the substrates of HDAC include non-histone proteins, some transcription factors, and cofactors [29]. As a consequence, histone deacetylases generally inhibit transcription. It is also interesting that they negatively regulate HIF-dependent transcriptions. Previous studies have shown that methylation of Reptin at lysine 67 in hypoxia condition by the methyltransferase G9a negatively regulates hypoxic responses [30]. Consequently, while in hypoxia condition, the chromatin remodeling factor HIF-1α Reptin binds to HDAC1, the target gene of HIF is involved in the supplementation of HDAC1, besides, HDAC4 and HDAC5 complement the expression of HIF target promoter in HIF-dependent transcription to become more active [31]. Moreover, histone deacetylase inhibitors could promote gene expression in the transcription of several HIF target promoters and induce inhibition of HIFdependent angiogenesis.

Hypoxia-induced histone methylation might be achieved by the partial inhibition of spherical JHDMS and the reduction of histone methylation of some hypoxia response promoters. Interestingly, Jumonji domain-containing protein 1A (JMJD1A) is the HIF target gene itself. Krieg and his colleagues suggested the regulation of feed-forward mechanism in which HIF might represent for the likely HIF-dependent gene expression of JMJD1A [36]. They suggested that JMJD1A maintained the apparent genetic pattern of the activities of the target promoters, thereby minimizing the required energy-supported expression. JMJD1A indicates that more consideration should be given to the induction of differential genes and other JHDMS involved in the activation of hypoxia-responsive genes. Further research is required to deter-

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Although there is a detailed study of hypoxia-induced conditions on JHDM, this kind of enzyme in hypoxia requires one or more RNA interference-silencing experiment targeting studies of JHDMS or multiple biological effects of JHDMS response. In the apparent regulation of hypoxia, histone modification and chromatin remodeling caused by relative enzymes also play a key role. Hypoxia-induced histone acetylation has become a highly suspected etiology of Alzheimer's disease and attention deficit hyperactivity disorder (ADHD) [37].

Evidence suggests that the increase of H3K9me2 is partly due to hypoxia-induced G9A methyltransferase. During hypoxia, H3K9me2 induces the increase of certain gene promoters as well. Further studies are required to assess the effects of hypoxia-induced epigenetic alterations on the organisms. To activate the gene transcription, a series of specific HIF-targeted genes promoter region is commonly regulated through histone methylation, acetylation, or alteration of chromatin structures. On the other hand, hypoxia could stimulate the inhibition of induced transcription, possibly by supporting the changes of the whole chromatin. Thus, it seems that hypoxia plays a dual role in the studies of epigenetic mechanisms of the genes as well as in controlling the induction and transcriptional downregulation of the HIF target

HIF will be activated when hypoxia occurs in vivo. As a consequence, transcription of more than 100 genes, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO), can be induced. However, the activation of HIF could be accompanied with significant decline in the activity in many other transcription factors. However, hypoxia-induced gene modulation is not limited to HIF activation. In contrast, epigenetic modification can be involved in this process. The epigenetic mechanisms play dual roles in hypoxia, meaning that they not only upregulate the HIF-controlled target genes but also downregulate the general transcrip-

In conclusion, hypoxia could induce extensive histone modifications that are usually associated with transcriptional repression or activation. Specifically, however, more research is needed to fully understand its biological functions and to identify the enzymes involved in signal transduction pathways. It provides holistic assessments regarding hypoxia on the epigenetic changes. Under hypoxia conditions, the following epigenetic changes were greatly noticed: (1) p300/CBP histone acetyltransferases interact with HIF and acetylate histones in HIF target promoters. HDAC4, HDAC5, or HDAC7 form a multi-protein complex with HIF-p300 increasing HIF transcriptional activity. HDAC4 and HDAC5 exert their effects by

tion factors. However, the specific mechanisms remain to be further explored.

mine whether this is true.

gene.

The mechanisms of the HDAC-dependent gene activation are still not well defined. However, it becomes increasingly apparent that the HIF C-TAD-p300/CBP interactions are necessary. There are evidences suggesting the targets of deacetylated p300/CBP and HIF. In this concept, HIF, P300 and HDAC4, HDAC5, or HDAC7 have been reported to form multi-polyprotein complexes [31, 32]. This also shows that HDAC4 and HDAC5 could promote the binding between HIF-1 and p300, thereby enhancing the expression of HIF target genes. However, the gene expressions regulated by epigenetic mechanisms that are involved in the hypoxia response are different. It is generally separated into following steps: (1) HIF recruit coactivator enhancing the expression of HIF. (2) The interaction between HIF-p300 and CBP by the inhibition of hypoxia to induce HIF-1 expression. (3) HDAC4 and HDAC5 can promote the interactions between HIF-1α and p300. (4) HDAC4 and HDAC5 promote the interaction between HIF-1α and p300. (5) SWI/SNF complexes alter the chromatin structures in some HIF target promoters and enhancers to benefit their expression [33].

Specifically, hypoxia promotes the changes in the status of the hypoxia-induced gene promoter histone methylation: activation of hypoxia histone demethylase and inhibition of JMJD1A histone demethylase, which can cause H3K9me2 reduction and H3K4me2 increase, respectively, thus enhancing the gene expression [34, 35]. In addition, hypoxia could increase the expression of some HIF target promoters H3K27me3 and H3K4me3. Under hypoxia conditions, the interactions of HIF1-α and Reptin are enhanced, leading to some HDAC1 supplementation of the HIF target genes and negative regulation of transcription; the changes in the status of histone methylation and acetylation promote hypoxia-inhibited gene. Hypoxia could increase the levels of H3K9me2 and H3K4me3, and decrease the levels of H3K27me3 and H3K9ac.

#### **4.2. Histone methylation**

Histone methylation, as another main epigenetic modification, is a stringent regulatory process, which relies on the activities of histone methyltransferase and histone demethylase. Histone demethylase induces the dynamic equilibrium of the histone methylation during hypoxia. During hypoxia, histone demethylase can increase the expression of these enzymes, and the decreased enzyme activity can be regulated completely or partly by oxygen deficiency. Some histone lysine methylation can be specifically prevented under hypoxic conditions. Hypoxia-induced histone methylation might be achieved by the partial inhibition of spherical JHDMS and the reduction of histone methylation of some hypoxia response promoters. Interestingly, Jumonji domain-containing protein 1A (JMJD1A) is the HIF target gene itself. Krieg and his colleagues suggested the regulation of feed-forward mechanism in which HIF might represent for the likely HIF-dependent gene expression of JMJD1A [36]. They suggested that JMJD1A maintained the apparent genetic pattern of the activities of the target promoters, thereby minimizing the required energy-supported expression. JMJD1A indicates that more consideration should be given to the induction of differential genes and other JHDMS involved in the activation of hypoxia-responsive genes. Further research is required to determine whether this is true.

deacetylases (HDACs), and the substrates of HDAC include non-histone proteins, some transcription factors, and cofactors [29]. As a consequence, histone deacetylases generally inhibit transcription. It is also interesting that they negatively regulate HIF-dependent transcriptions. Previous studies have shown that methylation of Reptin at lysine 67 in hypoxia condition by the methyltransferase G9a negatively regulates hypoxic responses [30]. Consequently, while in hypoxia condition, the chromatin remodeling factor HIF-1α Reptin binds to HDAC1, the target gene of HIF is involved in the supplementation of HDAC1, besides, HDAC4 and HDAC5 complement the expression of HIF target promoter in HIF-dependent transcription to become more active [31]. Moreover, histone deacetylase inhibitors could promote gene expression in the transcription of several HIF target promoters and induce inhibition of HIF-

The mechanisms of the HDAC-dependent gene activation are still not well defined. However, it becomes increasingly apparent that the HIF C-TAD-p300/CBP interactions are necessary. There are evidences suggesting the targets of deacetylated p300/CBP and HIF. In this concept, HIF, P300 and HDAC4, HDAC5, or HDAC7 have been reported to form multi-polyprotein complexes [31, 32]. This also shows that HDAC4 and HDAC5 could promote the binding between HIF-1 and p300, thereby enhancing the expression of HIF target genes. However, the gene expressions regulated by epigenetic mechanisms that are involved in the hypoxia response are different. It is generally separated into following steps: (1) HIF recruit coactivator enhancing the expression of HIF. (2) The interaction between HIF-p300 and CBP by the inhibition of hypoxia to induce HIF-1 expression. (3) HDAC4 and HDAC5 can promote the interactions between HIF-1α and p300. (4) HDAC4 and HDAC5 promote the interaction between HIF-1α and p300. (5) SWI/SNF complexes alter the chromatin structures in some HIF

Specifically, hypoxia promotes the changes in the status of the hypoxia-induced gene promoter histone methylation: activation of hypoxia histone demethylase and inhibition of JMJD1A histone demethylase, which can cause H3K9me2 reduction and H3K4me2 increase, respectively, thus enhancing the gene expression [34, 35]. In addition, hypoxia could increase the expression of some HIF target promoters H3K27me3 and H3K4me3. Under hypoxia conditions, the interactions of HIF1-α and Reptin are enhanced, leading to some HDAC1 supplementation of the HIF target genes and negative regulation of transcription; the changes in the status of histone methylation and acetylation promote hypoxia-inhibited gene. Hypoxia could increase the levels of H3K9me2 and H3K4me3, and decrease the levels of H3K27me3

Histone methylation, as another main epigenetic modification, is a stringent regulatory process, which relies on the activities of histone methyltransferase and histone demethylase. Histone demethylase induces the dynamic equilibrium of the histone methylation during hypoxia. During hypoxia, histone demethylase can increase the expression of these enzymes, and the decreased enzyme activity can be regulated completely or partly by oxygen deficiency. Some histone lysine methylation can be specifically prevented under hypoxic conditions.

target promoters and enhancers to benefit their expression [33].

dependent angiogenesis.

110 Myeloid Leukemia

and H3K9ac.

**4.2. Histone methylation**

Although there is a detailed study of hypoxia-induced conditions on JHDM, this kind of enzyme in hypoxia requires one or more RNA interference-silencing experiment targeting studies of JHDMS or multiple biological effects of JHDMS response. In the apparent regulation of hypoxia, histone modification and chromatin remodeling caused by relative enzymes also play a key role. Hypoxia-induced histone acetylation has become a highly suspected etiology of Alzheimer's disease and attention deficit hyperactivity disorder (ADHD) [37].

Evidence suggests that the increase of H3K9me2 is partly due to hypoxia-induced G9A methyltransferase. During hypoxia, H3K9me2 induces the increase of certain gene promoters as well. Further studies are required to assess the effects of hypoxia-induced epigenetic alterations on the organisms. To activate the gene transcription, a series of specific HIF-targeted genes promoter region is commonly regulated through histone methylation, acetylation, or alteration of chromatin structures. On the other hand, hypoxia could stimulate the inhibition of induced transcription, possibly by supporting the changes of the whole chromatin. Thus, it seems that hypoxia plays a dual role in the studies of epigenetic mechanisms of the genes as well as in controlling the induction and transcriptional downregulation of the HIF target gene.

HIF will be activated when hypoxia occurs in vivo. As a consequence, transcription of more than 100 genes, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO), can be induced. However, the activation of HIF could be accompanied with significant decline in the activity in many other transcription factors. However, hypoxia-induced gene modulation is not limited to HIF activation. In contrast, epigenetic modification can be involved in this process. The epigenetic mechanisms play dual roles in hypoxia, meaning that they not only upregulate the HIF-controlled target genes but also downregulate the general transcription factors. However, the specific mechanisms remain to be further explored.

In conclusion, hypoxia could induce extensive histone modifications that are usually associated with transcriptional repression or activation. Specifically, however, more research is needed to fully understand its biological functions and to identify the enzymes involved in signal transduction pathways. It provides holistic assessments regarding hypoxia on the epigenetic changes. Under hypoxia conditions, the following epigenetic changes were greatly noticed: (1) p300/CBP histone acetyltransferases interact with HIF and acetylate histones in HIF target promoters. HDAC4, HDAC5, or HDAC7 form a multi-protein complex with HIF-p300 increasing HIF transcriptional activity. HDAC4 and HDAC5 exert their effects by promoting association between HIF and p300. (2) SWI/SNF are complementary gene promoters of HIF-1α, which is a requirement for the expression of HIF-1α mRNA. The regulation of SWI/SNF could also describe the profound effects of HIF-dependent responses on hypoxia. On the other hand, the SWI/SNF complex alters the chromatin structure in some HIF target promoters or enhancers, thereby favoring their expression. (3) Hypoxia activates JMJD1A which promote a decrease in H3K9me2. In the meantime, oxygen deprivation also inhibits JARID1A histones demethylases which provoke an increase in H3K4me2 levels at their target promoters, thus enhancing gene expression. In addition, hypoxia increases H3K4me3 and H3K27me3 levels in some HIF target promoters, and hypoxia-inductive H3K4me3 seems to depend on the inhibitory effects of histone demethylase [38]. The hypoxia-inducible gene promoter was also observed in EPO, HMOX1, and DAF [39, 40]. (4) The interaction between Reptin and HIF1-α is enhanced in hypoxia, leading to recruitment of HDAC1 to some HIF target genes, negatively regulating their transcription (**Figure 2**). However, more research is still needed to fully understand its biological functions and to identify the enzymes involved in signal transduction pathways. It provides holistic assessment regarding hypoxia on the epigenetic modifications.

**5. HIF and epigenetic modification in myeloid leukemia**

[41, 42].

diagnostic methods and drug discovery.

Metabolic flexibility relies on the rewiring of the existing metabolic pathways, which are closely controlled by "pathway switch proteins," to efficient rerouting of metabolites selected by cellular needs. As discussed earlier, HIF controls many aspects of tumor in terms of location, size, cell type, or local invasion. Other aspects, like state of differentiation and hierarchical nature, were also regulated by HIF. Most tumor-initiating mutations occur in stem cell or progenitor populations. The expansion of these mutant cells with a more differentiated phenotype that usually characterizes individual cancers are responsible for the cause of pathogenesis. This was first described in 1997 for acute myeloid leukemia and subsequently extended to solid tumors, including melanoma, Glioblastoma (GBM), and pancreatic cancer

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Limited oxygen access is the common feature in solid tumor due to inadequate tissue perfusion, thus, cancer metabolism is heavily influenced by adaptation to highly hypoxic microenvironment. In many cases, HIF is involved as a master regulator. Recently, an increasing number of other proteins, regulated by HIF, are found to influence energy metabolism. In addition, a series of mutations in these proteins—for example, SDH, FH, isocitrate dehydrogenase (IDH), activation-induced cytosine deaminase (AID), or drive altered metabolism. These findings have supported the notion that HIF has a role to play in oncology, and affects

In hematological tumors, bone marrow and lymph nodes represent hypoxic environments. The quiescent hematopoietic stem cells (HSCs) in the bone marrow existing in the hypoxic niche utilization of predominantly glycolysis pathway are regulated by HIF-1α stabilization [21]. Hypoxia modulates mitochondrial respiration in an HIF-1α-dependent manner. HIF promoter, the expression of pyruvate dehydrogenase kinase-1 (PDK1), in turn, inhibits pyruvate dehydrogenase (PDH) [43]. In addition, PKD1 activation is also important in inducing pluripotent stem cell, as evidenced by four Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC) sufficient to upregulate PDK1, and initiates a Warburg-like metabolic rewiring which is closely linked with conversion of pluripotency [44]. On the other hand, metabolic reprograming initially triggered by HIF stabilizes HIF expression independently of oxygen to gain tumor survival advantage. As an example, imatinib-resistant cell expresses high levels of HIF-1αs and induces BCR-ABL upregulation [45]. Recent study further suggests that HIF is the potential cause to trigger gene translocation through limiting activation-induced cytosine deaminase (AID) expression [46, 47]. In the therapeutic point of view, the stem cell nature of cancer is also reflected in removing differentiation block therapy. For example, all-*trans* retinoic acid combined with cytotoxic drug was used in the clinical practice for the treatment of acute promyelocytic leukemia (APL) [48]. Other promoting differentiation agent aurora

kinase A inhibitors were shown effective in acute megakaryocytic leukemia [49].

Other feature of leukemia cell is attenuated metabolic pathway in glycolysis even in aerobic conditions [50]. Leukemic cells, other than solid tumors, have the advantage to access oxygen; however, levels of HIF-1α, GLUT1, GLUT3, and CA4 are still significantly enhanced compared to normal blood cells. Clinical evidence shows that higher glycolytic rate in leukemic cells

**Figure 2.** HIF regulated epigenetic changes in response to hypoxia.
