Reprogramming
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In biology, reprogramming refers to erasure and remodeling of
epigenetic In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are " ...
marks, such as DNA methylation, during mammalian development or in cell culture. Such control is also often associated with alternative covalent modifications of
histones In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are ...
. Reprogrammings that are both large scale (10% to 100% of epigenetic marks) and rapid (hours to a few days) occur at three life stages of mammals. Almost 100% of epigenetic marks are reprogrammed in two short periods early in development after
fertilization Fertilisation or fertilization (see spelling differences), also known as generative fertilisation, syngamy and impregnation, is the fusion of gametes to give rise to a new individual organism or offspring and initiate its development. Pro ...
of an
ovum The egg cell, or ovum (plural ova), is the female reproductive cell, or gamete, in most anisogamous organisms (organisms that reproduce sexually with a larger, female gamete and a smaller, male one). The term is used when the female gamete i ...
by a sperm. In addition, almost 10% of DNA methylations in neurons of the hippocampus can be rapidly altered during formation of a strong fear memory. After fertilization in mammals, DNA methylation patterns are largely erased and then re-established during early embryonic development. Almost all of the methylations from the parents are erased, first during early
embryogenesis An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sperm ...
, and again in
gametogenesis Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes. Depending on the biological life cycle of the organism, gametogenesis occurs by meiotic d ...
, with demethylation and remethylation occurring each time. Demethylation during early embryogenesis occurs in the preimplantation period. After a sperm fertilizes an
ovum The egg cell, or ovum (plural ova), is the female reproductive cell, or gamete, in most anisogamous organisms (organisms that reproduce sexually with a larger, female gamete and a smaller, male one). The term is used when the female gamete i ...
to form a
zygote A zygote (, ) is a eukaryotic cell formed by a fertilization event between two gametes. The zygote's genome is a combination of the DNA in each gamete, and contains all of the genetic information of a new individual organism. In multicell ...
, rapid DNA demethylation of the paternal DNA and slower demethylation of the maternal DNA occurs until formation of a
morula A morula (Latin, ''morus'': mulberry) is an early-stage embryo consisting of a solid ball of cells called blastomeres, contained in mammals, and other animals within the zona pellucida shell. The blastomeres are the daughter cells of the zygo ...
, which has almost no methylation. After the
blastocyst The blastocyst is a structure formed in the early embryonic development of mammals. It possesses an inner cell mass (ICM) also known as the ''embryoblast'' which subsequently forms the embryo, and an outer layer of trophoblast cells called th ...
is formed, methylation can begin, and with formation of the epiblast a wave of methylation then takes place until the implantation stage of the embryo. Another period of rapid and almost complete demethylation occurs during gametogenesis within the primordial
germ cell Germ or germs may refer to: Science * Germ (microorganism), an informal word for a pathogen * Germ cell, cell that gives rise to the gametes of an organism that reproduces sexually * Germ layer, a primary layer of cells that forms during embry ...
s (PGCs). Other than the PGCs, in the post-implantation stage, methylation patterns in somatic cells are stage- and tissue-specific with changes that presumably define each individual cell type and last stably over a long time.


Embryonic development

The mouse sperm
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...
is 80–90% methylated at its
CpG site The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
s in DNA, amounting to about 20 million methylated sites. After
fertilization Fertilisation or fertilization (see spelling differences), also known as generative fertilisation, syngamy and impregnation, is the fusion of gametes to give rise to a new individual organism or offspring and initiate its development. Pro ...
, the paternal chromosome is almost completely demethylated in six hours by an active process, before DNA replication (blue line in Figure). In the mature
oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The femal ...
, about 40% of its CpG sites are methylated. Demethylation of the maternal chromosome largely takes place by blockage of the methylating enzymes from acting on maternal-origin DNA and by dilution of the methylated maternal DNA during replication (red line in Figure). The
morula A morula (Latin, ''morus'': mulberry) is an early-stage embryo consisting of a solid ball of cells called blastomeres, contained in mammals, and other animals within the zona pellucida shell. The blastomeres are the daughter cells of the zygo ...
(at the 16 cell stage), has only a small amount of DNA methylation (black line in Figure). Methylation begins to increase at 3.5 days after fertilization in the
blastocyst The blastocyst is a structure formed in the early embryonic development of mammals. It possesses an inner cell mass (ICM) also known as the ''embryoblast'' which subsequently forms the embryo, and an outer layer of trophoblast cells called th ...
, and a large wave of methylation then occurs on days 4.5 to 5.5 in the epiblast, going from 12% to 62% methylation, and reaching maximum level after implantation in the uterus. By day seven after fertilization, the newly formed
primordial germ cells Primordial may refer to: * Primordial era, an era after the Big Bang. See Chronology of the universe * Primordial sea (a.k.a. primordial ocean, ooze or soup). See Abiogenesis * Primordial nuclide, nuclides, a few radioactive, that formed before t ...
(PGC) in the implanted
embryo An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sperm ...
segregate from the remaining
somatic cell A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells co ...
s. At this point the PGCs have about the same level of methylation as the somatic cells. The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast toward the gonadal ridge. Now the cells are rapidly proliferating and beginning demethylation in two waves. In the first wave, demethylation is by replicative dilution, but in the second wave demethylation is by an active process. The second wave leads to demethylation of specific loci. At this point the PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle
t embryonic day 13.5 (E13.5), see the second figure in this section T, or t, is the twentieth letter in the Latin alphabet, used in the modern English alphabet, the alphabets of other western European languages and others worldwide. Its name in English is ''tee'' (pronounced ), plural ''tees''. It is de ...
After fertilization some cells of the newly formed embryo migrate to the germinal ridge and will eventually become the
germ cell Germ or germs may refer to: Science * Germ (microorganism), an informal word for a pathogen * Germ cell, cell that gives rise to the gametes of an organism that reproduces sexually * Germ layer, a primary layer of cells that forms during embry ...
s (sperm and oocytes) of the next generation. Due to the phenomenon of genomic imprinting, maternal and paternal genomes are differentially marked and must be properly reprogrammed every time they pass through the germline. Therefore, during the process of
gametogenesis Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes. Depending on the biological life cycle of the organism, gametogenesis occurs by meiotic d ...
the primordial germ cells must have their original biparental DNA methylation patterns erased and re-established based on the sex of the transmitting parent. After fertilization, the paternal and maternal genomes are demethylated in order to erase their epigenetic signatures and acquire totipotency. There is asymmetry at this point: the male pronucleus undergoes a quick and active demethylation. Meanwhile the female pronucleus is demethylated passively during consecutive cell divisions. The process of DNA demethylation involves
base excision repair Base excision repair (BER) is a cellular mechanism, studied in the fields of biochemistry and genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from t ...
and likely other DNA-repair-based mechanisms. Despite the global nature of this process, there are certain sequences that avoid it, such as differentially methylated regions (DMRS) associated with imprinted genes,
retrotransposons Retrotransposons (also called Class I transposable elements or transposons via RNA intermediates) are a type of genetic component that copy and paste themselves into different genomic locations ( transposon) by converting RNA back into DNA throug ...
and centromeric
heterochromatin Heterochromatin is a tightly packed form of DNA or '' condensed DNA'', which comes in multiple varieties. These varieties lie on a continue between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a rol ...
. Remethylation is needed again to differentiate the embryo into a complete organism. ''In vitro'' manipulation of pre-implantation embryos has been shown to disrupt methylation patterns at imprinted loci and plays a crucial role in cloned animals.


Learning and Memory

Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either accumulated slowly (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. Rats subjected to one instance of contextual fear conditioning create an especially strong long-term memory. At 24 h after training, 9.17% of the genes in the rat genomes of hippocampus neurons were found to be differentially methylated. This included more than 2,000 differentially methylated genes at 24 hours after training, with over 500 genes being demethylated. The hippocampus region of the brain is where contextual fear memories are first stored (see figure of the brain, this section), but this storage is transient and does not remain in the hippocampus. In rats contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just 1 day after conditioning, but rats retain a considerable amount of contextual fear when a long delay (28 days) is imposed between the time of conditioning and the time of hippocampectomy.


Molecular stages

Three molecular stages are required for reprogramming the DNA methylome. Stage 1: Recruitment. The enzymes needed for reprogramming are recruited to genome sites that require demethylation or methylation. Stage 2: Implementation. The initial enzymatic reactions take place. In the case of methylation, this is a short step that results in the methylation of cytosine to 5-methylcytosine. Stage 3: Base excision DNA repair. The intermediate products of demethylation are catalysed by specific enzymes of the base excision DNA repair pathway that finally restore cystosine in the DNA sequence. The Figure in this section indicates the central roles of ten-eleven translocation methylcytosine dioxygenases (TETs) in the demethylation of 5-methylcytosine to form cytosine. As reviewed in 2018, 5mC is very often initially oxidized by TET dioxygenases to generate 5-hydroxymethylcytosine (5hmC). In successive steps (see Figure) TET enzymes further hydroxylate 5hmC to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Thymine-DNA glycosylase (TDG) recognizes the intermediate bases 5fC and 5caC and excises the glycosidic bond resulting in an apyrimidinic site (AP site). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by
APOBEC image:Apobec.J.Steinfeld.D.png, 300px, upExample of a member of the APOBEC family, APOBEC-2. A cytidine deaminase from ''Homo sapiens''.; ; rendered usinPyMOL APOBEC ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide") is a family o ...
(AID/APOBEC) deaminases to form 5-hydroxymethyluracil (5hmU) or 5mC can be converted to
thymine Thymine () (symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine n ...
(Thy). 5hmU can be cleaved by TDG, SMUG1,
NEIL1 Endonuclease VIII-like 1 is an enzyme that in humans is encoded by the ''NEIL1'' gene. NEIL1 belongs to a class of DNA glycosylases homologous to the bacterial Fpg/Nei family. These glycosylases initiate the first step in base excision repair by c ...
, or MBD4. AP sites and T:G mismatches are then repaired by base excision repair (BER) enzymes to yield cytosine (Cyt).


TET family

The isoforms of the TET enzymes include at least two isoforms of TET1, one of TET2 and three isoforms of TET3. The full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and primordial germ cells (PGCs). The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes and neurons designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and neurons and was not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes, but is nearly absent at the 2-cell stage. It is possible that TET3o, high in neurons, oocytes and zygotes at the one cell stage, is the major TET enzyme utilized when very large scale rapid demethylations occur in these cells.


Recruitment of TET to DNA

The TET enzymes do not specifically bind to 5-methylcytosine except when recruited. Without recruitment or targeting, TET1 predominantly binds to high CG promoters and CpG islands (CGIs) genome-wide by its CXXC domain that can recognize un-methylated CGIs. TET2 does not have an affinity for 5-methylcytosine in DNA. The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons, binds most strongly to CpGs where the C was converted to 5-carboxycytosine (5caC). However, it also binds to un-methylated CpGs. For a TET enzyme to initiate demethylation it must first be recruited to a methylated
CpG site The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
in DNA. Two of the proteins shown to recruit a TET enzyme to a methylated cytosine in DNA are OGG1 (see figure Initiation of DNA demthylation) and EGR1.


OGG1

Oxoguanine glycosylase 8-Oxoguanine glycosylase, also known as OGG1, is a DNA glycosylase enzyme that, in humans, is encoded by the ''OGG1'' gene. It is involved in base excision repair. It is found in bacterial, archaeal and eukaryotic species. Function OGG1 is th ...
(OGG1) catalyses the first step in base excision repair of the oxidatively damaged base 8-OHdG. OGG1 finds 8-OHdG by sliding along the linear DNA at 1,000 base pairs of DNA in 0.1 seconds. OGG1 very rapidly finds 8-OHdG. OGG1 proteins bind to oxidatively damaged DNA with a half maximum time of about 6 seconds. When OGG1 finds 8-OHdG it changes conformation and complexes with 8-OHdG in the binding pocket of OGG1. OGG1 does not immediately act to remove the 8-OHdG. Half maximum removal of 8-OHdG takes about 30 minutes in HeLa cells ''in vitro'', or about 11 minutes in the livers of irradiated mice. DNA oxidation by reactive oxygen species preferentially occurs at a guanine in a methylated CpG site, because of a lowered ionization potential of guanine bases adjacent to 5-methylcytosine. TET1 binds (is recruited to) the OGG1 bound to 8-OHdG (see figure). This likely allows TET1 to demethylate an adjacent methylated cytosine. When human mammary epithelial cells (MCF-10A) were treated with H2O2, 8-OHdG increased in DNA by 3.5-fold and this caused large scale demethylation of 5-methylcytosine to about 20% of its initial level in DNA.


EGR1

The gene ''early growth response protein 1'' ('' EGR1'') is an immediate early gene (IEG). The defining characteristic of IEGs is the rapid and transient up-regulation—within minutes—of their mRNA levels independent of protein synthesis. EGR1 can rapidly be induced by neuronal activity. In adulthood, EGR1 is expressed widely throughout the brain, maintaining baseline expression levels in several key areas of the brain including the medial prefrontal cortex, striatum, hippocampus and amygdala. This expression is linked to control of cognition, emotional response, social behavior and sensitivity to reward. EGR1 binds to DNA at sites with the motifs 5′-GCGTGGGCG-3′ and 5'-GCGGGGGCGG-3′ and these motifs occur primarily in promoter regions of genes. The short isoform TET1s is expressed in the brain. EGR1 and TET1s form a complex mediated by the C-terminal regions of both proteins, independently of association with DNA. EGR1 recruits TET1s to genomic regions flanking EGR1 binding sites. In the presence of EGR1, TET1s is capable of locus-specific demethylation and activation of the expression of downstream genes regulated by EGR1.


History

The first person to successfully demonstrate reprogramming was John Gurdon, who in 1962 demonstrated that differentiated somatic cells could be reprogrammed back into an embryonic state when he managed to obtain swimming tadpoles following the transfer of differentiated intestinal epithelial cells into enucleated frog eggs. For this achievement he received the 2012
Nobel Prize in Medicine The Nobel Prize in Physiology or Medicine ( sv, Nobelpriset i fysiologi eller medicin) is awarded yearly by the Nobel Assembly at the Karolinska Institute, Nobel Assembly at the Karolinska Institute for outstanding discoveries in physiology or ...
alongside
Shinya Yamanaka is a Japanese stem cell researcher and a Nobel Prize laureate. He serves as the director of Center for iPS Cell ( induced Pluripotent Stem Cell) Research and Application and a professor at the Institute for Frontier Medical Sciences at Ky ...
. Yamanaka was the first to demonstrate (in 2006) that this somatic cell nuclear transfer or oocyte-based reprogramming process (see below), that Gurdon discovered, could be recapitulated (in mice) by defined factors (
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
, Sox2, Klf4, and
c-Myc ''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' ( MYC), ''l-myc'' ( MYCL), and ''n-myc'' ( MYCN). ''c-myc'' (also sometimes re ...
) to generate
induced pluripotent stem cell Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed ...
s (iPSCs). Other combinations of genes have also been used, including LIN25 and Homeobox protein NANOG.


Phases of reprogramming

With the discovery that cell fate could be altered, the question of what progression of events occurs signifies a cell undergoing reprogramming. As the final product of iPSC reprogramming was similar in morphology, proliferation,
gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. ...
, pluripotency, and
telomerase Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most e ...
activity, genetic and morphological markers were used as a way to determine what phase of reprogramming was occurring. Reprogramming is defined into three phase: initiation, maturation, and stabilization.


Initiation

The initiation phase is associated with the downregulation of cell type specific genes and the upregulation of pluripotent genes. As the cells move towards pluripotency, the
telomerase Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most e ...
activity is reactivated to extend
telomeres A telomere (; ) is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature mos ...
. The cell morphology can directly affect the reprogramming process as the cell is modifying itself to prepare for the gene expression of pluripotency. The main indicator that the initiation phase has completed is that the first genes associated with pluripotency are expressed. This includes the expression of Oct-4 or Homeobox protein NANOG, while undergoing a mesenchymal–epithelial transition (MET), and the loss of apoptosis and
senescence Senescence () or biological aging is the gradual deterioration of functional characteristics in living organisms. The word ''senescence'' can refer to either cellular senescence or to senescence of the whole organism. Organismal senescence inv ...
. If the cell is directly reprogrammed from one
somatic cell A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells co ...
to another, the genes associated with each cell type begin to be upregulated and downregulated accordingly. This can either occur through direct cell reprogramming or creating a intermediate, such as a iPSC, and differentiating into the desired cell type. The initiation phase is completed through one of three pathways: nuclear transfer,
cell fusion Cell fusion is an important cellular process in which several uninucleate cells (cells with a single nucleus) combine to form a multinucleate cell, known as a syncytium. Cell fusion occurs during differentiation of myoblasts, osteoclasts and tr ...
, or defined factors (
microRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. m ...
,
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
, epigenetic markers, and other small molecules).


Somatic cell nuclear transfer

An
oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The femal ...
can reprogram an adult nucleus into an embryonic state after somatic cell nuclear transfer, so that a new organism can be developed from such cell. Reprogramming is distinct from development of a somatic epitype, as somatic epitypes can potentially be altered after an organism has left the developmental stage of life. During somatic cell nuclear transfer, the oocyte turns off tissue specific genes in the somatic cell nucleus and turns back on embryonic specific genes. This process has been shown through cloning, as seen through John Gurdon with the tadpoles and Dolly the Sheep. Notably, these events have shown that cell fate is a reversible process.


Cell fusion

Cell fusion Cell fusion is an important cellular process in which several uninucleate cells (cells with a single nucleus) combine to form a multinucleate cell, known as a syncytium. Cell fusion occurs during differentiation of myoblasts, osteoclasts and tr ...
is used to create a multi nucleated cell called a
heterokaryon A heterokaryon is a multinucleate cell that contains genetically different nuclei. Heterokaryotic and heterokaryosis are derived terms. This is a special type of syncytium. This can occur naturally, such as in the mycelium of fungi during sexual ...
. The fused cells allow for otherwise silenced genes to become reactivated and expressive. As the genes are reactivated, the cells can re-differentiate. There are instances where transcriptional factors, such as the Yamanaka factors, are still needed to aid in
heterokaryon A heterokaryon is a multinucleate cell that contains genetically different nuclei. Heterokaryotic and heterokaryosis are derived terms. This is a special type of syncytium. This can occur naturally, such as in the mycelium of fungi during sexual ...
cell reprogramming.


Defined factors

Unlike nuclear transfer and cell fusion, defined factors do not require a full genome, only reprogramming factors. These reprogramming factors include
microRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. m ...
,
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
, epigenetic markers, and other small molecules. The original transcription factors, that lead to iPSC development, discovered by Yamanaka include
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
, Sox2, Klf4, and
c-Myc ''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' ( MYC), ''l-myc'' ( MYCL), and ''n-myc'' ( MYCN). ''c-myc'' (also sometimes re ...
(OSKM factors). Although the OSKM factors have been shown to induce and aid in pluripotency, other transcription factors such as Homeobox protein NANOG, LIN25, TRA-1-60, and C/EBPα aid in the efficiency of reprogramming. The use of
microRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. m ...
and other small molecule-driven processes has been utilized as a means of increasing the efficiency of the differentiation from somatic cells to pluripotency.


Maturation

The maturation phase begins at the end of the initiation phase, when the first pluripotent genes are expressed. The cell is preparing itself to be independent from the defined factors, that started the reprogramming process. The first genes to be detected in iPSCs are
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
, Homeobox protein NANOG, and Esrrb, followed later by Sox2. In the later stages of maturation,
transgene A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
silencing marks the start of the cell becoming independent from the induced
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
. Once the cell is independent, the maturation phase ends and the stabilization phase begins. As reprogramming efficiency has proven to be a variable and low efficiency process, not all the cells complete the maturation phase and achieve pluripotency. Some cells that undergo reprogramming still remain under apoptosis at the beginning of the maturation stage from
oxidative stress Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal re ...
brought on by the stresses of gene expression change. The use of
microRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. m ...
, proteins, and different combinations of the OSKM factors have started to lead towards a higher efficiency rate of reprogramming.


Stabilization

The stabilization phase refers to the processes in the cell that occur after the cell reaches pluripotency. One genetic marker is the expression of Sox2 and
X chromosome The X chromosome is one of the two sex-determining chromosomes ( allosomes) in many organisms, including mammals (the other is the Y chromosome), and is found in both males and females. It is a part of the XY sex-determination system and XO se ...
reactivation, while epigenetic changes include the
telomerase Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most e ...
extending the
telomeres A telomere (; ) is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature mos ...
and loss of the cell’s epigenetic memory. The epigenetic memory of a cell is reset by the changes in DNA methylation, using activation-induced cytidine deaminase (AID), TET enzymes (TET), and
DNA methyltransferase In biochemistry, the DNA methyltransferase (DNA MTase, DNMT) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosy ...
(DMNTs), starting in the maturation phase and into the stabilization stage. Once the epigenetic memory of the cell is lost, the possibility of differentiation into the three germ layers is achieved. This is considered a fully reprogrammed cell as it can be passaged without reverting to its original somatic cell type.


In cell culture systems

Reprogramming can also be induced artificially through the introduction of exogenous factors, usually
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s. In this context, it often refers to the creation of
induced pluripotent stem cell Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed ...
s from mature cells such as adult
fibroblast A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of ...
s. This allows the production of
stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...
s for
biomedical research Medical research (or biomedical research), also known as experimental medicine, encompasses a wide array of research, extending from " basic research" (also called ''bench science'' or ''bench research''), – involving fundamental scienti ...
, such as research into
stem cell therapies Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition. , the only established therapy using stem cells is hematopoietic stem cell transplantation. This usually takes the form of a bone-marrow transplantation, b ...
, without the use of embryos. It is carried out by the
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to de ...
of stem-cell associated genes into mature cells using
viral vector Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism ('' in vivo'') or in cell culture (''in vitro''). Viruses have evolved specialized mole ...
s such as
retrovirus A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...
es.


Transcription factors

One of the first transacting factors discovered to change a cell was found in a myoblast when the
complementary DNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA ( mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a sp ...
(cDNA) coding for MyoD was expressed and converted a
fibroblast A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of ...
to a myoblast. Another transacting factor that directly transformed a lymphoid cell into a myeloid cell was C/EBPα. MyoD and C/EBPα are examples of a small number of single factors that can transform cells. More often, a combination of transcription factors work in conjunction to reprogram a cell.


OSKM

The OSKM factors (
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
, Sox2, Klf4, and
c-Myc ''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' ( MYC), ''l-myc'' ( MYCL), and ''n-myc'' ( MYCN). ''c-myc'' (also sometimes re ...
) were initially discovered by Yamanaka in 2006, by the induction of a mouse fibroblast into an
induced pluripotent stem cell Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed ...
(iPSCs). Within the following year, these factors were used to induce human fibroblasts into iPSCs.
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
is part of the core regulatory genes needed for pluripotency, as it is seen in both
embryonic stem cells Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre- implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they cons ...
and tumors. The use of Oct4 even in small increases allows for the start differentiation into pluripotency. Oct4 works in conjecture with Sox2 for the expression of
FGF4 Fibroblast growth factor 4 is a protein that in humans is encoded by the ''FGF4'' gene. The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activi ...
which could aid in differentiation. Sox2 is a gene used in maintaining pluripotency in stem cells. Oct4 and Sox2 work together to regulate hundreds of genes utilized in pluripotency. However, Sox2 is not the only possible Sox family member to participate in gene regulation with Oct4 –  Sox4, Sox11, and Sox15 also participate, as the Sox protein is redundant throughout the stem cell
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...
. Klf4 is a transcription factor used in
proliferation Proliferation may refer to: Weapons *Nuclear proliferation, the spread of nuclear weapons, material, and technology *Chemical weapon proliferation, the spread of chemical weapons, material, and technology * Small arms proliferation, the spread of ...
, differentiation, apoptosis, and
somatic cell A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells co ...
reprogramming. When being utilized in cellular reprogramming, Klf4 prevents cell division of damaged cells using its apoptotic ability, and aids in
histone acetyltransferase Histone acetyltransferases (HATs) are enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form ε-''N''-acetyllysine. DNA is wrapped around histones, and, by transferring an ...
activity.
c-Myc ''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' ( MYC), ''l-myc'' ( MYCL), and ''n-myc'' ( MYCN). ''c-myc'' (also sometimes re ...
is also known as an oncogene, and in certain conditions can become cancer causing. In cellular reprogramming, c-Myc is used for
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA ( DNA replication) and some of its organelles, and sub ...
progression, apoptosis, and cellular transformation for further differentiation.


NANOG

Homeobox protein NANOG (NANOG) is a transcription factor used to aid in the efficiency of generating iPSCs by maintaining pluripotency and suppressing cell determination factors. NANOG works by promoting
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important ...
accessibility through repression of
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn ar ...
markers, such as H3K27me3. NANOG aids recruitment of
Oct4 Oct-4 ( octamer-binding transcription factor 4), also known as POU5F1 ( POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. ...
, Sox2, and Esrrb used in transcription, while also recruiting Brahma-related gene-1 (BRG1) for
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important ...
accessibility.


C/EBPα

CEBPA is a commonly used factor when reprogramming cells into not only iPSCs, but also other cells. C/EBPα has shown itself to be a single transacting factor during direct reprogramming of a lymphoid cell into a myeloid cell. C/EBPα is considered a 'path breaker' to aid in preparing the cell for intake of the OSKM factors and specific transcription events. C/EBPα has also been shown to increase the efficiency of the reprogramming events.


Variability

The properties of cells obtained after reprogramming can vary significantly, in particular among iPSCs. Factors leading to variation in the performance of reprogramming and functional features of end products include genetic background, tissue source, reprogramming factor stoichiometry and stressors related to cell culture.


See also

* Induced stem cells * Epigenome editing


References

{{reflist DNA Epigenetics Induced stem cells