Demethylases are enzymes that remove methyl (CH3) groups from nucleic acids, proteins (particularly histones), and other molecules. Demethylases are important epigenetic proteins, as they are responsible for transcriptional regulation of the genome by controlling the methylation of DNA and histones, and by extension, the chromatin state at specific gene loci.
Histone lysine demethylation
Histone methylation was initially considered an effectively irreversible process as the half-life of the histone methylation was approximately equal to the histone half-life. Histone lysine demethylase LSD1 (later classified as KDM1A) was first identified in 2004 as a nuclear amine oxidase homolog. Two main classes of histone lysine demethylases exist, defined by their mechanisms: flavin adenine dinucleotide (FAD)-dependent amine oxidases and α-ketoglutarate-dependent hydroxylase.
- FAD-dependent amine oxidase domains containing the active catalytic site of LSD1/KDM1
- Jumonji-C domains containing the active catalytic site of KDM2 through KDM8
- Jumonji-N domains responsible for Jumonji-C domain conformation stability
- SWIRM (SWI3P, RSC8P and Moira) domains proposed as an anchor site for histone substrates and responsible chromatin stability
- PHD, CXXC and C5HC2 zinc finger domains responsible for histone recognition and binding
Histone lysine demethylases are classified according to their domains and unique substrate specificities. The lysine substrates and identified according to their position in the corresponding histone amino acid sequence and methylation state (for example, H3K9me3 refers to trimethylated histone 3 lysine 9.)
- The KDM1 homologs include KDM1A and KDM1B. KDM1A (also referred to as LSD1/AOF2/BHC110) demethylates H3K4me1/2 and H3K9me1/2, and KDM1B (also referred to as LSD2/AOF1) demethylates H3K4me1/2. KDM1 activity is critical to embryogenesis and tissue-specific differentiation, as well as oocyte growth. KDM1A was the first demethylase identified and thus the most well understood.:Deletion of the gene for KDM1A can have effects on the growth and differentiation of embryonic stem cells and is universally lethal in knockout mice. KDM1A is also believed to play a role in cancer, as KDM1A gene expression is observed to be upregulated in some cancers. KDM1A inhibition has therefore been considered a possible epigenetic treatment for cancer.:KDM1B, however, is mostly involved in oocyte development. Deletion of this gene leads to maternal effect lethality in mice. Orthologs of KDM1 in D. melanogaster and C. elegans appear to function similarly to KDM1B rather than KDM1A.
- The KDM2 homologs include KDM2A and KDM2B. KDM2A (also referred to as JHDM1A/FBXL11) and KDM2B (also referred to as JHDM1B/FBXL10) demethylate H3K4me3 and H3K36me2/3. KDM2A has roles in either promoting or inhibiting tumor function, and KDM2B has roles in oncogenesis.:KDM2A contains a CXXC zinc finger domain responsible for binding to unmethylated CpG uislands, and it is believed that KDM2A may bind to many gene regulatory elements in the absence of sequence-specific transcription factors.:Overexpressed KDM2B has been observed in human lymphoma and adenocarcinoma, and underexpressed KDM2B has been observed in human prostate cancer and glioblastoma. KDM2B has been additionally shown to prevent senescence in some cells through ectopic expression.
- The KDM3 homologs include KDM3A, KDM3B and KDM3C. KDM3A (also referred to as JHDM2A/JMJD1A/TSGA), KDM3B (also referred to as JHDM2B/JMJD1B) and KDM3C (also referred to as JMJD1C/TRIP8) demethylate H3K9me1/2.\:KDM3A has roles in spermatogenesis and metabolic functions, and the roles are of KDM3B and JMJD1C are not specifically known.:Knockdown studies of KDM3A in mice resulted in male infertility and adult onset-obesity. Additional studies have indicated that KDM3A may play a role in regulation of androgen receptor-dependent genes as well as genes involved in pluripotency, indicating a potential role for KDM3A in tumorigenesis.
- The KDM4 homologs include KDM4A, KDM4B, KDM4C, and KDM4D. KDM4A (also referred to as JMDM3A/JMJD2A), KDM4B (also referred to as JMDM3B/JMJD2B) and KDM4C (also referred to as JMDM3C/JMJD2C) demethylate H3K9me2/3 and H3K36me2/3, and KDM4D (also referred to as JMDM3D/JMJD2D) demethylates H3K9me2/3.:KDM4A, KDM4B and KDM4C have roles in tumorigenesis, and the role of KDM4D is unknown. KDM4C amplification has been documented in oesophageal squamous carcinoma, medulloblastomas= and breast cancer; amplification of KDM4B has also been found in medulloblastomas. Other gene expression data has also suggested KDM4A, KDM4B, and KDM4C are over-expressed in prostate cancer.
- The KDM5 family includes KDM5A, KDM5B, KDM5C, and KDM5D. These are also referred to as JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, and JARID1D/SMCY, respectively. These enzymes can act on di- and trimethylated H3K4.
- KDM5 protein family appear to play key developmental functions. The deletion of the JmjC domain of retinoblastoma binding protein related 2 (RBR-2) in C. elegans express defects in vulva formation. Mutations to the JmjC domain in Drosophila causes either lethal effects on larval or many developmental defects in those that survive.
- KDM5A in cell culture systems have also shown links to regulation of differentiation, mitochondrial function, cell cycle progression. KDM5B and KDM5C have also shown to interaction with PcG proteins, which are involved in transcriptional repression. KDM5C mutations (found on the X-chromosome) have also been found in patients with X-linked mental retardation. Depletion of KDM5C homologs in D. rerio have shown brain-patterning defects and neuronal cell death.
- The KDM6 family includes KDM6A, KDM6B, and UTY. KDM6A (also referred to as UTX) and KDM6B (also referred to as JMJD3) act on di- and trimethylated H3K27 and have roles in development; the substrate and role of UTY is unknown. On the whole, both KDM6A and KDM6B possess tumor-suppressive characteristics. KDM6A knockdowns in fibroblasts lead to an immediate increase in fibroblast population. KDM6B expressed in fibroblasts induces oncogenes of the RAS_RAF pathway. Deletions and point mutations of KDM6A have been identified as one cause of Kabuki Syndrome, a congenital disorder resulting in intellectual disability.
- Other possible roles have been suggested for KDM6B. Specifically in one study, mutating homologs of KDM6B disrupted gonadal development in C.elegans. Other studies have shown that KDM6B expression is up-regulated in activated macrophages and dynamically expressed during differentiation of stem cells.
- On the other hand, depletion of homologs of KDM6A in D. rerio have shown decreased expression of HOX genes, which play a role in regulating body patterning during development. In mammalian studies, KDM6A has been shown to regulate HOX genes as well.
Another example of a demethylase is protein-glutamate methylesterase, also known as CheB protein (EC 188.8.131.52), which demethylates MCPs (methyl-accepting chemotaxis proteins) through hydrolysis of carboxylic ester bonds. The association of a chemotaxis receptor with an agonist leads to the phosphorylation of CheB. Phosphorylation of CheB protein enhances its catalytic MCP demethylating activity resulting in adaption of the cell to environmental stimuli. MCPs respond to extracellular attractants and repellents in bacteria like E. coli in chemotaxis regulation. CheB is more specifically termed a methylesterase, as it removes methyl groups from methylglutamate residues located on the MCPs through hydrolysis, producing glutamate accompanied by the release of methanol.
CheB is of particular interest to researchers as it may be a therapeutic target for mitigating the spread of bacterial infections.
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