RNA polymerase II (Pol II) is a 12 subunit protein complex from yeast to human that is required for gene expression. Gdown1 containing Pol II [Pol II (G)] is a special form of Pol II that is catalytically inactive and heavily depends on the 30-subunit Mediator complex for its activator and basal dependent function in vitro. Here we report for the first time, the identification and the generation of a 15-subunit human Mediator complex via the novel multibac baculovirus expression system that is fully responsive to Pol II (G). Our results show complete recovery of Pol II (G) dependent transcription both with full 30-subunit Mediator and also with 15-subunit recombinant Mediator that we synthesized. Moreover, we also show that the recombinant Mediator interacts with Pol II (G) as well. These results enlighten us towards understanding how a certain population of Pol II that is involved in selected gene regulation is activated by Mediator complex. Mediator complex is a multisubunit coactivator complex consisting of different modules and is the chief regulator of the transcription by RNA polymerase II (Pol II) . It was first discovered as a purified complex in yeasts . Studies have shown that Mediator complex has an essential protein composition and structure which is largely conserved from yeast to human . Human Mediator complex was initially annotated as thyroid hormone receptor-associated protein (TRAP) complex . Subsequently, it was found that the Mediator complex has a critical role in not only activator dependent transcription but also basal transcription. Furthermore, Mediator complex-depleted nuclear extracts have been shown to be defective in terms of RNA Pol II transcription function in both TATA promoters and TATA-less promoters .Human Mediator is a dynamic coactivator complex consisting of 30 subunits with a molecular size of 2MDa . It consists of 4 modules: head, middle, tail and kinase . In humans, the head and middle modules, together with MED14 and MED26, constitute the active core Mediator complex . The tail and kinase modules are connected dynamically to the core Mediator . The Mediator complex primarily transmits signals from general transcription factors and activators to Pol II, regulating the function of the enzyme in the preinitiation, initiation, elongation and reinitiation stages .The eukaryotic Pol II, consisting of 12 evolutionally conserved subunits, is responsible for transcription of all protein-encoding genes . It has been found that Pol II also has a 13th subunit called Gdown1 (encoded by POL2RM), and the Pol II form containing this subunit is called Pol II (G) . Gdown1 persists to bind tightly to Pol II, even in the presence of high concentrations of salts or urea at low molarity. This protein is metazoan specific and is expressed in a wide number of human tissue samples . Therefore, Gdown1 is thought to play important roles in transcriptional regulation. Also, in vitro studies have shown that the Gdown1 acts as a repressor by inhibiting the transcription activity of Pol II in the absence of the Mediator complex . In the presence of the Mediator, however, the inhibitory effects of Gdown1 on both activator-dependent and -independent  transcription disappear . Unfortunately, the biological role of Gdown1-mediated repression and how the Mediator reverses this repression are not fully understood yet. However, it was found that in the presence of Gdown1, TFIIF’s (an important factor on PIC assembly) binding to Pol II was inhibited, leading to a blockage of PIC assembly . Also, Gdown1 directly binds to RPB1 and RPB5 subunits of Pol II and competes with TFIIF. In another interaction study, it has been suggested that the region  where Gdown1 interacts with Pol II coincides with TFIIF and TFIIB binding sites on Pol II . Binding of Gdown1 to Pol II inhibits transcription by preventing TFIIF and TFIIB interaction with Pol II in the absence of Mediator . Gdown1 also plays roles in the elongation phase of transcription. Since Gdown1 and TFIIF interaction sites on Pol II overlap, Gdown1 prevents the stimulation of elongation by TFIIF and blocks termination activity of TTF2. Phosphorylation of Gdown1 (Ser-270) by kinase decreases the binding of Pol II and reverses the inhibition of TTF2 and TFIIF . As this form of Pol II is responsive to Mediator and requires it for function, it is critical to understand which subunits of the Mediator are required to overcome this Gdown1 negative affect on Pol II. In this study, in vitro transcription assays using RNA polymerase II enzyme in the form of Pol II (G) purified from nuclear extracts and Mediator complexes with varying compositions  produced with multibac system were performed. Our results have shown for the first time that both the recombinant Mediator with 15 subunits and the full natural Mediator complex containing 30 subunits exerted a full recovery of transcription in the presence of Pol II (G). Moreover, we also show that this function of the core Mediator comes from its ability to interact with Pol II (G) that finally results in recruiting Pol II to promoters .HEK293 nuclear extracts were prepared from FLAG-tagged Gdown1 stable cell line and dialyzed in different concentration of TGEA buffer  and fractionated on a DE52 column. The fractionated nuclear extract (TGEA 300) dialysate was then subjected to M2 agarose beads and eluted with 0.5 mg/mL FLAG peptide.The input, flow-through, and the eluates obtained with various concentrations of the elution buffer in the purification steps were analyzed with Western blot, in which antibodies to Gdown1, RPB1 and RPB6 were used. Polyacrylamide gel electrophoresis was performed to check the purity after FLAG peptide elution and the gel was analyzed by silver staining.General transcription factors were purified as described before . Basically, while the recombinant TFIIB, TFIIE and TFIIE were expressed in bacteria and purified, baculovirus expresse TFIIA as well as the activators Gal4VP16, TRa and RXRa were purified from insect cells and purified . Full natural Mediator, TFIID, TFIIH and Pol II were purified from HeLa cell lines stably expressing FLAG-tagged subunit of the corresponding protein complexes .The Head+Middle+14+26 complex was reconstituted as described by Cevher et al. (2014). The cDNAs encoding different Mediator subunits which were tagged with either FLAG, Myc, 6xHis or HA tags were cloned into pFBDM transfer vectors. Head module subunits contained pFBDM  and the middle module subunits contained pUCDM . The plasmids were then integrated into the bacmid DNA and the viruses were amplificated by transfecting Sf9 cells. After that, Hi5 cells were infected for the production of H+M and H+M+14 complex. Protein extraction was performed using BC500  from Hi5 cells 60 h after infection and the lysates were washed with BC300 . Subsequently, the extract was incubated with anti-FLAG M2 agarose beads and the proteins were purified by eluting with 0.5 mg/mL FLAG peptide and BC300. Coomassie brilliant blue stain was used to confirm the presence of proteins in the final elution.Purified factor- or nuclear extract-based systems were used in the in vitro transcription assays as described previously . For the transcription reaction, 50 ng template was used. All templates containing the G-less cassettes downstream of the adenovirus major late (ML) core promoter were used for transcription analysis. The pG5ML template contained five Gal4 binding sites; the p4TRE template contained four T3 response element regions; p4ERED53 template contained four estrogen response element regions and TRE3.Purified general transcription factors  and PC4 are used for the assay. Gal4-Vp16, T3/TR/RxR and TR/RxR were added to the reaction mixtures containing the pG5ML, p4TRE and TRE3 templates, respectively, and labelled nucleotide triphosphates either [α-32P]UTP or [α-32P]CTP. The mixtures were incubated at 30 ºC for 50 min. Electrophoresis was then performed and analyzed by autoradiography.RNA Pol II consists of twelve evolutionarily conserved subunits and is required for transcription of protein coding genes. Some fraction of purified Pol II contains a tightly associated and metazoan specific thirteenth subunit called Gdown1 protein. This protein inhibits transcription of Pol II in the absence of the Mediator complex. Here we show the stepwise purification of recombinant human Mediator complex that removes the negative effect of Gdown1. For this, we use the novel multibac baculovirus expression system and recombinantly generate the largest human protein complex that has ever been reconstituted. We basically cloned the human head module subunits  and human middle module subunits  along with MED14 into pFBDM and pUCDM transfer vectors . Later, we verified the cloned subunits within these plasmids by PCR . Upon verification of the cloned subunits, we transformed them in to DH10Multibac containing bacteria. We later purified the DH10 Multibac bacmid from bacteria, verified that the transfer vectors integrated in to the bacmid via transposition as well as Cre-lox P recombination and then transfected insect Sf9 cells with the purified bacmid . Finally, we extracted proteins from insect cells and purified our recombinant Mediator proteins via affinity purification (against FLAG tag) followed by Superose 6 gel filtration (size exclusion chromatography) to get pure and near homogenous proteins . Hek293 cell extracts were stably expressed with f:Gdown1 protein in order to saturate Pol II with Gdown1 and purify large quantity of Pol II with saturated Gdown1. As the tag is on Gdown1, purification of Pol II via FLAG tag ensures that 100% of purified Pol II contains Gdown1 protein. The cell extract was prepared and dialyzed against TGEA with 100 mM salt. Later, the sample was loaded to DE52 anion exchange column . The bound proteins were eluted and the fraction with 300 mM TGEA was collected and put to M2 agarose for FLAG tag affinity purification. The purified protein was then resolved in SDS page and silver stain showing all subunits of Pol II along with Gdown1 was observed .Prior to checking the activity of our recombinant Mediator with Pol II (G), we characterized its function with HeLa nuclear extracts where the source of transcription comes from HeLa extracts. Basically, we immunodepleted Mediator from HeLa nuclear extracts and performed in vitro transcription reactions by supplementing the Mediator depleted nuclear extracts with our recombinant Mediator complexes. As the nuclear extract has a population of Pol II and Pol II (G), we did in vitro transcription with nuclear extracts and characterized if our recombinant Mediators could recover transcription. As can be seen in Figure 3a, neither head, nor middle nor head+middle could recover transcription (lanes 1–8). However, our human core Mediator complex could recover transcription to its full potential (lane 9). Later, we checked if the transcription was dose dependent on the recombinant human core Mediator complex and saw that as we increased the concentration of this protein complex, we observed more and more transcripts . Finally, we checked if our recombinant Mediator could recover activated transcription with VP16. For this, we used the natural 30-subunit Mediator complex as a positive control as it fully responds to VP16. As VP16 does not interact with the core-Mediator complex, we expected not to see an activated recovery of transcription but basal transcription . This way we verified that our recombinant Mediator is fully functional.Human Mediator was initially purified from liganded thyroid hormone receptor (TR/RXR). As Mediator strongly interacts with this activator and fully responds in transcription, we recombinantly generated and purified thyroid hormone receptor and its partner retinoid X receptor . We next tested if this liganded activator responds to Mediator dependent transcription in the presence  and absence of Mediator . For that we used either Mediator depleted or not depleted nuclear extracts. As can be seen, the activator TR/RXR (lanes 3 vs. 7) and VP16 (lanes 9 vs. 10) fully responded to Mediator dependent transcription. Once we optimized the working conditions for Mediator and the activator in nuclear extracts that partially has Pol II (G), we next purified all the general transcription factors , PC4, Mediator and Pol II (G) and optimized transcription with varying amounts of Pol II (G) in the presence and absence of Mediator and TR/RXR . With the Mediator, the negative effect of Gdown1 was fully recovered for both basal (lanes 1 vs. 3 and 5 vs. 7) and activated transcription (lanes 2 vs. 4 and 6 vs. 8). Finally, we wanted to characterize if our recombinant human core Mediator complex could revert the negative effect of Gdown1 on Pol II. In order to do these kinds of experiments, our lab is equipped with unique recombinant purified system where we can dissect in to purified proteins  to understand the necessity/function of each factor in the in vitro transcription system. Therefore, we purified all GTFs , PC4 and recombinant Mediator variants among with Pol II (G)  and tested if the recombinant Mediator could revert the negative effect of Gdown1 on Pol II just like a full natural 30-subunit Mediator can. Remarkably, for the first time, we can see from Figure 5a that the recombinant human core Mediator could recover basal transcription of Pol II (G) to the same extent as full natural 30-subunit Mediator. This suggests us that the subunits within this 16 subunit core Mediator could revert the negative effect of Gdown1 and may not need the tail and the kinase modules to overcome this effect. Next, we checked if the recombinant human core Mediator could recover TR/RXR activated transcription. Here, as we know that our human core Mediator does not interact with TR/RXR, we do not expect it to recover activated transcription but we expect it to only recover basal transcription. As can be seen from Figure 5b, while the natural Mediator could recover both activated and basal transcription (lanes 1–4), the recombinant core Mediator could only recover basal transcription (lanes 5–8) proving once again that our human core Mediator fully responds to Pol II (G) and removes the negative effect. Mediator exerts its function mainly by binding to RNA Pol II and recruiting it to promoters. Finally, in order to understand the mechanism of how the recombinant human core Mediator activates Pol II (G) transcript, we checked if this recombinant Mediator could interact with Pol II (G). Remarkably, for the first time, we see that this form of the Mediator interacts to its full extent with Pol II (G)  and telling us this is how it can recruit this form of Pol II to promoters .Mediator complex is critical for both basal and activated transcription as it serves as a hub between enhancer bound activators and promoter bound general transcription factors. In fact, Mediator was first isolated in yeast with Pol II and thus was called a holoenzyme. Its strong association with Pol II is a key property of Mediator as it brings Pol II to promoters to facilitate transcription . Later, Mediator was purified from human cell extracts through liganded thyroid hormone receptor and hence was initially named as thyroid hormone receptor-associated protein complex (TRAP). Later, we recombinantly generated the core-Mediator complex composing of head+middle+MED14 to bind to Pol II and recruit Pol II to promoters to facilitate transcription. A long standing question was in what is the Mediator component that could revert the negative effect of Gdown1 protein in Pol II (G). Previously, we reconstituted the largest protein complex up to date and showed that the recombinant human Mediator can fully activate Pol II mediated transcription . Here, in order to see if our recombinant core could recover transcription of Pol II (G) and compare it to the full natural Mediator complex, we tested our recombinant human core-Mediator complex for in vitro transcription and coimmunoprecipitation assays. Remarkably, our in vitro transcription assays as well as coimmunoprecipitation show that the recombinant Mediator not only can bind to Pol II (G) but also can revert its negative effect on transcription just like the natural Mediator can. Our findings now open-up new avenues towards understanding the Mediators role at its subunit, modular and multimodular level in detailed understanding of not only how enhancer bound activated genes are activated  but also how the negative effects are overcome by the Mediator complex.


Doc5 transposable element, which retains a heterochromatin insertion pattern in the melanogaster species complex, can be bound by chromatin proteins, and thus be involved in the organization of heterochromatic domains. Using the Yeast One Hybrid approach, we found Rpl22 as a potential interacting protein of Doc5. We further tested in vitro the observed interaction through Electrophoretic Mobility Shift Assay, uncovering that the N-terminal portion of the protein is sufficient to interact with Doc5. However, in situ localization of the native protein failed to detect Rpl22 association with chromatin. The results obtained are discussed in the light of the current knowledge on the extra-ribosomal role of ribosomal protein in eukaryotes, which suggests a possible role of Rpl22 in the determination of the heterochromatin in Drosophila.Chromatin is a highly dynamic biological entity that allows for both the control of gene expression and the stabilization of chromosomal domains. Given the high degree of plasticity observed in model and non-model organisms, it is not surprising that new chromatin components are frequently described. In this work, we tested the hypothesis that the remnants of the  Chromatin  is a nucChromatin is found in two fundamental states during the cell cycle, the loosely condensed euchromatin and the highly compacted heterochromatin. A huge number of DNA–protein and protein–protein interactions contribute to the maintenance of these two structures, the plasticity of which is tightly regulated at the epigenetic level.Many proteins act as structural components or regulators of the chromatin state, and post-translational modifications of many chromatin components play a fundamental role in maintaining the dynamic state of different chromatin domains. The ongoing ENCODE projects ,6 aim toSeveral pieces of observation suggest that ribosomal proteins (RPs) could have an active role in chromatin dynamics. First, RNA-mediated processes have a functional role in regulating chromatin structure and gene expression through the action of non-coding RNA molecules ,8,9. SecThird, the presence of RPs in the nucleus is well-recognized since RPs are imported into the nucleus and assembled into pre-ribosomes in the nucleolus .Therefore, a subset of RPs could be co-opted as chromatin components to perform additional functions under either physiological or exceptional conditions.D. melanogaster, several hundreds of genes have been mapped in the constitutive heterochromatin, thus demonstrating its importance in the physiology of cells, tissues, and organs in the fly ATP , 1X Klenow reaction buffer and 2U of Klenow fragment .In total, 5 μg of the pT/Doc5 plasmid was EcoRI-digested and the released fragment was gel-purified using the QIAquick Gel Extraction Kit (Qiagen). A filling-in reaction was performed to end-label the target DNA. A total of 50 ng of the eluted fragment was incubated with [αLabeled fragments were purified using Sephadex G50 exclusion chromatography columns.A total of 2 ng of the labeled fragment was incubated with the appropriate protein  in binding buffer as described in   were performed according to James et al.  using thSequencing of the cloned fragments was performed at the BMR Genomics sequencing facility .Global alignments were performed using DNA Strider . Local ahttp://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi (accessed on 1 March 2021)) [https://nucpred.bioinfo.se/cgi-bin/single.cgi (accessed on 2 March 2021)) [NLS signals were searched with cNLS Mapper (h 2021))  using a h 2021)) .Bari1 cluster in the heterochromatin of 2R chromosome of D. melanogaster [Doc5/Porto1 element, a highly repeated non-LTR retrotransposon in the heterochromatin of D. melanogaster [Doc5/Porto1 element is shown in Doc5.We have previously identified a 596 bp DNA sequence duplication (formerly named DRM8) at both sides of the nogaster . Specifinogaster . The simDoc5 can be found in the reference genome of D. melanogaster  applied to identify positive clones ensures that the false positive rate is minimized.Twenty-four positive clones, selected on selective media lacking histidine, were further tested with the β-galactosidase activity . Many ofDoc5-His and Doc5-lacZ cassette, data not shown) to confirm the bait–pray interaction.The positive clones obtained from the first round of screening were further validated by independent transformation of the isolated plasmids into the bait-containing yeast strains .To further solidify this result, we assayed the Rpl22–As can be observed in Doc5 interaction , piRH. sapiens and model organisms, respectively. The outcome of these huge projects led to the birth of epigenomics that aims to link cell-type-specific gene expression to chromatin structure. The specific features of chromatin domains are also of critical importance for genome evolution since the propensity of certain loci to be converted and relocated from the euchromatin to the heterochromatin is probably determined by the ancestral epigenetic marks [The stabilization of large chromosomal domains containing extended repeat blocks essentially depends on the chromatin architecture that wraps these loci. Both the Encode  and modEic marks . For thiHowever, chromatin is a highly dynamic biological entity, and for this reason, it is difficult to provide a definitive and exhaustive description. Unbiased approaches, i.e., not focused on a particular developmental stage or specific tissue, allow for a near-to-complete characterization of chromatin-associated proteins. It follows that the elucidation of the changing state of chromatin in the most diverse cellular types is of particular importance toward the complete understanding of physiological and pathological conditions .Doc5 transposon, a non-autonomous TE family enriched in the heterochromatin of D. melanogaster and closely related species [Drosophila, the direct binding of protein to TEs, especially involving retrotransposons, has been previously reported [D. melanogaster expression library with Doc5 as bait and found Rpl22 as the best candidate interacting protein. We have further validated the DNA–protein interaction with a series of EMSA experiments that confirmed the results of the experiments in yeast. We further demonstrated that the NH-terminal domain (H1–H5 domain) of the protein is both necessary and sufficient to bind DNA. Furthermore, the assays performed in vitro show that the Doc5–Rpl22 interaction depends on the amount of protein input. We cannot dismiss the hypothesis that this behavior could depend both on the presence of multiple binding sites on the target (which we have not investigated), and on the ability of Rpl22 to multimerize or to form homogeneous aggregates. In addition, the net charge density of the expressed H1-H5 domain is greater than that of the wild-type Rpl22 protein , which can account for the increased shift of the H1-H5/Doc5 complex if compared to the wild-type Rpl22/Doc5 complex  and cl-8  cell cultures, and it is associated with chromatin, resulting in gene suppression. Immunofluorescent staining and chromatin immunoprecipitation (ChIP) analyses demonstrated that RpL22 and H1 are both associated with condensed chromatin. In the same study, it was demonstrated that the overexpression of RpL22 caused the transcriptional repression of two-thirds of the genes suppressed by histone H1. By contrast, RpL22 depletion caused the up-regulation of the transcription of several tested genes, supporting a role for RpL22 as transcriptional repressor [Rpl22 is a ribosomal protein with a prevailing cytoplasmic localization. Past-published reports claimed that Rpl22 also localizes to the nucleus of borators  demonstrepressor . These oDoc5 transposon in the D. melanogaster genome. Non-autonomous TEs often acquire new functions in complex genomes, over evolutionary time. Many examples of evolutionarily inactive TEs that have been co-opted, exapted, or domesticated are described in the scientific literature [However, Rpl22 has not been previously identified in surveys aimed at the identification of chromatin structure. This can be due to an experimental bias when searching histone modifications . On the terature ,66.Doc5 is under the control of the piRNA pathway [Doc5 copies are found, these findings suggest that the short RNAs generated from Doc5 could have alternative roles in the regulation of gene expression, or alternatively in the regulation of the transposition of other, unrelated, TEs.It has been demonstrated that  pathway ,68. SincDoc5 could mark a chromatin domain with a structural function that prevents the excessive expansion of the Bari1 cluster. This hypothesis could be extended to other species-specific heterochromatic repeats, since the Bari1 cluster is unique to the D. melanogaster species, while Doc5 is present in the genome of sibling species [Alternatively,  species .In contrast with previously reported results, we were not able to demonstrate/reproduce a pan-nuclear localization of Rpl22 in S2R+ cells. Our experiments only revealed a nucleolar localization of the protein, without any detectable association to chromatin. This contrasting result could be explained considering the limitations of the immunolocalization technique, which would not allow for the detection of a small amount of chromatin proteins. Moreover, the differences between the cell lines used in our experimental setup (S2R+) and in previous studies (Kc) should be taken into account. Kc are male derived, while S2R+ derive from females. Kc have a plasmatocyte-like phenotype, while S2R+ combines properties of plasmatocytes and crystal cells . FinallySimilarly, we did not detect Rpl22 signals on the polytene chromosome arms, nor in the chromocenter. The limitation in terms of resolution using the polytene chromosome to assess the presence of DNA–protein interactions in heterochromatin is due to the under-replicated nature of the chromocenter ,72. MoreDrosophila S2 cells in an RNAi screen [S. pombe revealed the presence of ribosomal protein complexes at transcription sites with unexpected peaks at centromeres, raising the intriguing hypothesis that RP complexes are involved in tRNA biogenesis and possibly centromere functions [Despite the lack of localization to chromosomes and chromatin, several additional observations support the hypothesis of an involvement of Rpl22 in chromatin dynamics. There are 12 out of 91 Rpl22-interacting proteins that suggest its involvement in chromatin-related processes. Furthermore, RpL22 has been identified as one of the two hundred genes required for mitotic spindle assembly in i screen , and theunctions .D. melanogaster. However, experiments in vivo do not confirm the results of experiments in vitro, suggesting that further investigation is needed to reveal the physiological role of Rpl22 in the context of the chromosome structure.We have presented in vitro evidence of the interaction between a typical heterochromatic sequence and a ribosomal protein in Doc5 element has been co-opted to absolve further functions in the heterochromatin, several suggestive hypotheses could be proposed. Doc5 could act as a bidirectional promoter that allows for the transcription of the Bari1 cluster in order to activate the piRNA-mediated repression of the transposition. In this hypothesis, the ribosomal protein Rpl22 could help in the transcriptional activation from this promoter [Bari1 elements tested so far are transpositionally active [D. melanogaster laboratory strains [Genes) also demonstrated that Rpl22 binds in vitro to a transposable element-derived consensus sequence. This observation leads to the hypothesis that ribosomal proteins could be also involved in controlling the activity of TEs in Drosophila, not only at the translation level [While further studies are needed to understand if the promoter  or in thpromoter . Considey active ,21,75 any active ,77, this strains . In a coon level  but also


Ribosomal transcription constitutes the major energy consuming process in cells and is regulated in response to proliferation, differentiation and metabolic conditions by several signalling pathways. These act on the transcription machinery but also on chromatin factors and ncRNA. The many ribosomal gene repeats are organised in a number of different chromatin states; active, poised, pseudosilent and repressed gene repeats. Some of these chromatin states are unique to the 47rRNA gene repeat and do not occur at other locations in the genome, such as the active state organised with the HMG protein UBF whereas other chromatin state are nucleosomal, harbouring both active and inactive histone marks. The number of repeats in a certain state varies on developmental stage and cell type; embryonic cells have more rRNA gene repeats organised in an open chromatin state, which is replaced by heterochromatin during differentiation, establishing different states depending on cell type. The 47S rRNA gene transcription is regulated in different ways depending on stimulus and chromatin state of individual gene repeats. This review will discuss the present knowledge about factors involved, such as chromatin remodelling factors NuRD, NoRC, CSB, B-WICH, histone modifying enzymes and histone chaperones, in altering gene expression and switching chromatin states in proliferation, differentiation, metabolic changes and stress responses. Ribosomal genes, both the nucleolar 47S genes and the 5S rRNA genes, are highly transcribed and contribute to the major part of all transcripts. The expression is tightly coupled to proliferation and cell growth with a high demand for protein synthesis ,2,3 in mRibosomal biogenesis takes place in the nucleolus, a membrane-less nuclear body that forms around active Nuclear Organising Regions (NORs), after each mitosis . The NORThe nucleolus is also the target of several stress responses, such as heat shock, hypoxia, and DNA damage, all of which affect the expression of the 47S rRNA transcription ,7,8. In The human haploid genome harbours approximately 200 gene repeats found in clusters located on five acrocentric chromosomes, chromosome 13, 14, 15, 21 and 22 ,8. Not aThe active 47S rRNA gene repeats are organised with the high motility group (HMG) protein UBF, which binds at the at the enhancer element, UCE, in the promoter, but also in the gene body and the upstream enhancer region ,33,35,37The boundary element functions as an insulator between chromatin configurations and may also represent a nucleation site for assemble factor that are responsible for switching chromatin structures . SeveralInitiation and elongation of the 47S rRNA transcription are rate limiting steps in responses to environmental cues without changing the chromatin state ,51,52,53Directly upstream of the UCE in the promoter, a binding site, T0, for transcription termination factor 1 (TTF-1) is located , and a sSimilar to histones, UBF is acetylated by histone acetyl-transferases (HATs), particular p300/CBP, and deacetylated by histone deacetylases (HDACs) to regulate the 47S rRNA expression ,65. RiboThe 47S rRNA gene repeats are maintained in and open chromatin state early in development, and then subsequent silencing of gene repeats is an important process during differentiation . In the The repressed state established by NoRC is characterised by repressive chromatin marks, heterochromatin factors, and methylated DNA ,74,75,76Histone variants of the canonical histones have been associated with rDNA. The DNA-methylated state binds macroH2A, which is associated with silent chromatin . It accuThe switch between active and DNA methylated inactive states involves the NoRC complex, which replaces UBF for nucleosomes in a process that occurs mainly during differentiation, embryonic development and malignant transformation. The switch employs specific transcription factors, chromatin remodelling and histone PTMs  in cells that need to develop a migratory cellular program, such as neural crest progenitor cells in early development. A temporary increased ribosomal transcription in G1/S-phase is required for EMT and is executed by the EMT-specific transcription factor Snail and the mTORC2 component Rictor . The incLittle is known about the switch between rRNA repeats silenced with the histones variants macroH2A and H1 and active genes. If these gene repeats also exhibit further attributes of NoRC silenced chromatin than DNA-methylation is not known. Nevertheless, nucleolin counteracts the silent state imposed by macroH2A by increasing UBF loading along the rRNA gene body and increasing the presence of active histone marks at the promoter ,93. NuclThe rRNA gene promoters adopt further chromatin states than the active and repressed gene states referred to as the poised and pseudo-silent states, which in contrast to the repressed promoters silenced by NoRC, assemble on unmethylated DNA. The exact compositions of these states are largely unknown and they may constitute a group of differently organised nucleosomal gene repeats ,49,101 wThe poised “closed” marked nucleosome at the promoter is moved by the ATPase CSB to an active position (−157 to −2), compatible with TIF-1A/RRN3 and RNA pol I binding Many factors have been associated with the switch between the permissive poised state and the active state. In mouse cells, the NoRC, NuRD and CSB complexes bind to TTF-1 and are recruited to the promoter region where they remodel the chromatin ,101. TTFHow TTF-1 regulate its interactions with chromatin remodellers and affects gene expression is not fully understood. Nucleolin reduces the binding of TTF-1 to T0, which decreases the association of TIP5 with the promoter  but whetThe 47S rRNA gene repeats are organised in several in different chromatin states and differently regulated to meet the need from the environment. In mammalian cells, the active genes are organised with UBF in an open chromatin state, constitutively repressed genes are heterochromatinised on methylated-DNA, and further gene repeats are in the less defined permissive poised state or pseudo silent state B. It is Several ncRNA from both the rDNA and outside of the nucleolus are involved in the regulation of 47S rRNA gene transcription. These work in two ways, either by influencing the chromatin structure at the promoter or by binding proteins influencing the structure of the nucleolus A,B. Thes


The roles of the two isoforms of ErbB3-binding protein 1 (Ebp1) in cellular function and its regulation in disease and development is a stimulating area in current fields of biology, such as neuroscience, cancer biology, and structural biology. Over the last two decades, a growing body of studies suggests have suggested different functions for the EBP1 isoforms in various cancers, along with their specific binding partners in the ubiquitin-proteasome system. Owing to the specific cellular context or spatial/temporal expression of the EBP1 isoforms, either transcriptional repression or the activation function of EBP1 has been proposed, and epigenetic regulation by p48 EBP1 has also been observed during in the embryo development, including in brain development and neurologic disorders, such as schizophrenia, in using an Ebp1 knockout mouse model. Here, we review recent findings that have shaped our current understanding of the emerging function of EBP1 isoforms in cellular events and gene expression, from development to disease. PA2G4 gene can undergo enzymatic processing to yield two different protein products, p42 EB1 and p48 EB1. These proteins differ by the presence or absence of 54 amino acids at one end, and Jee-Yin Ahn at the Sungkyunkwan University School of Medicine, Suwon, South Korea, and colleagues have reviewed current insights into the functional consequences of this difference. The two proteins bind to distinct sets of molecular partners. The p48 form appears to regulate a host of genes involved in brain development, but also appears to drive cancerous growth in various tumors. In contrast, p42 is scarcer during development, and appears to inhibit tumor formation.A pair of proteins that originate from a common gene exert strikingly different effects on embryonic development as well as tumor growth and progression. RNA transcripts generated from the  The Ebp1 gene (PA2G4) is composed of ten exons and encodes two splice variants, p48 EBP1 and p42 EBP1 (lacking 54 amino acids at the N-terminus)2. The difference in the N-terminus between p48 and p42 EBP1 directs unique function and association with different binding partners and their regulation3. The longer form of p48 Ebp1 is the predominant form that is highly expressed in mammalian cells during embryogenesis and is maintained at a certain level after birth. p42 EBP1 can be detected after birth when the p48 EBP1 level is decreased relative to its level at the embryonic period4. In several types of cancer cells, including glioblastoma multiforme (GBM) and lung cancer cells, p48 EBP1 expression was notably high, which was noteworthy because of its oncogenic function. p48 EBP1 enhances cancerous growth and cell cycle progression and prevents cell death through the regulation of HDM2 activity for p537. Conversely, p42 EBP1 is barely detectable in cancer cells, and its ubiquitin-proteasome system (UPS)-dependent degradation acts as a tumor suppressor. This leads to the UPS-dependent degradation of the p85 subunit of PI3K, inhibiting its activity9.ErbB3-binding protein 1 (Ebp1) is a member of the PA2G4 family of proteins, which regulate proliferation10. Cryo-EM reconstruction studies have suggested that EBP1 can potently regulate translation as it binds to DNA11 and an array of RNA targets, including FMDV-IRES12, the 3′-UTR region of bcl2 mRNA13, various RNAs14, and the mature human 80S ribosome15. However, such data cannot address the specific roles of EBP1 in translational regulation. In addition, although experimental validation is lacking, p42 EBP1 is assumed to not bind to ribosomes when it lacks the 54 amino acids that are involved in recognition of the cs1 element of ES27L-B15. Nonetheless, studies suggest that two of the EBP1 isoforms could have different properties in terms of overall conformation, folding, and differential interacting partners, including ribosome binding.The X-ray structure of p48 EBP1 confirms the presence of a methionine aminopeptidase fold; it has also been highlighted that p42 EBP1 is missing 1.5 helices at the amino terminus16. Overexpression of EBP1 disrupts muscle progenitors, leading to neurogenic-like states. EBP1 protein is healthily expressed in myoblasts during development and regenerative myogenesis. This expression controls the balance between proliferation and differentiation in the chick embryo model17. In Xenopus, loss of Ebp1 leads to downregulation of the neural border zone, neural crest, and cranial placode genes by interacting with the transcription factor Six118. The only Ebp1 knockout mouse, which was generated to study embryonic development, came from our lab, and it showed that embryos of Ebp1(−/−) mice exhibited developmental abnormalities, including brain malformation and severe hemorrhage development with massive cell death and failure in cell cycle progression19. Notably, we suggest that an essential role for EBP1 in development is the repression of the gene-silencing unit Suv39H1/DNMT1, with the suppression here leading to target gene expression.Recent studies have proposed a potent role for EBP1 in embryonic development. For instance DPPA4 expression is restricted to early embryos and diminishes at E13.5. Furthermore, it interacts with p48 EBP1 in pluripotent stem cells yet not in healthy, nonpluripotent stem cells; further, this interaction was reduced upon differentiation of pluripotent stem cellsThe precise molecular mechanisms of how EBP1 contributes to different steps in cellular processes are not well defined, despite independent studies over the last two decades that demonstrate that EBP1 regulates various cellular events, including proliferation, survival, and differentiation, either in embryogenesis or human disease   comprises ten exons and extends over 8.4 kb. The ATG initiation codon is located in exon 1, and the STOP codon is located in exon 10. One Ebp1 isoform has been reported; it encodes 394 amino acids , although it encodes two alternatively spliced EBP1 isoforms, p48 (394 a.a.) and p42 (340 a.a.). Pa2g4 is located ~1.3 kb downstream of Erbb3, and it encodes the human epidermal growth factor receptor heregulin. Furthermore, Erbb3 overlaps with the Ebp1 putative regulatory region. Although EBP1 was identified as a binding partner of ERBB320, only p42 EBP1 binds ErbB3; p48 EBP1 did not bind to ErbB32.The ErbB3-binding protein 1 mouse Ebp1 are ~2.2 and 1.7 kb in size22. Ebp1 contains three in-frame ATG codons in its N-terminus. Translation of the longer transcript initiates at the first ATG in exon 1 and generates a 394 amino acid protein, p48 EBP1. Alternative splicing of pre-mRNA presumably leads to the omission of the 29 nucleotides that contain the second ATG, resulting in a smaller transcript. The frameshift that is caused by this mutation leads to translation of the smaller mRNA, which is initiated at the third ATG in exon 2b and the generation of p42 EBP1. p48 EBP1 migrates at an apparent molecular weight of 48 kDa, while p42 EBP1 migrates at an apparent molecular weight of 42 kDa, as shown by SDS/PAGE2.In humans, the two mRNA transcripts of 14, and a peptidase m24 domain (104–120 a.a.). The X-ray structure of EBP1 (8–360 a.a.) revealed that it possesses the predicted methionine aminopeptidase (MetAP) fold (type II); however, it did not possess catalytic activity, which may be due to its shorter N-terminus (~15- residue missing) and a C-terminal extension of 50 residues harboring a highly positively charged lysine patch. Although the lysine-rich C-terminal region was not part of the crystallization model , the positive surface charge of EBP1 was found to cluster on the convex side and included a charged surface loop (62–72 amino acid) and C-terminal extension (338–360) that can interact with proteins and RNA23. Unlike the prediction with sequence analysis that suggests EBP1 contains two conserved RNA binding domains as a consensus sequence with a sigma 70-like motif (46–64 amino acid) and ds RNA binding domain (91–156)24, neither of these domains is observed in the crystal structure of EBP1. Rather, a lysine-rich 364–373 amino acid sequence is likely to be the key determinant for RNA binding. This was highlighted by the deletion of this region that severely impairs EBP1 binding to RNA or DNA targets and mediates the interaction with internal ribosome entry site (IRES) of the foot and mouth disease virus (FMDV), exhibiting sequence-specific stimulation of FMDV-IRES-mediated translation initiation10. The predicted crystal structure of p42 lacks 1.5 alpha-helices, and a large hydrophobic surface of p42 is exposed to the solvent. This exposure may cause destabilization of p42 Ebp1, accounting for its significantly lower levels of expression in many types of cancer cells25.The amino acid M55 in p48 corresponds to M1 in p42; for simplicity, we use M55 hereafter to refer to both p42 and p48. p48 Ebp1 (394 amino acid) contains one initiator methionine (this residue is removed), two nucleolar localization signals in exon 1 (lysine 20 and 22 residues) that are necessary for nucleolar localization15. Moreover, EBP1 occupancy on the ribosome was not found to be different when comparing healthy cells or cells derived from cancer cells (HEK293 cells vs. HeLa cells). Despite the cell proliferation ability of p48 EBP1 in cancer cells26, neither of the experiments revealed a direct effect of EBP1 on translational inhibition. This may be due to the different roles of the EBP1 isoforms in cell growth. Predictably, p42 EBP1 may have impaired binding to the ribosome, as the missing 54 amino acids are involved in recognition of ES27L and are an integral feature of the MetAP-like fold. Therefore, EBP1 binding to the ribosome is likely isoform-specific and perhaps correlates to tumor progression15. However, the translational inhibitory role of p48 EBP1 or its mechanistic study has not yet been experimentally validated.A recent high-tech cryo-EM reconstruction of EBP1 highlighted that EBP1 binds to the human mature 80S ribosome and occupies the exit site of the ribosome and recruits the rRNA expansion segment ES27L, which is one of the longest tentacle-like dsRNA insertions. However, the biological impact of EBP1 in the EBP1-ribosome complex is not clarified, as EBP1 binding did not alter the translational elongation cycle or any conformational changes in the ribosomal proteins and rRNA in the peptidyl transferase center27. Before we reported two isoforms of EBP12, early studies of EBP1 from Dr Hamburger’s group suggested that EBP1 was a binding partner of ErbB3, and the assumption was that it was p42 EBP1. A series of studies have proposed that EBP1 is a tumor suppressor in breast and prostate cancer cells following the study of p42 EBP1, in which translation starts only at the third in-frame ATG. In support of this observation, the pBK/Ebp1 expression plasmid was used throughout their study; the plasmid encoded a 372 amino acid protein lacking N-terminal 22 amino acids33. Human Ebp1 cDNA encodes a protein predicted to have 394 amino acids; as expected, the protein was found to have 394 amino acids when it was empirically analyzed. In agreement with the tumor-suppressive activity of EBP1, our studies also demonstrated that p42 EBP1 acts as a tumor suppressor in GBM and non-small lung cancer (NSCLC) cells34. Strikingly, p42 EBP1, and not p48 EBP1, interacts with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and couples the p85 subunit to heat shock protein 70 (HSP70). The carboxy terminus of Hsp70 interacting protein (CHIP)-mediated UPS thereby promotes a reduction of p85 subunits, consequently leading to inhibition of its lipid kinase activity in both in vitro and in vivo models7. Moreover, p42 EBP1 also suppressed AKT kinase activation, resulting in inhibition of tumorigenic properties of NSCLC34. Furthermore, in salivary adenoid cystic carcinoma35, the tumor suppression activity of EBP1 was demonstrated using a pcDNA-ebp1 plasmid obtained from Dr. Hamburger. This work suggests that this tumor suppression effect comes from p42 EBP1.We have identified two isoforms of the EBP1 protein, p48 EBP1 and p42 EBP1, that are translated and expressed in humans, rats, and mice, but only p42 EBP1 can bind to ErbB334. These results are in agreement with the finding that only p42 EBP1 is polyubiquitinated and degraded in human cancer cells via hBre125. Interestingly, in colorectal cancer cells, both p48 EBP1 and p42 EBP1 bind to FBXW7 (F-box and WD40 domain protein 7), a substrate recognition subunit of the SCF (SKP1/CUL1/F-box protein) E3 ligase complex, and p48 EBP1 is ubiquitinated and degraded in a GSK-3b-dependent manner, whereas p42 EBP1 does not undergo degradation. In contrast, a tumor suppressor function of FBXW7 was accelerated by p42 EBP1, enhancing the association of FBXW7 and its oncogenic targets, including c-Myc, Aurora-A, and cyclin E. At the same time, p48 EBP1 contributes to the relocalization of FBXW7 from the nucleus to the cytoplasm, where it is not able to bind to its oncogenic nuclear targets36. Moreover, only p42 Ebp1 is sumoylated on lysine (K) residues 93 and 298 by translocation in liposarcoma/FUS, and this process that is regulated by PKC-δ-mediated S360 phosphorylation. p42 sumoylation mediates its nucleolar translocation and is required for its stability. In turn, sumoylated p42 is associated with pRB, which thereby blocks E2F-1 transcriptional activity and inhibits cell proliferation37. These studies provide insight into the mechanisms underlying tumor suppressor p42 downregulation in cancer cells.p42 EBP1 protein levels were notably lower in GBM patients than they were in healthy tissues obtained from the same patient, while p48 EBP1 was highly expressed in GBM and NSCLC cells38, p48 EBP1 is expressed at high levels in cancer cells25, including GBM6, anaplastic large cell lymphoma39, colorectal cancer40 and acute myeloid leukemia (AML)41. Moreover, patients with breast cancer who express high levels of PA2G4 have poor clinical outcomes42, and patients with GBM or pancreatic ductal adenocarcinoma who have high protein levels of p48 also have poor clinical outcomes43. In oral squamous cell carcinoma, Ebp1 is upregulated. It binds to the podoplanin promoter, which leads to a dramatic increase in both RNA and protein levels of podoplanin. Podoplanin is highly expressed in various human cancers and is associated with tumor progression as well as the pathogenesis of oral cancer44.In contrast to the tumor-suppressive activity of p42 EBP1, reflecting the anti-apoptotic and growth-promoting activities of p486. In addition, phosphorylated HDM2 is confined to the nucleus, and enhanced Akt activity prevents HDM2 self-ubiquitination7. In addition to inhibiting the tumor suppressor activity of p53, p48 exerts oncogenic activity through cyclin-dependent kinase 2 (CDK2)-dependent phosphorylation of its N-terminal domain at serine 34 residue, which is absent from p42. Mutating this phosphorylation site to alanine (S34A) prevents its phosphorylation. The loss of phosphorylation abrogates the ability of p48 to accelerate tumor cell growth both in vitro and in vivo, suggesting that CDK2-mediated phosphorylation of serine 34 of p48 is critical for the tumorigenic activity of p48 in human cancer cells26. The details of the protein domain and post-translational modifications involved in the functions of EBP1 isoforms are depicted in Fig. In our effort to identify the mechanism underlying the oncogenic activity of p48, we found that p48 overexpression in human glioma cells significantly stimulated the tumorigenic properties of cancer cells and enhanced tumor growth in mouse xenograft models. These oncogenic effects resulted from EBP1-dependent UPS regulation. P48 EBP1, and not p42 EBP1, binds to the E3 ligase domain of HDM2, which is famous for p53-mediated tumor suppression and the enhancement of the association of HDM2 and p53. The promotion of p53 poly-ubiquitination and the degradation leads to reduced levels and activity of p53. In addition to negatively regulating p53 by abrogating its interaction with HDM2, we proposed an alternative mechanism by which p48 negatively regulates p53. Specifically, we found that p48 stimulates Akt-mediated HDM2 phosphorylation, which leads to reduced protein levels of HDM227. This also correlated with the ErbB3 expression profile, which is extremely limited in the developing CNS of mice. ErbB3 mRNA was not detectable during embryogenesis, but it started to be detected at P745, which is coincident with expression of p42 EBP1 during the postnatal development period. These findings reflect that p48 EBP1 does not bind to ErbB3, and only p42 EBP1 is a binding to ErbB3, suggesting that p48 EBP1 could contribute to embryonic development independent of ErbB3.Despite independent studies from the past 20 years that have demonstrated that EBP1 is an essential regulator of various cellular functions in cancers and in the development of mammalian cells, the precise molecular mechanisms of how EBP1 contributes to the multiple steps of cellular processes are not well defined. Few studies are available that provide in vivo evidence of EBP1 functions due to a lack of an appropriate mammalian model systems. We have previously demonstrated that p48 EBP1 is the major isoform in the developing brain neurons of rats and during murine brain development, whereas there is no detectable p42 EBP1 expression during the embryonic stage17. Moreover, in adult mice, EBP1 expression was undetectable in quiescent satellite cells and myogenic stem cells. However, it was induced during the activation of satellite cells. Furthermore, in chick embryos, specific depletion of EBP1 inhibited muscle progenitor differentiation, indicating a conserved function of EBP1 in the regulation of embryonic muscle progenitors and adult stem cells17. In addition, Ebp1 mRNA levels were increased from E9 to E20 in chicken embryonic muscle. Moreover, in cultured myoblasts, Ebp1 was upregulated in proliferating cells and decreased at the early stage of myogenic differentiation, revealing that EBP1 contributes to myoblast differentiation by inhibiting SMAD2/3 signaling46. In flies, overexpression of EBP1 disrupts muscle progenitors, leading to neurogenic-like states47.Indeed, only p48 EBP1 was highly expressed during developmental myogenesis as well as regenerative myogenesis in mice, as it controlled the balance between proliferation and differentiation in the chick embryo model18 suggested that EBP1 binds to the homeodomain-containing transcription factor Six1 protein and affects the formation of the complex containing Six1 with its cofactor Eya1 in HEK293 cells. In a Xenopus fibroblast-like cell line (XTC-2), EBP1 activates gene transcription and modestly modulates the transcriptional activity of the Six1-Eya1 complex. In contrast, in HEK293 cells, EBP1 represses transcription of both an artificial and a natural reporter, revealing that EBP1 might regulate gene expression either positively or negatively, depending on cell context. The authors also demonstrated that overexpression of p48 EBP1 does not affect proliferation, whereas its reduction elicits reduced proliferation and apoptosis in the Xenopus embryo. In healthy cells, p48 EBP1 maintains cell growth and survival and has balanced expression with its isoforms, which is postulated not to be the case in cancer cells. EBP1 contributes to craniofacial development for proper formation of the neural border zone and its cranial derivatives, neural crest, and cranial placode genes. While six1 is increased upon EBP1 knockdown in a mouse myoblast cell line17, six1 expression was regulated in an EBP1-dose-dependent manner in the preplacodal ectoderm of an embryo, indicating the differential role of EBP1 in myogenesis and craniofacial development. This may be due to different binding partners or differential transcriptional regulatory functions of EBP1 in specific cellular contexts.A recent elaborate study from16. However, DPPA4 is associated with transcriptionally active chromatin along with both phosphorylated RNA polymerase II and H3K4 trimethylation in mouse ESCs48, Knoepfler and his colleagues have shown that DPPA4 has transcriptional repression activity49. They reported that EBP1 inhibits the transcriptional repression of DPPA4, thereby enhancing stem cell-related transcriptional regulation at specific genomic loci or cooperating in activating transcription at native targets16.In human embryonic stem cells, only p48 EBP1 interacts with developmental pluripotency-associated 4 (DPPA4), and this interaction is reduced upon differentiation of pluripotent cellsEbp1 knockout mouse model was not available, a previously50 generated gene trap of the Ebp1 gene was generated via an insertion at intron 2, and the homozygous Ebp1(−/−) mice were viable but were 30% smaller than wild-type littermates and exhibited transient growth retardation. For more than 10 years, no other report was presented using that model. In our study, we generated conditional deletion of Ebp1 exons 6–10 to avoid targeting Ebp1 exon 1 because the short distance between the Ebp1 and Erbb3 genes (~1.3 kb) could lead to the deregulation of ErbB3 expression and affect ErbB3 putative regulatory elements present in the 3′ region of the ErbB3 gene19. Unlike the only previous report of Ebp1-deficient mice carrying a gene trap insertion that showed that Ebp1 knockout mice were viable50, our Ebp1 knockout mouse model resulted in embryonic death between E13.5 and E15.5. Moreover, knockout embryos exhibited prominently dilated vessels and hemorrhages throughout the entire body, particularly in the brain, and they displayed dilated cartilage primordium and deficient brain organogenesis as well as having a much smaller body and whole-brain volume compared with those of the control mice.As the Ebp1(+/+) WT mice after 2 months. Genetic ablation of Ebp1 causes massive cell death and dysregulation of the epigenetic repression unit SUV39H1/DNMT1. Ebp1(−/−) mice demonstrated the upregulation of Suv39H1-dependent H3K9 trimethylation and activation of DNMT1, revealing markedly increased global DNA methylation. EBP1 binds directly to the promoter region of DNNT1 and represses its transcription.Moreover, heterozygous mice appeared to be smaller , yet they were able to gain bodyweight comparable to that of Survivin promoter enhances gene expression. Hence, we highlight in vivo evidence for an essential role of EBP1 in epigenetic regulation during embryonic development19  revealed that it represses the transcription of E2F1-mediated genes , in addition to E2F132, through recruitment of HDAC2 or through interaction with retinoblastoma (Rb). However, p42 EBP1 still inhibits the transcription of cyclin E Rb-null cells. This leaves questions to be answered regarding a specific network of p42 EBP1 in the repression for E2F-mediated transcription32. p42 EBP1 has also been shown to suppress androgen-mediated gene transcription, tumorigenesis of prostate cancer cells and metastasis of salivary adenoid carcinoma cells in mice51. However, due to the limitation of p42 EBP1 expression during the prenatal period, it is not yet known what the exact role is of p42 EBP1 in the embryonic development of mice or in brain development.Emerging evidence highlights that EBP1 can modulate transcriptional activity in many types of cancer and stem cells16 Fig. . p42 EBP27, which account for the loss of further cell proliferation in neurons. In addition, EBP1 activates gene transcription in XTC-2 cells, whereas p48 EBP1 represses transcription in HEK293 cells, indicating that transcriptional regulation of p48 EBP1 is dependent in the cellular context18. Moreover, in a Xenopus embryo, p48 EBP1 acts as a corepressor of the six1 transcription factor and is required for neural crest and optic development. Furthermore, p48 EBP1 represses the transcriptional repression of DPPA4 in a pluripotent cell-specific context16. EBP1 suppressed HNF4a (hepatocyte nuclear factor 4a)-mediated transcription of its target genes that are implicated in insulin secretion in pancreatic β-cells through binding to HNF4a in the same binding pocket as other well-known coactivators, such as SRC1 or PGC1a52.In addition to cancer cells, the developing hippocampal neurons from rats also exhibits strong association of p42 EBP1 with Rb, while p48 EBP1, especially the S360 phosphorylated form, barely binds to Rb. It is thought this may allow tight interactions between E2F1 and Rb, which thereby lead to E2F1 inactivation and cell cycle arrest in the G1 phaseEbp1(−/−) mouse embryos and conditional homozygous mutant  analysis, we have shown that Ebp1 expression is negatively correlated with genes associated with transcriptional repression, such as Dmnt1, Suv39H1, Ezh1, or Prdm5, whereas a positive correlation was seen with the transcription activation-related gene Dot1. Moreover, we found higher levels of H3K9 trimethylation in Ebp1(−/−) MEFs than in WT MEFs and a lack of EBP1 association with heterochromatin. In particular, p48 EBP1 directly binds to the Dnmt1 promoter region and represses Dnmt1 expression, thereby relieving Dnmt1 target genes from promoter methylation. In addition, p48 EBP1 competes with Dnmt1 for target gene DNA binding, disrupting Dnmt1-mediated promoter methylation. Collectively, our study suggests that EBP1 is a potent regulator of gene expression that may influence the regulation of multiple genes by modulating epigenetic regulators during development19. Intriguingly, in postnatal heterozygous Ebp1(+/−) mice, we found reduced hippocampal volume and mRNA levels of GAD67, which are both popular features of schizophrenia (SZ). Ebp1(+/−) mice also showed SZ-like behaviors. Accordingly, both Dnmt1 and EBP1 bind to the promoter region of histone deacetylase 1 (HDAC1), which leads to histone deacetylation of GAD67 and suppresses HDAC1 expression. Thus, the loss of EBP1 can facilitate the development of cognitive and social impairments in SZ patients via dysregulation of epigenetic repressors and reduction of GAD67 expression in the hippocampus53. Further investigation is required for the dissection of the epigenetic machinery in various regions of the brain and developmental and neurological diseases. Such studies may inform therapeutic strategies to restore the balance of gene expression.Employing conventional 12, the 3′-untranslated region of bcl2 mRA13, rRNA14 and ss/ds RNA10. EBP1 has also been implicated in ribosome biogenesis via interaction with NPM9. In NPM1-mutated AML, HOXB-AS3, a lncRNA embedded in the HOXB locus, binds to p48 EBP1 and guides it to rDNA, where it augments the interaction of EBP1 with NPM in the nucleolus, ultimately contributing to efficient protein translation54. In primary T cells, p48 EBP1 is involved in rRNA synthesis by regulating TIF1A55.In addition to its role in controlling transcription, EBP1 has been thought to be involved in protein synthesis, as EBP1 interacts with an array of RNA targets, including FMDV-IRESSaccharomyces cerevisiae, yet no evidence for functional homology has been reported56. However, a functional disconnect between the homologs would not surprising as EBP1 does not possess structural features that are important for ribosome biogenesis factor interaction in yeast Arx1, and it does not bind to nucleoporins that are required for pre-60S nuclear export factor58. In particular, EBP1 binds to nontranslating human 80S ribosome, competing with MetAP and blocking exit from the ribosome because of significant interactions with general uL23/uL29 docking sites for nascent chain-associated factors. However, it is not known whether EBP1 binding arrests protein synthesis. The precise role of two isoforms of EBP1 in translational regulation and molecular mechanisms underlying specific cellular contexts pathologically and physiologically will provide insight into the contribution of EBP1 in various cellular functions and diseases.Considering the structural similarity of p48 EBP1 with methionine aminopeptidase (MetAP), which cleaves the first methionine from the growing polypeptide chain, EBP1 lacks catalytic activity. It is not surprising to find EBP1 in complex with ribosomes. EBP1 has been identified as the human homolog of the 60S ribosomal nuclear export factor Arx1 from As we state in this manuscript, the two EBP1 isoforms perform different functions in many aspects of cellular events, including cell proliferation and survival, regulation of gene expression, and protein synthesis in the development of the disease. Many questions regarding isoform specificity in cells remain unanswered and have raised confusion and/or controversial results. The two isoforms have different spatial/temporal expression patterns; during embryogenesis, only p48 EBP1 is expressed, and p48 EBP1 is dominantly expressed in cancers and many types of cell lines, which is in agreement with the protein instability of p42 EBP1, as mediated by UPS, and our experience in the difficulty of detecting endogenous p42 EBP1.9. Through the N-terminal domain, which is absent in p42 EBP1, p48 EBP1 interacts specifically with HDM2 for p53 degradation and binds to CDK4 to mediate S34 phosphorylation26. Interestingly, p42 EBP1 specifically interacts with the p85 subunit of PI3K and HSP70/CHIP E3 ligase complex through the C-terminal domain, which is present in both p42 and p48 EBP17. A balanced expression of two isoforms may be essential for maintaining normal cellular growth, as both EBP1 isoforms are involved in the linkage of ubiquitin to the target of E3 ligases.These isoforms interact with a variety of signaling molecules, transcriptional/translational machinery proteins, and UPS proteins. While they share some binding partners, they also have specific binding partners. For example, both isoforms of EBP1 can interact with NPM to regulate cell cycle progression18. Moreover, no apparent translational inhibitory effect of p48 EBP1 or the association of p42 EBP1 with the 80S ribosome has been determined15. For instance, during development, the uniquely expressed p48 EBP1 binds to the Dnmt1 or survivin promoters and may coordinate with the regulation of multiple genes by modulating epigenetic regulators19. However, we do not yet know in the case of cancers or other developmental/neurologic disease conditions, whether the two isoforms are differentially expressed and/or are altered in DNA binding ability by binding partners. To date, only a few studies have investigated in detail the transcriptional/translational regulation of two EBP1 isoforms. However, future studies will likely reveal critically important functions for EBP1 isoforms in development and human diseases.It will be important to elucidate whether the two isoforms distinctively bind to epigenetic factors or transcriptional regulators in different contexts. In particular, isoform specificity may be relevant to transcriptional regulation or translational regulation, as EBP1 demonstrated both transcriptional repression and activation function