NPAS2
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Neuronal PAS domain protein 2 (NPAS2) also known as member of PAS protein 4 (MOP4) is a
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
that in humans is encoded by the ''NPAS2''
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
. NPAS2 is paralogous to
CLOCK A clock or chronometer is a device that measures and displays time. The clock is one of the oldest Invention, human inventions, meeting the need to measure intervals of time shorter than the natural units such as the day, the lunar month, a ...
, and both are key proteins involved in the maintenance of circadian rhythms in mammals. In the
brain The brain is an organ (biology), organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It consists of nervous tissue and is typically located in the head (cephalization), usually near organs for ...
, NPAS2 functions as a generator and maintainer of mammalian
circadian rhythm A circadian rhythm (), or circadian cycle, is a natural oscillation that repeats roughly every 24 hours. Circadian rhythms can refer to any process that originates within an organism (i.e., Endogeny (biology), endogenous) and responds to the env ...
s. More specifically, NPAS2 is an activator of transcription and
translation Translation is the communication of the semantics, meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The English la ...
of core clock and clock-controlled genes through its role in a
negative feedback loop Negative feedback (or balancing feedback) occurs when some function of the output of a system, process, or mechanism is fed back in a manner that tends to reduce the fluctuations in the output, whether caused by changes in the input or by o ...
in the
suprachiasmatic nucleus The suprachiasmatic nucleus or nuclei (SCN) is a small region of the brain in the hypothalamus, situated directly above the optic chiasm. It is responsible for regulating sleep cycles in animals. Reception of light inputs from photosensitive r ...
(SCN), the brain region responsible for the control of circadian rhythms.


Discovery

The mammalian and mouse ''Npas2'' gene was first
sequenced In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...
and characterized in 199
Dr. Steven McKnight's
lab and published by Yu-Dong Zhou et al. The gene’s cDNAs encoding mouse and human forms of NPAS2 were isolated and sequenced. RNA blotting assays were used to demonstrate the selective presence of the gene in brain and
spinal cord The spinal cord is a long, thin, tubular structure made up of nervous tissue that extends from the medulla oblongata in the lower brainstem to the lumbar region of the vertebral column (backbone) of vertebrate animals. The center of the spinal c ...
tissues of mice.
In situ hybridization ''In situ'' hybridization (ISH) is a type of Hybridisation (molecular biology), hybridization that uses a labeled complementary DNA, RNA or modified nucleic acid strand (i.e., a Hybridization probe, probe) to localize a specific DNA or RNA seq ...
indicated that the pattern of Npas2 mRNA distribution in
mouse brain A mouse (: mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus'' ...
is broad and complex, and is largely non-overlapping with that of Npas1. Using
Immunohistochemistry Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of Antibody, antibodies binding specifically to antigens in biological tissues. Alber ...
of human testis, Ramasamy et al. (2015) found the presence of NPAS2 protein in both
germ cell A germ cell is any cell that gives rise to the gametes of an organism that reproduces sexually. In many animals, the germ cells originate in the primitive streak and migrate via the gut of an embryo to the developing gonads. There, they unde ...
s within the tubules of the testes and in the interstitial space of
Leydig cell Leydig cells, also known as interstitial cells of the testes and interstitial cells of Leydig, are found adjacent to the seminiferous tubules in the testicle and produce testosterone in the presence of luteinizing hormone (LH). They are polyhedral ...
s.


Structure


In humans

The ''Npas2'' gene resides on
chromosome A chromosome is a package of DNA containing part or all of the genetic material of an organism. In most chromosomes, the very long thin DNA fibers are coated with nucleosome-forming packaging proteins; in eukaryotic cells, the most import ...
2 at the band q13. The gene is 176,679 bases long and contains 25 exons. The predicted 824-amino acid human NPAS2 protein shares 87% sequence identity with mouse ''Npas2''.


In mice

The ''Npas2'' gene has been found to reside on chromosome 1 at 17.98 centimorgans and is 169,505 bases long.


Function


In the brain

The NPAS2 protein is a member of the
basic helix-loop-helix Basic or BASIC may refer to: Science and technology * BASIC, a computer programming language * Basic (chemistry), having the properties of a base * Basic access authentication, in HTTP Entertainment * ''Basic'' (film), a 2003 film * Basic, one ...
(bHLH)-PAS
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
family and is expressed in the SCN. NPAS2 is a
PAS domain A Per-Arnt-Sim (PAS) domain is a protein domain found in all kingdoms of life. Generally, the PAS domain acts as a molecular sensor, whereby small molecules and other proteins associate via binding of the PAS domain. Due to this sensing capabilit ...
-containing protein, which binds other proteins via their own protein-protein (PAS) binding domains. Like its paralogue,
CLOCK A clock or chronometer is a device that measures and displays time. The clock is one of the oldest Invention, human inventions, meeting the need to measure intervals of time shorter than the natural units such as the day, the lunar month, a ...
(another PAS domain-containing protein), the NPAS2 protein can dimerize with the
BMAL1 Basic helix-loop-helix ARNT-like protein 1 or aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL), or brain and muscle ARNT-like 1 is a protein that in humans is encoded by the ''BMAL1'' gene on chromosome 11, region p15.3. It's ...
protein and engage in a transcription/translation negative feedback loop (TTFL) to activate transcription of the mammalian ''Per'' and ''Cry'' core clock genes. NPAS2 has been shown to form a
heterodimer In biochemistry, a protein dimer is a macromolecular complex or multimer formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ...
with BMAL1 in both the brain and in cell lines, suggesting its similarity in function to the CLOCK protein in this TTFL. Compensation is a key feature of TTFLs that regulate circadian rhythms. BMAL1 compensates for CLOCK in that if CLOCK is absent, BMAL1 will upregulate to maintain the mammalian circadian rhythms. NPAS2 has been shown to be analogous to the function of CLOCK in CLOCK-deficient mice. In ''Clock'' knockout mice, NPAS2 is upregulated to keep the rhythms intact. ''Npas2''-mutant mice, which do not express functional NPAS2 protein, still maintain robust circadian rhythms in locomotion. However, like CLOCK-deficient mice in the CLOCK/BMAL1 TTFL, ''Npas2''-
mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It i ...
mice (in the NPAS2/BMAL1 TTFL) still have small defects in their circadian rhythms such as a shortened circadian period and an altered response to changes in the typical light-dark cycle. In addition, ''Npas2''
knockout A knockout (abbreviated to KO or K.O.) is a fight-ending, winning criterion in several full-contact combat sports, such as boxing, kickboxing, Muay Thai, mixed martial arts, karate, some forms of taekwondo and other sports involving striking, ...
mice show sleep disturbances and have decreased expression of '' mPer2'' in their
forebrain In the anatomy of the brain of vertebrates, the forebrain or prosencephalon is the rostral (forward-most) portion of the brain. The forebrain controls body temperature, reproductive functions, eating, sleeping, and the display of emotions. Ve ...
s. Mice without functional alleles of both ''Clock'' and ''Npas2'' became arrhythmic once placed in constant darkness, suggesting that both genes have overlapping roles in maintaining circadian rhythms. In both
wild-type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, " ...
and ''Clock'' knockout mice, ''Npas2'' expression is observed at the same levels, confirming that ''Npas2'' plays a role in maintaining these rhythms in the absence of ''Clock''.


In other tissues

''Npas2'' is expressed everywhere in the periphery of the body. Special focus has been given to its function in
liver The liver is a major metabolic organ (anatomy), organ exclusively found in vertebrates, which performs many essential biological Function (biology), functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of var ...
tissues, and its
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
is upregulated in ''Clock''-mutant mice. However, studies have shown that ''Npas2'' alone is unable to maintain circadian rhythms in peripheral tissues in the absence of CLOCK protein, unlike in the SCN. One theory to explain this observation is that
neuron A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...
s in the brain are characterized by intercellular coupling and can thus respond to deficiencies in key clock proteins in nearby neurons to maintain rhythms. In peripheral tissues such as the liver and
lung The lungs are the primary Organ (biology), organs of the respiratory system in many animals, including humans. In mammals and most other tetrapods, two lungs are located near the Vertebral column, backbone on either side of the heart. Their ...
, however, the lack of intercellular coupling does not allow for this compensatory mechanism to occur. A second theory as to why NPAS2 can maintain rhythms in CLOCK-deficient SCNs but not in CLOCK-deficient peripheral tissues, is that there exists an additional unknown factor in the SCN that is not present in peripheral tissues.


Non-circadian function

NPAS2-deficient mice have been shown to have
long-term memory Long-term memory (LTM) is the stage of the Atkinson–Shiffrin memory model in which informative knowledge is held indefinitely. It is defined in contrast to sensory memory, the initial stage, and short-term or working memory, the second stage ...
deficits, suggesting that the protein may play a key role in the acquisition of such memories. This theory was tested by inserting a reporter gene (lacZ) that resulted in the production of an NPAS2 protein lacking the bHLH domain. These mice were then given several tests, including the cued and contextual fear task, and showed long-term memory deficits in both tasks.


Interactions

NPAS2 has been shown to interact with: * ARNTL (also known as BMAL1). Like Clock, Npas2 mRNA cycles with a similar phase to that of Bmal1, with both peaking 8 hours before the peak of Per2 mRNA expression''.'' This is consistent with the observation that NPAS2 forms a
heterodimer In biochemistry, a protein dimer is a macromolecular complex or multimer formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ...
with BMAL1 to drive ''Per2'' expression. *
EP300 Histone acetyltransferase p300 also known as p300 HAT or E1A-associated protein p300 (where E1A = adenovirus early region 1A) also known as EP300 or p300 is an enzyme that, in humans, is encoded by the ''EP300'' gene. It functions as histone ace ...
. NPAS2 and EP300 interact in a time-dependent, synchronized manner. EP300 is recruited to NPAS2 as a coactivator of clock gene expression. *
Retinoic acid receptor alpha Retinoic acid receptor alpha (RAR-α), also known as NR1B1 (nuclear receptor subfamily 1, group B, member 1), is a nuclear receptor that in humans is encoded by the ''RARA'' gene. ''NR1B1'' is a gene with a protein product and has a chromosomal ...
(RARα) and
retinoid X receptor alpha Retinoid X receptor alpha (RXR-alpha), also known as NR2B1 (nuclear receptor subfamily 2, group B, member 1) is a nuclear receptor that in humans is encoded by the ''RXRA'' gene. Function Retinoid X receptors (RXRs) and retinoic acid receptors ...
(RXRα). In peripheral clocks, RARα and RXRα interact with NPAS2 by inhibiting the NPAS2:BMAL1 heterodimer-mediated expression of clock genes. This interaction depends upon humoral signaling by
retinoic acid Retinoic acid (simplified nomenclature for all-''trans''-retinoic acid) is a metabolite of vitamin A1 (all-''trans''-retinol) that is required for embryonic development, male fertility, regulation of bone growth and immune function. All-''trans ...
and serves to phase-shift the clock. *
Small heterodimer partner The small heterodimer partner (SHP) also known as NR0B2 (nuclear receptor subfamily 0, group B, member 2) is a protein that in humans is encoded by the ''NR0B2'' gene. SHP is a member of the nuclear receptor family of intracellular transcription ...
(SHP). In the liver circadian clock, NPAS2 and SHP engage in a TTFL: NPAS2 controls the circadian rhythms of SHP by rhythmically binding to its promoter, while SHP inhibits transcription of ''Npas2'' when present.


Clinical significance

''Npas2''
genotype The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the alleles or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a ...
s can be determined through tissue samples from which genomic
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
is extracted and assayed. The assay is performed under PCR conditions and can be used to determine specific mutations and polymorphisms.


Polymorphisms and tumorigenesis

Mounting evidence suggests that the NPAS2 protein and other circadian genes are involved in
tumorigenesis Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abn ...
and tumor growth, possibly through their control of cancer-related biologic pathways. A missense polymorphism in NPAS2 (Ala394Thr) has been shown to be associated with risk of human tumors including breast cancer. These findings provide evidence suggesting a possible role for the circadian ''Npas2'' gene in cancer prognosis. These results have been confirmed in both breast and colorectal cancers.


NPAS2 and mood disorders

Current research has revealed an association between seasonal affective disorder (SAD) and general mood disorder related to NPAS2, ARNTL, and
CLOCK A clock or chronometer is a device that measures and displays time. The clock is one of the oldest Invention, human inventions, meeting the need to measure intervals of time shorter than the natural units such as the day, the lunar month, a ...
polymorphisms. These genes may influence seasonal variations through metabolic factors such as body weight and appetite. Associated with a connection to mood disorders, NPAS2 has been found to be involved with dopamine degradation. This was first suggested by the observation that the clock components BMAL1 and NPAS2 transcriptionally activated a
luciferase Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'' ...
reporter driven by the murine monoamine oxidase A (''MAOA'') promoter in a circadian fashion. This suggested that these two clock components (BMAL1 and NPAS2) directly regulated ''MAOA'' transcription. Subsequent findings discovered positive transcriptional regulation of BMAL1/NPAS2 by PER2. In mice lacking PER2, both ''MAOA'' mRNA and MAOA protein levels were decreased. Therefore, dopamine degradation was reduced, and dopamine levels in the
nucleus accumbens The nucleus accumbens (NAc or NAcc; also known as the accumbens nucleus, or formerly as the ''nucleus accumbens septi'', Latin for ' nucleus adjacent to the septum') is a region in the basal forebrain rostral to the preoptic area of the hypo ...
were increased. These findings indicate that degradation of
monoamines Monoamine neurotransmitters are neurotransmitters and neuromodulators that contain one amino group connected to an aromaticity, aromatic ring by a two-carbon chain (such as -CH2-CH2-). Examples are dopamine, norepinephrine and serotonin. All m ...
is regulated by the circadian clock. It is very likely that the described clock-mediated regulation of monoamines is relevant for humans, because single-nucleotide polymorphisms in '' Per2,
Bmal1 Basic helix-loop-helix ARNT-like protein 1 or aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL), or brain and muscle ARNT-like 1 is a protein that in humans is encoded by the ''BMAL1'' gene on chromosome 11, region p15.3. It's ...
'', and ''Npas2'' are associated in an additive fashion with seasonal affective disorder or winter depression.


See also

* ''Clock'' (gene) * ''Bmal1/Arntl'' (gene) * Suprachiasmatic nucleus (SCN) * ''Per'' (gene) * Steven McKnight (scientist)


References


External links

*
Steven McKnight
the first scientist to implicate ''Npas2'' as a contributor to circadian rhythms {{Transcription factors, g1 Transcription factors PAS-domain-containing proteins