The term epigenetics refers to the study of inherited changes in the phenotype of a cell that do not result from changes in DNA sequence. For example, during the differentiation of bone marrow stem cells into the several different types of blood cells, a hematopoietic stem cell divides into two daughter cells, one of which continues to have the properties of a hematopoietic stem cell, including the potential to differentiate into all the different types of blood cells. But the other daughter cell becomes either a lymphoid progenitor cell or a myeloid progenitor cell. Lymphoid pro genitor cells generate daughter cells that differentiate into lymphocytes, which perform many of the functions involved in immune responses to pathogens. Myeloid progenitor cells divide into daughter cells that are commit ted to differentiating into red blood cells, different kinds of phagocytic white blood cells, or the cells that generate platelets involved in blood clotting. Lymphoid and myeloid progenitor cells both have the same DNA sequence as the zygote (generated by fertilization of an egg cell by a sperm cell) from which they developed, but they have restricted developmental potential because of epigenetic differences between them.

Such epigenetic changes are initially the consequence of the expression of specific master transcription factors that are regulators of cellular differentiation, controlling the ex pression of other genes that encode transcription factors and proteins involved in cell-cell communication in com plex networks of gene control, and which are currently the subject of intense investigation. Changes in gene expression initiated by transcription factors are often reinforced and maintained over multiple cell divisions by post-translational modifications of histones and methylations of DNA at position 5 of the cytosine pyrimidine ring that are maintained and propagated to daughter cells when cells divide. Consequently, the term epigenetic marks is used to refer to such post-translational modifications of histones and 5-methyl C modification of DNA.

DNA Methylation Represses Transcription

As mentioned earlier, most promoters in mammals fall into the CpG island class. Active CpG island promoters have Cs in their CG sequences that are unmethylated. Un methylated CpG island promoters have reduced affinity for histone octamers, but nucleosomes immediately neighboring the unmethylated promoters are modified by histone H3 lysine 4 di- or trimethylation and are associated with Pol II molecules that are paused during transcription of both the sense and antisense template DNA strands, as dis cussed earlier. Recent research indicates that methylation of histone H3 lysine 4 occurs in mouse cells because a protein named Cfp1 (CXXC fin ger protein 1) binds unmethylated CpG-rich DNA through a zinc-finger domain (CXXC) and associates with a his tone methylase specific for histone H3 lysine 4 (Setd1). Chromatin-remodeling complexes and the general transcription factor TFIID, which initiates Pol II preinitiation complex assembly, associate with nucleosomes bearing the H3 lysine 4 trimethyl mark, promoting Pol II transcription initiation.

In differentiated cells, however, a small percentage of specific CpG island promoters, depending on the cell type, have CpGs marked by 5-methyl C. This modification of CpG island DNA triggers chromatin condensation. A family of proteins that bind to DNA that is rich in 5-methyl C–modified CpGs (called methyl CpG-binding proteins, or MBDs) bind to the marked promoters and associate with histone deacetylases and repressive chromatin-remodeling complexes that condense chromatin, resulting in transcriptional repression. The 5-methyl C is added to the CpGs by DNA methyl transferases named DNMT3a and DNMT3b. They are referred to as de novo DNA methyl transferases because they methylate an unmethylated C. Much remains to be learned about how DNMT3a and b are directed to specific CpG islands. But once they have methylated a DNA sequence, methylation at that C is passed on through DNA replication through the action of the ubiquitous maintenance methyl transferase DNMT1:

(red indicates daughter strands). As a consequence, once a CpG island promoter is methylated by DNMT3a or b, it continues to be methylated by DNMT1 in subsequent daughter cells. Consequently, the promoter remains repressed in all subsequent daughter cells through interactions with MBDs, even after the stimulus for the initial C-methylation by DNMT3a or b has ceased. Therefore, repression of C-methylated promoters is inherited through cell division. This mechanism of epigenetic repression is being intensely investigated because tumor-suppressor genes encoding proteins that function to suppress the development of cancer are often inactivated in cancer cells by abnormal CpG methylation of their promoter regions, as discussed further in Chapter 24.

Methylation of Specific Histone Lysines Is Linked to Epigenetic Mechanisms of Gene Repression

 Figure 1b summarized the different types of post- translational modifications that are found on histones, including acetylation of lysines and methylation of lysines on the nitrogen atom of the terminal ε-amino group of the lysine side chain. Lysines can be modified by the addition of one, two, or three methyl groups to this terminal nitrogen atom, generating mono-, di-, and trimethylated lysine, all of which carry a single positive charge.

Fig1. An in vivo transfection assay measures transcription activity to evaluate proteins believed to be transcription factors. The assay system requires two plasmids. One plasmid contains the gene encoding the putative transcription factor (protein X). The second plasmid contains a reporter gene (e.g., luciferase) and one or more binding sites for protein X. Both plasmids are simultaneously introduced into cells that lack the gene encoding protein X. The production of reporter-gene RNA transcripts is measured; alternatively, the activity of the encoded protein can be assayed. If reporter-gene transcription is greater in the presence of the X-encoding plasmid than in its absence, then the protein is an activator; if transcription is less, then it is a repressor. By use of plasmids encoding a mutated or rearranged transcription factor, important domains of the protein can be identified.

The acetylation state at a specific histone lysine on a particular nucleosome results from a dynamic equilibrium between acetylation and deacetylation by histone acetylases and histone deacetylases, respectively. Acetylation of histones in a localized region of chromatin predominates when local DNA bound activators transiently bind histone acetylase complexes. Deacetylation predominates when repressors transiently bind histone deacetylase complexes. Pulse-chase radiolabeling experiments have shown that acetyl groups on histone lysines turn over rapidly through the sequential actions of histone acetylases and histone deacetylases. In contrast, methyl groups on histones are much more stable. Histone lysine methyl groups can be removed by histone lysine demethylases. But the resulting turnover of histone lysine methyl groups is much slower than the turnover of histone lysine acetyl groups, which makes methylation the more appropriate post-translational modification for propagating epigenetic information.

Several other post-translational modifications of histones have been characterized (see Figure 1b). These modifications all have the potential to positively or negatively regulate the binding of proteins that interact with the chromatin fiber to regulate transcription as well as other processes, such as chromosome folding into the highly condensed structures that form during mitosis. A picture of chromatin has emerged in which histone tails extending as random coils from the chromatin fiber are post-translationally modified to generate one of many possible combinations of modifications that regulate transcription and other processes by regulating the binding of a large number of different protein complexes. This control of the interactions of proteins with specific regions of chromatin that results from the combined influences of various post translational modifications of histones has been called a his tone code. Some of these modifications, such as histone lysine acetylation, are rapidly reversible, whereas others, such as histone lysine methylation, can be templated through chromatin replication, generating epigenetic inheritance in addition to inheritance of DNA sequence. Table 1 summarizes the influence that post-translational modifications of specific histone amino acid residues usually have on transcription.

Table1. Histone Post-Translational Modifications Associated with Active and Repressed Genes

Histone H3 Lysine 9 Methylation in Heterochromatin In most eukaryotes, some co-repressor complexes contain his tone methyl transferase subunits that methylate histone H3 at lysine 9, generating di- and trimethyl lysines. These methylated lysines are binding sites for isoforms of HP1 protein that function in the condensation of heterochromatin, as dis cussed in Chapter 8. For example, the KAP1 co-repressor complex functions with a class of more than 200 zinc-finger transcription factors encoded in the human genome. This co-repressor complex includes an H3 lysine 9 methyl transferase that methylates nucleosomes over the promoter regions of repressed genes, leading to HP1 binding and repression of transcription. An integrated transgene in cultured mouse fibroblasts that was repressed through the action of the KAP1 co-repressor was associated with heterochromatin in most cells, whereas the active form of the same transgene was associated with euchromatin (Figure 2). Chromatin immunoprecipitation assays showed that the repressed gene was associated with histone H3 methylated at lysine 9 and with HP1, whereas the active gene was not.

Fig2. Association of a repressed transgene with hetero chromatin. Mouse fibroblasts were stably transformed with a transgene that contained binding sites for an engineered repressor. The repressor was a fusion between a DNA-binding domain, a repression domain that interacts with the KAP1 co-repressor complex, and the ligand-binding domain of a nuclear receptor that allows the nuclear import of the fusion protein to be controlled experimentally. DNA was stained blue with the dye DAPI. Brighter-staining regions are regions of heterochromatin, where the DNA concentration is higher than in euchromatin. The transgene was detected by hybridization of a fluorescently labeled complementary probe (green). When the recombinant repressor was retained in the cytoplasm, the transgene was transcribed (left) and was associated with euchromatin in most cells. When hormone was added so that the recombinant repressor entered the nucleus, the transgene was repressed (right) and associated with heterochromatin. Chromatin immunoprecipitation assays (see Figure 9-18) showed that the repressed gene was associated with histone H3 methylated at lysine 9 and HP1, whereas the active gene was not. [From Ayyanathan, K. et al., “Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation,” Genes and Development, 2003,17:1855–1869. Courtesy of Frank Rauscher; republished with permission from Cold Spring Harbor Laboratory Press.]

Importantly, H3 lysine 9 methylation is maintained following chromosome replication by the mechanism diagrammed in Figure 3. When a methylated region of DNA is replicated in S phase, the histone octomers associated with the parent DNA are randomly distributed to the daughter DNA molecules. New histone octamers that are not methylated on lysine 9 also associate randomly with the new daughter chromosomes, but since the parent histone octomers are associated with both daughter chromosomes, approximately half of the daughter chromosomes’ nucleosomes are methylated on lysine 9. Association of histone H3 lysine methyl transferases (directly or indirectly) with the parent methylated nucleosomes leads to methylation of the newly assembled histone octamers. Repetition of this process with each cell division results in maintenance of H3 lysine 9 methylation of this region of the chromosome.

Fig3. Maintenance of histone H3 lysine 9 methylation during chromosome replication. When chromosomal DNA is replicated, the parent histones randomly associate with the two daughter chromosomes, while unmethylated histones synthesized during S phase are assembled into other nucleosomes in those same daughter chromosomes. Association of histone H3 lysine 9 methyl transferases (H3K9 HMT) with parent nucleosomes bearing the histone 3 lysine 9 di- or trimethylation mark methylates the newly added unmodified nucleosomes. Consequently, histone H3 lysine 9 methylation marks are maintained during repeated cell divisions unless they are specifically removed by a histone demethylase.

Epigenetic Control by Polycomb and Trithorax Complexes

 Another type of epigenetic mark that is essential for repression of genes in specific cell types in multicellular animals and plants involves a set of proteins known collectively as Polycomb proteins and a counteracting set of proteins known as Trithorax proteins. These names were derived from the phenotypes of mutations in the genes encoding these proteins in Drosophila, in which they were first discovered. The Polycomb repression mechanism is essential for maintaining the repression of genes in specific types of cells, and in all the subsequent cells that develop from them, throughout the life of an organism. Important genes regulated by Poly comb proteins include the Hox genes, which encode master regulatory transcription factors. Different combinations of Hox transcription factors help to direct the development of specific tissues and organs in a developing embryo. Early in embryogenesis, expression of Hox genes is controlled by typical activator and repressor proteins. However, the expression of these activators and repressors stops at an early point in embryogenesis. Correct expression of the Hox genes in the descendants of the early embryonic cells is then maintained throughout the remainder of embryogenesis and on into adult life by the Polycomb proteins, which maintain the repression of specific Hox genes. Trithorax proteins perform the opposite function, maintaining the expression of the Hox genes that were expressed in a specific cell early in embryogenesis in all the subsequent descendants of that cell. Polycomb and Tri thorax proteins control thousands of genes, including genes that regulate cell growth and division (i.e., the cell cycle). Polycomb and Trithorax genes are often mutated in cancer cells, contributing importantly to the abnormal properties of these cells.

Remarkably, virtually all cells in the developing embryo and adult express a similar set of Polycomb and Trithorax proteins, and all cells contain the same set of Hox genes. Yet only the Hox genes in cells where they were initially repressed in early embryogenesis remain repressed, even though the same Hox genes in other cells remain active in the presence of the same Polycomb proteins. Consequently, as in the case of the yeast silent mating-type loci, the expression of Hox genes is regulated by a process that involves more than specific DNA sequences interacting with proteins that diffuse through the nucleoplasm.

A current model for repression by Polycomb proteins is depicted in Figure 4. Most Polycomb proteins are sub units of one of two classes of multiprotein Polycomb repressive complexes: PRC1 and PRC2. The PRC2 complexes are thought to act initially by associating with the repression domains of specific repressors bound to their cognate DNA sequences early in embryogenesis, or with ribonucleoprotein complexes containing long noncoding RNAs, as discussed in a later section. The PRC2 complexes contain histone deacetylases that inhibit transcription, as discussed above. They also contain a subunit [E(z) in Drosophila, EZH2 in mammals] with a SET domain, which is the catalytic domain of several histone methyl transferases. This SET domain in PRC2 complexes methylates histone H3 on lysine 27, generating di- and trimethyl lysines. A PRC1 complex then binds the methylated nucleosomes through dimeric Pc subunits (CBXs in mammals), each containing a methyl lysine–binding do main (called a chromodomain) specific for methylated H3 lysine 27. Binding of the dimeric Pc to neighboring nucleosomes is proposed to condense the chromatin into a structure that inhibits transcription. This proposal is supported by electron microscopy studies showing that PRC1 complexes cause nucleosomes to associate in vitro (Figure 4d, e).

Fig4. Model for repression by Polycomb complexes. (a) During early embryogenesis, repressors associate with the PRC2 complex. (b) This association results in methylation (Me) of neighboring nucleosomes on histone H3 lysine 27 (K27) by the SET domain containing subunit E(z). (c) The PRC1 complex binds nucleosomes methylated at H3 lysine 27 through a dimeric, chromodomain containing subunit Pc. The PRC1 complex condenses the chromatin into a repressed chromatin structure. PRC2 complexes associate with PRC1 complexes to maintain H3 lysine 27 methylation of neighboring histones. As a consequence, PRC1 and PRC2 association with the region is maintained when expression of the repressor proteins in (a) ceases. (d, e) Electron micrograph of a 1-kb fragment of DNA bound by four nucleosomes in the absence (d) and presence (e) of one PRC1 complex per five nucleosomes. See A. H. Lund and M. van Lohuizen, 2004, Curr. Opin. Cell Biol. 16:239; and N. J. Francis, R. E. Kingston, and C. L. Woodcock, 2004, Science 306:1574. [Parts (d) and (e) republished with permission of AAAS, from Francis, N.J. et al., “Chromatin compaction by a poly comb group protein complex, “ Science, 2004, 306(5701):1574–7; permission conveyed through Copyright Clearance Center, Inc.]

PRC1 complexes also repress transcription through additional mechanisms. The PRC1 complex contains a ubiquitin ligase that monoubiquitinylates histone H2A at lysine 119 in the H2A C-terminal tail. This modification of H2A inhibits elongation by inhibiting a histone chaperone that removes histone octamers from DNA as Pol II transcribes through a nucleosome, then replaces them as the polymerase passes. PRC1 also associates with a histone demethylase that specifically removes methyl groups from lysine 4 of histone H3, an activating mark discussed above.

PRC2 complexes associate with nucleosomes bearing the histone H3 lysine 27 trimethylation mark, maintaining methylation of H3 lysine 27 in nucleosomes in the region. This methylation results in association of the chromatin with PRC1 and PRC2 complexes even after expression of the initial repressor proteins shown in Figure 4a, b has ceased. This association maintains H3 lysine 27 methylation by a mechanism analogous to that diagrammed in Figure 3. This mechanism is a key feature of Polycomb repression, which is maintained through successive cell divisions for the life of an organism (~100 years for some vertebrates, 2000 years for a sugar cone pine!).

Trithorax proteins counteract the repressive mechanism of Polycomb proteins, as shown in studies of expression of the Hox transcription factor Abd-B in the Drosophila embryo (Figure 5). Abd-B is normally expressed only in posterior segments of the developing embryo. When the Polycomb system is defective, Abd-B is expressed in all cells of the embryo. When the Trithorax system is defective and cannot counteract repression by the Polycomb system, Abd-B is repressed in most cells, except those in the very posterior of the embryo. Trithorax complexes include a histone methyl transferase that trimethylates histone H3 lysine 4, a histone methylation that is associated with the promoters of actively transcribed genes. This histone modification creates a binding site for histone acetylase and for chromatin remodeling complexes that promote transcription, as well as for TFIID, the general transcription factor that initiates preinitiation-complex assembly. Nucleosomes with H3 lysine 4 methylation are also binding sites for specific histone demethylases that remove H3 histone K9 and K27 methylation, preventing the binding of HP1 and the Polycomb repressive complexes. Nucleosomes marked with H3 lysine 4 methylation are also thought to be distributed to both daughter DNA molecules during DNA replication, resulting in maintenance of this epigenetic mark by a strategy similar to that diagrammed in Figure 3.

Fig5. Opposing influence of Polycomb and Trithorax complexes on expression of the Hox transcription factor Abd-B in Drosophila embryos. At the stage of Drosophila embryogenesis shown, Abd-B is normally expressed only in posterior segments of the developing embryo, as shown at the top (wt) by immunostaining with a specific anti–Abd-B antibody. In embryos with homozygous mutations of Scm, a Polycomb gene (PcG) encoding a protein associated with the PRC1 complex, Abd-B expression is derepressed in all embryo segments. In contrast, in homozygous mutants of trx, a Trithorax gene (trxG), Abd-B repression is increased so that the protein is expressed at high concentrations only in the most posterior segment. [From Klymenko, T., and Muller, J., “The histone methyltransferases Trithorax and Ash1 pre vent transcriptional silencing by Polycomb group proteins,” EMBO Reports ©2004 John Wiley and Sons. Reproduced with permission of Wiley-VCH.]

Long Noncoding RNAs Direct Epigenetic Repression in Metazoans

Repressive complexes have been discovered that are com posed of multiple repressing proteins bound to RNAs many kilobases in length that do not contain long open reading frames and are consequently called long noncoding RNAs or lncRNAs. In some cases, these lncRNA-protein complexes re press genes on the same chromosome from which the RNA is transcribed, as in the case of X-chromosome inactivation in female mammals. In other cases, these repressive RNA-protein complexes act in trans, repressing genes on chromosomes other than those from which the lncRNA is transcribed.

X-Chromosome Inactivation in Mammals The phenomenon of X-chromosome inactivation in female mammals is one of the most intensely studied examples of epigenetic re pression mediated by a lncRNA. X inactivation is controlled by a roughly 100-kb domain on the X chromosome called the X-inactivation center. Remarkably, this region encodes several lncRNAs required for the random inactivation of one entire X chromosome early in the development of female mammals. The functions of these lncRNAs are only partially understood. The most intensively studied are transcribed from the complementary DNA strands near the middle of the X- inactivation center: the 40-kb TSIX lncRNA and the XIST RNA, which is spliced and polyadenylated into an RNA of about 17 kb that is not exported to the cytoplasm (Figure 6a).

Fig6. The Xist long noncoding RNA encoded in the X-inactivation center coats the inactive X chromosome in cells of mammalian females, repressing transcription of most genes on the inactive X. (a) The region of the human X-inactivation center encoding the noncoding RNAs Xist (transcribed from the inactive X), and Tsix (transcribed from the active X). Numbers are base pairs from the left end of the X chromosome. (b) A cultured fibroblast from a human female was analyzed by in situ hybridization with a probe complementary to Xist RNA labeled with a red fluorescent dye (left), a chromo some paint set of probes for the X chromosome labeled with a green fluorescent dye (center), and an overlay of the two fluorescent micro graphs. The condensed inactive X chromosome is associated with Xist RNA. (c) Model for the spreading of the Xist lncRNA-protein complex on the inactive X chromosome during early differentiation of female embryonic stem cells. See E. Heard and A.-V. Gendrel, 2014, Annu. Rev. Cell Dev. Biol. 30:561. (d) Proteins associated with Xist lncRNA. Question marks indicate that it is not yet known how PRC2 complexes associate with HDAC3 and the RNA-binding protein SHARP. See C. A. McHugh et al., 2015, Nature 521:232. [Part (b) ©1996 C. M. Clemson et al., The Journal of Cell Biology, 132:259–275. doi: 10.1083/jcb.132.3.259.]

In differentiated female cells, the inactive X chromosome is associated with XIST RNA-protein complexes along its entire length (Figure 6b). Targeted deletion of the Xist gene in cultured embryonic stem cells showed that it is required for X inactivation. Unlike most protein-coding genes on the inactive X chromosome, the Xist gene is actively transcribed. The XIST RNA-protein complexes do not diffuse to interact with the active X or other chromosomes, but remain associated with the inactive X chromosome. Since the full length of the inactive X becomes coated by XIST RNA-protein complexes (see Figure 6b), these complexes must spread along the chromosome from the X-inactivation center where XIST is transcribed. In contrast to XIST, TSIX is transcribed from the active X chromosome, not from the inactive X chromosome.

In the early female mouse embryo, made up of embryonic stem cells capable of differentiating into all cell types, genes on both X chromosomes are transcribed, and the 40-kb TSIX lncRNA (see Figure 6a) is transcribed from both copies of the X chromosome. Experiments employing engineered deletions in the X-inactivation center showed that TSIX transcription prevents significant transcription of the XIST RNA from the complementary DNA strand. Later in development, as cells begin to differentiate, TSIX transcription is repressed on one of the X chromosomes. This repression occurs randomly in different cells on the X chromosome derived from the sperm (X_p) or on the X chromosome derived from the egg (X_m). This inhibition of TSIX transcription determines which of the X chromosomes will be inactivated as the cells differentiate further because inhibition of TSIX transcription allows transcription of the XIST lncRNA on that chromosome.

The transcribed XIST RNA contains RNA sequences that, by unknown mechanisms, cause it to spread along the X chromosome. Recent studies indicate that XIST lncRNA-protein complexes first associate with regions of the X chromosome localized near the X-inactivation center in the three-dimensional, folded structure of the future inactive X (Figure 6c), as shown by chromosome conformation capture assays. These initial sites of XIST association are in gene-rich regions of the X chromosome and are postulated to serve as “entry sites” where additional copies of the XIST lncRNA-protein complexes first bind and then spread to neighboring regions. The mechanism of spreading is not currently understood. The inactive X chromosome also becomes associated with PRC2 complexes, which catalyze the trimethylation of histone H3 lysine 27. This methylation results in association of the PRC1 complex and transcriptional repression, as discussed above. These mechanisms of transcriptional repression must be redundant, however, because repression still occurs in the absence of the Polycomb proteins essential for the assembly of PRC1 and PRC2. At the same time, continued transcription of TSIX from the other, active X chromosome continues, represses XIST transcription from that X chromosome, and consequently prevents XIST-mediated repression of the active X. XIST and PRC1 and 2 complexes are then observed to associate with gene-poor regions of the inactive X chromosome as well as with gene-rich regions.

Recent analysis by protein mass spectrometry (see Chapter 3) of proteins associated with XIST lncRNA during the initiation phase of X inactivation in cultured mouse embryonic stem cells revealed that SMRT, a protein first characterized as a co-repressor that interacts with the thyroid hormone nuclear receptor in the absence of hormone, is part of the protein complex that interacts with XIST RNA. SMRT, in turn, interacts with a histone deacetylase (HDAC3). Subsequent knockdown experiments with siRNAs directed against SMRT and HDAC3 showed that they are required for X inactivation, as are other identified RNA- and chromatin-binding proteins that link SMRT to XIST RNA and are required for the association of XIST RNA and PRC2 with the inactive X chromosome (Figure 6d). A short time later in development, the DNA of the inactive X also becomes methylated at most of its CpG is land promoters. Specialized histone octamers in which histone H2A is replaced by a paralog of H2A called macroH2A also become associated with the inactive X. DNA methylation and macroH2A contribute to the stable repression of the inactive X through the multiple cell divisions that occur later during embryogenesis and throughout adult life.

Trans Repression by Long Noncoding RNAs Another example of transcriptional repression by a long noncoding RNA was discovered recently by researchers studying the function of noncoding RNAs transcribed from a region encoding a cluster of Hox genes, the HOXC locus, in cultured human fibroblasts. Depletion of a 2.2-kb noncoding RNA expressed from the HOXC locus by siRNA unexpectedly led to derepression of the HOXD locus, a roughly 40-kb region on another chromosome encoding several Hox proteins and multiple other noncoding RNAs, in these cells. Assays similar to chromatin immunoprecipitation showed that this noncoding RNA, named HOTAIR (for Hox Antisense Intergenic RNA), associates with the HOXD loci and with PRC2 complexes. This association results in histone H3 lysine 27 di- and trimethylation, PRC1 association, histone H3 lysine 4 demethylation, histone H2A monoubiquitinylation, and transcriptional re pression. This process is similar to the recruitment of Polycomb complexes by Xist RNA, except that Xist RNA functions in cis, remaining in association with the chromosome from which it is transcribed, whereas HOTAIR leads to Polycomb repression in trans on both copies of another chromosome. Once again, redundant mechanisms for repression of these HOXD loci must exist, because extensive, but less complete, repression at the HOXD locus continues in the appropriate cells in mouse embryos with homozygous HOTAIR knockout mutations.

Cis Activation by Long Noncoding RNAs Examples of lncRNAs involved in gene activation have been characterized recently. For example, HOTTIP lncRNA, which is transcribed from the 5′ end of the HOXA locus, is proposed to coordinate the activation of HOXA genes by binding to a histone H3 lysine 4 methylase. In addition, nascent transcripts of lncRNA genes have been reported to activate transcription from promoters several kilobases away by interacting with the Mediator complex and delivering it to the promoter by looping of the intervening chromatin.

In humans, but not in mice, a lncRNA called XACT has been discovered to associate with multiple sites along the full length of the active X chromosome and is postulated to contribute to maintenance of gene activity on that chromo some. XACT is also remarkable for being one of the longest characterized RNAs: 252 kb! It is mostly unspliced.

In Drosophila, equal expression of genes encoded on the X chromosome in males and females (dosage compensation) does not result from inactivating one X chromosome in females. Rather, a generalized twofold increase in transcriptional activation of genes on the single X chromosome in males is controlled by two lncRNAs, roX1 and roX2, transcribed from the X chromosome in males only. The roX1 and roX2 RNAs associate with several proteins encoded by MSL (male-specific-lethal) genes and spread over the X chromosome specifically, much as Xist lncRNA-protein complexes spread over the inactive X in mammals.

Recently, sequencing of total cellular RNA in multiple types of human cells identified roughly 15,000 human lncRNAs. Many of these lncRNAs have sequences that are evolutionarily conserved in most mammals, and about 5000 are found only in primates. This conservation of sequence strongly suggests that these lncRNAs, like XIST, HOTAIR, and HOTTIP, have important functions. Multiple lncRNAs are expressed only in specific cell types at specific times during development. For example, multiple lncRNAs are expressed primarily in differentiating red blood cells. Knockdown of several of these lncRNAs inhibits normal red blood cell development, but precisely how these lncRNAs perform their essential functions is not yet clear. The study of these conserved long noncoding RNAs and how they influence gene expression is another area of intense current investigation.

ENCODE (Encyclopedia of DNA Elements) encompasses a consortium of international research groups organized and funded by the US National Human Genome Research Institute with the goal of building a comprehensive, publically available database of human DNA control elements and the transcription factors that bind to them in different cell types, histone post-translational modifications mapped by ChIP-seq and other related methods, DNase I hypersensitive sites, and regulatory lncRNAs and their sites of association in the genome, as well as newly discovered regulatory elements “that control cells and circumstances in which a gene is active.” Data sets from human cells and cells of model organisms that are too large to be published are also made publically available at a site called GEO (Gene Expression Omnibus) maintained by the US National Center for Bioinformatics (NCBI). Most journals that publish research based on genomic methods such as RNA-seq and ChIP-seq require that authors upload their original data to GEO. Worldwide public access to these data sets is greatly accelerating the pace of discovery in the area of gene regulation.

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مواضيع ذات صلة

Chromatin-Remodeling Complexes Help Activate or Repress Transcription

Activators Can Direct Histone Acetylation at Specific Genes

Repressors Can Direct Histone Deacetylation at Specific Genes

Formation of Heterochromatin Silences Gene Expression at Telomeres, near Centromeres, and in Other Regions

Multiprotein Complexes Form on Enhancers

Transcription Factor Interactions Increase Gene-Control Options

Structurally Diverse Activation and Repression Domains Regulate Transcription

Electrophoresis of Nucleic Acids

Regulation of Transcription Factor Activity

Activators Are Composed of Distinct Functional Domains

DNase I Footprinting and EMSA Detect Protein-DNA Interactions

Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements