Promoter KDM5A is also important in cell differentiation.

Promoter and enhancer region (JMJC
demethylase, NO66)

NO66, a JmjC
domain-containing protein, catalyzes the removal of mono-, di-, and tri-methyl
marks at H3K4, as well as di- and tri-methyl marks at H3K36. NO66
commonly involves in gene silencing and heterochromatin formation.


NO66 has been found in all
the developing bones, such as the E15.5 vertebrae and mandible and E18.5 femur,
tibia, and fibula, indicating that NO66 plays a role in osteogenesis. Both NO66
and osterix (OSX) can be found present in the promoter of bone sialoprotein
(BSP). NO66 inhibit OSX-mediated promoter activation of Bsp gene, repressing transcriptional activity and gene expression,
via demethylation of H3K4me3. This is further
supported by knockdown of NO66 in preosteoblasts accelerates osteoblast
differentiation and maturation, and significantly increases the expression
levels of OSX-dependent matrix-forming genes (Bsp). Thus, NO66
represses OSX target genes and inhibits osteogenesis. Mechanistically, NO66
also interacts with OSX to inhibit OSX transcriptional activity, which
later affects genes expression.



protein 2 (RBP2), also known as KDM5A, catalyzes the removal of mono-, di-, and
tri-methyl marks at H3K4.

KDM5A plays an important role in neural
cells proliferation. In neural progenitor cells (NPCs), KDM5A represses
astrocyte differentiation through suppressing astroglia gene (Gfap) expression. Thus, astrocytes
differentiation in NPCs is restrained. KDM5A represses astrocytogenesis via
downregulating H3K4me3 and maintain H3K9me2 at Gfap promoter. This suggests that KDM5 at the promoter region is
crucial for the repression of astrocyte differentiation in NPCs, via H3K4me3

KDM5A is also important in cell
differentiation. For example, in innate immune system, NK cells are activated by the
JAK-STAT signaling pathway and rapidly express IFN-?. However, JAK-STAT pathway is
repressed by suppressor of cytokine signaling 1 (Socs1). Recently, KDM5A plays a crucial role in
activation of natural killer (NK) cells as deficiency of KDM5A has impaired
activation of natural killer (NK) cells, with decreased of IFN-g
production. KDM5A carried out this action with a functional role of suppressing
SOCS1 expression via maintaining a low level of H3K4me3 at the promoter region
of Socs1. Thus, KDM5A is essential in activation of NK cells, via suppressing Socs1 expression through decreasing
H3K4me3 at the promoter region.


Although KDM5A may have beneficial action in normal cells, it is
also a critical transcriptional regulator in tumorigenesis and drug tolerance
in cancer cells. For example, in vivo,
KDM5A is crucial for breast cancer metastasis to the lung. KDM5A positively
regulate numerous metastasis genes, such as TNC.
Loss of KDM5A also suppresses tumor formation in transgenic mice. Thus,
demonstrating that negative impact of KDM5A in metastasis and it has been
proposed that KDM5A can be the potential therapeutic target for metastasis and
progression in future. However, KDM5A function differently in cells, other
targeting strategy is required for inhibition of breast cancer metastasis at



KDM5C co-occupy a large set of active enhancers
including almost all super-enhancers and function as general negative
regulators of enhancers. For example, through deletion of RACK7, recruitment of
KDM5C to active enhancer requires RACK7. RACK7
negatively regulate active enhancer via decreasing H3K4me3 active mark
significantly at active enhancer. Loss of KDM5C increases transcription of
targeted genes too. Thus, KDM5C inactivate enhancer region via H3K4me3


Reader’ proteins

Readers recruits various
components of the nuclear signaling network to chromatin, mediating fundamental
processes such as gene transcription, DNA replication and recombination, DNA
damage response and chromatin remodeling. Misreading
of epigenetic marks has been shown to underlie a host of human diseases,
including autoimmune and developmental abnormalities and cancer.

methyl readers are characterised into groups depending on individual
recognition of residues (Table 3). Readers of methyllysine are the most thoroughly characterized
group, and those involves in H3K4me reading include 1) plant homeodomain (PHD),
2) tandem tudor domain (TTD) and others in table 3. These domains
are located within the chromatin modifying proteins themselves, but are also
found in chromatin remodelers and adaptor proteins that respond to the histone





PHD fingers of numerous
proteins make extensive binding to H3K4me3 with high affinity (Table 4),
imparting a high degree of specificity. Thus, PHD is a well-characterized
reader of H3K4me3. For example, a subunit of basal transcription complex TFIID,
TAF3, binds to H3K4me3 through its PHD finger and activate transcription
activity. In contrast, recognition of H3K4me3 by the PHD finger of ING2, a
subunit of the mSin3a histone deacetylase complex, associates with
post-translational modification to repress gene expression.


PHD finger of histone
methylase is able to mediate enzymatic activity of these proteins. For
instance, PHD finger of the PHF8 histone demethylase recognises and binds to
H3K4me3, and result in chromatin-modifying mechanism. In KDM5B, a demethylase of H3K4me1/2/3, has 3
PHD fingers (PHD1/2/3). KDM5B is stabilised at promoters and enhancers of
target genes through binding of its PHD1 and PHD 3 finger with unmodified H3
(H3K4me0) and H3K4me1/2/3 (prefers H3K4me3) respectively. No binding of PHD2
to histone tails was observed. Consistently, knockdown of KDM5B substantially
increases gene expression. These simply imply that PHD-dependent KDM5B result
in transcriptionally inactive chromatin is via H3K4me1/2/3 demethylation at
promoter and enhancer region.  Similarly, histone demethylase LSD1
enriched at promoters of target genes through binding of the PHD finger of the
BHC80 subunit with unmodifed H3, correlates with gene repression.


However, histone acetylase
PHF20 PHD finger is highly selective for H3K4me2 and showed weak interaction
with H3K4me3, mark by MLL1. Through PHD finger mutant E662K, binding of PHF20
PHD finger to H3K4me2 is required for PHF20-dependent H4K16 acetylation at
promoter region and transcriptional activation of target genes. Thus, this
shows that PHD finger upregulate gene expression via targeted H3K4me2, instead
of H3K4me3. Similarly, recognition
of H3 by the JADE1 PHD1 finger recruit the activating HBO1 HAT complex at
promoter region and correlates with upregulated gene expression level. HBO1
co-localises with H3K4me3 too.


these studies demonstrate that the downstream effect of the interaction between
a PHD finger and histone tail depends on the enzymatic function of the host
protein or the host complex.



of H3K4me by Tandem
tudor domain; TTD)

TTD of SGF29, is a subunit of SAGA.
SAGA is a large chromatin-modifying complex that regulates gene expression.
SGF29 subunit is required for the recruitment of SAGA to gene promoters and for
the acetylation of histone H3 by SAGA. In human and Saccharomyces cerevisiae,
SGF29 recognizes H3K4me2/3 marks (preferred H3K4me3), by means of tandem Tudor
domains (SGF29-TT). Consistently, using ChIP sequencing, Sgf29 at gene promoters
overlapped with H3K4me3 mark and knockdown of Sgf29 resulted in loss of H3K4me3
binding. Therefore, SGF29-TT
is responsible for facilitating the binding of SAGA to promoter and regulate
transcriptional activity.

The structure of human Spindlin1
revealed a tandem of Tudor domains (Tudor 1 and Tudor 2) tightly packed against
a third Tudor domain (Tudor 3) via hydrophobic interactions, giving rise to a
circular arrangement of three Tudor domains. In the crystal structure of
Spindlin1 in complex with H3K4me3, via Tudor 2. Spindlin1 mutant with impaired
H3K4me3 binding reduced rRNA expression. Given that H3K4me are enriched at
promoters, these studies imply that Tudor 2 domain regulate promoters via
recruiting Spindlin1 to H3K4me3.  Within
Spindlin1, H3R8me2 is also recognised by Tudor 1 domain (Trp62, Trp72, Tyr91,
and Tyr98), which is stabilised by Phe25 residue from Tudor 3 domain. The
co-recognition of H3K4me3 and H3R8me2a by the Tudor domains of Spindlin1
illustrates a double binding mode that leads to a high affinity
interaction. For example, the structure of Spindlin1–H3K4me3R8me2a is useful to study
Spindlin1 functions. Consistently, Spindlin1, H3K4me3, and H3R8me2a are all
enriched at the promoter region of Wnt target genes. Thus, suggesting a role of
Tudor domain in mediating binding of Spindlin1 to H3K4me3 at promoter and
regulate transcription of these genes.

of histone modifiers in cancer

Recent studies has notably shown that
misregulation of HMs such as histone methylation leads to diseases including
cancer. For example, gaining or losing of H3K4me1 at enhancers clearly
distinguishes normal colon crypts and colorectal cancer. This suggests that
dysregulation of transcriptional activity consequently changes cellular
phenotype drastically. Therefore, histone modifiers play an important role in
regulating transcriptional activity.

mutation in catalytic domains of histone methyltransferases affect histone
methylation.  For example, N4848S
mutation (found in cancers of endometrium, central nervous system) in catalytic SET domain of MLL3,
abolished the catalytic activity of enzyme. In contrast, Y4884C mutation (found
in colorectal
cancer) have MLL3 gain-of-function, whereby
catalytic properties of MLL3 changes from a monomethyltransferase with H3K4me0
as a preferred substrate to a trimethyltransferase with higher catalytic
activity towards H3K4me1. This leads to increase of H3K4me2/3, indicating that
mutations of MLL3 contributes to carcinogenesis.


Interestingly, alteration with histone
modifiers are not the only option in driving dysregulation of gene
transcription. Recently, Lentivirus is shown to induce overexpression of NO66,
which preferentially expressed in colorectal cancer tissue, while expressed
weakly in normal tissues (fig 17). NO66
is also highly expressed in 80% of lung cancer tissue and rarely in normal
tissues. In addition, overexpression of NO66 conferred anti-apoptotic activity,
which further contributes to hallmark of cancer.


Reader proteins function can also be
affected by mutations that aborogate binding to histone mark. Within or near
the H3K4me3 binding site of ING1 PHD finger is targeted by mutations (, N216S,
V218I and G221V). All three mutation impairs binding of ING1 to H3K4me3,
leading to inability of inducing apoptotic activity and nucleotide repair.



mechanism is particularly important during development and also in acquiring
cancerous phenotype. These finding suggest that methylation of histones is an
important component of epigenetic machinery that the role of histone modifiers
in modulating modifications at histone level is important in transcriptional
activity. Dysregulation of these proteins will result in diseases, including
various cancer (eg. endometrial,
colorectal and lung cancer etc.). As
epigenetic mechanisms are reversible and can be targeted by small molecule
inhibitors. Thus, epigenetics is an appealing target for cancer treatment.
Several inhibitors of histone methyltransferase are also under clinical trials.
The only complication is of how inhibitors targeting only cancer cells as
epigenetics are essential for normal cell function. Thus, further studies are
required in histone modifiers and epigenetic mechanism.




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