Although the situation is evolving, current methods have various difficulties with the accurate mapping of loops even in mammalian Hi-C data, and most of them fail to identify chromatin loops in animal species with substantially different genome architecture. This paper presents the loop and significant contact annotation (LASCA) pipeline, which uses Weibull distribution-based modeling to effectively identify loops and enhancer–promoter interactions in Hi-C data from evolutionarily distant species: from yeast and worms to mammals. Available at:  These megabase-scale compartments are partitioned into self-interacting structures, termed topologically associating domains (TADs). The distinguishing feature of TADs is that spatial contacts of remote genomic elements are more frequent within TADs than between individual TADs4. Boundaries between individual TADs are enriched with cohesin complex and CCCTC-binding factor (CTCF). Recent data show that TADs are formed via dynamic DNA loop extrusion and may harbor smaller contact domains, some of which are chromatin loops6. Along with chromatin compartments and TADs, chromatin loops represent one of the major levels of hierarchical folding of the genome. Although most of the loops in mammals are anchored by CTCF and cohesin, several other proteins can mediate long-distance genomic interactions: Yin Yang 1 (YY1), zinc finger protein 143 (ZNF143), LIM domain-binding factor 1 (LDB1)7. All the three are involved in establishing enhancer-promoter interactions either by direct binding and bridging specific genomic sites (YY1 and ZNF143)9, or by binding to a subset of transcription factors (LDB1)10. Species that lack CTCF-dependent loops can however utilize DNA extruding complexes such as condensin and cohesin to organize their genomes or genome parts into consecutive similar-sized chromatin loops. Specifically, Caenorhabditis elegans X chromosome contains dozens of loops associated with a condensin-like dosage compensation complex (DCC)12. In budding yeast, S-phase chromatin forms consecutive loops which base points often colocalize with binding sites of cohesin protein Scc113.Techniques exploiting the proximity ligation procedure  have significantly improved our understanding of the spatial (3D) genome organization. C-methods have confirmed the existence of chromosomal territories that are spatially compartmentalized into active and repressed chromatin domains, referred to as A and B compartmentscis-regulatory elements14. Hence, the identification of loops is an essential part of most studies that involve Hi-C-based 3D genome analyses. Methods that have been developed to detect chromatin loops and/or statistically significant genomic interactions from Hi-C contact maps comprise two groups: (1) statistical/probabilistic model-based methods , and (2) peak-calling methods . All of them are primarily focused on mapping the loops in Hi-C datasets from mammals, and experience some (in most cases insurmountable) challenges working with Hi-C datasets from animal species with substantially different genome architecture. Here, we present the LASCA pipeline that uses Weibull distribution-based modeling to effectively identify chromatin loops, including enhancer-promoter interactions, in Hi-C data from different animal species . Our results demonstrate that LASCA-detected loops are (1) reproducible, (2) highly supported by aggregate peak analyses and genomic/epigenomic correlates of loop formation, (3) validated by protein-centric chromatin conformation methods (ChIA-PET and HiChIP). We have compared LASCA with the most commonly used methods from each of the abovementioned groups (HiCCUPS and Fit-Hi-C) and with a very recent approach MUSTACHE. Working with mammalian Hi-C data, LASCA showed very similar results to HiCCUPS and MUSTACHE, and even outperformed HiCCUPS in detecting CTCF-independent loops. In contrast to methods compared, LASCA could also detect chromatin loops in C. elegans and S. cerevisiae, which makes it an omni-purpose approach.In contrast to chromatin compartments and TADs, chromatin loops have clear biological roles, such as bringing gene promoters to their cognate 22 to identify these significant interactions. Comparison of several statistical distributions  performed in Sanyal et al.20 clearly showed that the Weibull distribution fits Hi-C interaction frequency the best. We further analyzed the quality of the Weibull distribution fitting of Hi-C interaction frequency at different distance ranges and found that its good performance is distance-independent . Adjacent significant pixels are grouped into clusters; for each cluster, the center is defined, and its coordinates are retrieved and considered as the loop coordinates. Identified loops may be filtered according to their aggregate peak analysis (APA)17 or peak analysis (PA)11 scores, signal intensity, and signal enrichment over random signals located at the same distance from the central diagonal. The loops should also display a signal decay from the central loop pixel. These filters are optional and should be used depending on the animal species under study. Specifically, using these filters with mammalian Hi-C data will enhance the accuracy of loop annotation, whereas utilizing the filters with the yeast genome appears to be less useful.In Hi-C heatmaps, loops appear as bright dots located at different distances from a central diagonal. Here, we applied Weibull distribution-based modelingrix Fig. . The p-vH. sapiens (GM12878 cells17), M. musculus (CH12.LX cells17), C. elegans24, and S. cerevisiae (S-phase cells13). We managed to annotate a significant number of loops in each case  and HiChIP (68%) loops . Nevertheless, we found that most of the LASCA-identified chromatin loops belonged to significant genomic contacts detected by Fit-Hi-C . LASCA identified approximately the same number of loops as MUSTACHE and two times more loops than HiCCUPS in Hi-C datasets from both human and mouse  was not mapped by HiCCUPS Fig. c. To finThe LASCA pipeline is suitable for the identification of enhancer-promoter interactions Fig. a. In thiC. elegans and S. cerevisiae distinguishes LASCA from most other chromatin loop callers. LASCA, the protocol, and suite of scripts, are publicly available at https://github.com/ArtemLuzhin/LASCA_pipeline.LASCA allows mapping of chromatin loops as well as enhancer–promoter interaction in Hi-C datasets obtained from animal species with different genome size and genome organization complexity, such as human, mouse, worm, and yeast. To annotate representative loops/contacts, LASCA requires minimal adjustments and filtering, particularly when analyzing worm or yeast data. High-quality performance with Hi-C data from p-values are calculated as the probability of finding a model pixel with the same or higher intensity, and FDR correction of the p-values is performed to obtain corresponding q-values (default argument: 0.1). Additionally, q-values may be corrected in accordance with the scaling of a particular chromosome (default argument: turned off). Briefly, for the selected range of diagonals of the Hi-C matrix, the average value of the contact frequency for each diagonal is calculated. Then, all values in the obtained set of average values of the contact frequencies are divided by the average value of the contact frequency in the first diagonal, thus forming a set of normalization coefficients with a value = 1 in the first diagonal. The q-values in each diagonal are divided by the corresponding normalization coefficient determined for this particular diagonal. The q-value threshold is defined by a user (default argument: 0.1) to determine significant pixels (contacts).The LASCA pipeline consists of three main steps Fig. . In the On the second step, adjacent significant pixels are grouped into clusters using a density-based spatial clustering of applications with noise (DBSCAN) algorithm (default argument: minimum cluster size is three pixels). Pixels are assigned as neighbors related to a particular cluster if the maximum Euclidean distance between these two pixels = 1. The size of the cluster (in pixels) may be specified by a user. For each cluster, the center is defined (default argument: the brightest pixel), and its coordinates are retrieved and considered as the loop coordinates. The cluster center is defined as either the arithmetic mean of the x and y coordinates of pixels in a cluster or a pixel in a cluster possessing maximum intensity (default option).On the third step, identified loops may be subjected to various filters, such as the enrichment of signal over background , signal intensity, signal decay from the center of a cluster, and signal enrichment over random signals at the same distance. All steps and parameters in the LASCA pipeline can be turned on/off and adjusted by the user (default argument: turned off).17 was calculated as the ratio of the intensity of the central pixel of the loop to the average intensity of the right corner of the 11 × 11 pixel-circle around the center of the loop. PA-score11 was calculated as the average ratio between the nearest pixels to the center of the loop and the average intensity of the right corner of the 11 × 11 pixel-circle around the center of the loop.APA-scoreC. elegans, loops were annotated at 5 and 10 kb resolution with a subsequent merging of overlapped loops. The following parameters were used: q = 0.95, adjust_by_scale = False, q_value_trhd = 0.05, min_cluster = 2, filter_zeros = 2, filter_PA = 1, filter_APA = 1.7, filter_intensity = 0.1. Finally, for S. cerevisiae, loops were mapped with following settings: q = 0.95, adjust_by_scale = False, q_value_trhd = 0.01, min_cluster = 3, filter_zeros = 2, filter_intensity = 0.1.Using the LASCA pipeline, we identified loops in several organisms. For human cell line GM12878 Hi-C maps, loops were annotated at 5 and 10 kb resolution followed by merging of overlapped loops and filtering them by signal enrichment over random signals located at the same distance. The following parameters were used for 5 and 10 kb Hi-C maps: q = 0.95, adjust_by_scale = True, q_value_trhd = 0.2, scaling_q_value_trhd = 0.2, min_cluster = 2, filter_zeros = 2, filter_PA = 1, filter_APA = 1.9, filter_intensity = 0.3. For mouse cell line CH-12LX, loops were identified at 10 kb resolution with the following parameters: q = 0.95, adjust_by_scale = True, q_value_trhd = 0.1, scaling_q_value_trhd = 0.05, min_cluster = 3, filter_zeros = 2, filter_PA = 1, filter_APA = 1.8, filter_intensity = 0.1. For 17 (GSE63525). To identify loops in GM12878 cells using MUSTACHE, we annotated the loops separately on the 5 and 10 kb maps with the following parameters: -pt 0.05, -st 0.88, -sz 1.6, -oc 2, -i 10. Then we merged the loop lists found at different resolutions. Significant contacts in GM12878 cells and S. cerevisiae were also annotated using FitHiC2 with default settings, except for the -U option . We cut off significant contacts by q <  = 10−9.HiCCUPS loops for GM12878 (primary) and CH12-LX cells were obtained from Rao et al.We used Bedtools to count the overlapping of the loops. We considered the loops overlapped if at least 70% of the reciprocal intersection of regions between loop base points was observed.25 with default settings, except –pad parameter, which was 10 for S. cerevisiae and 50 for C. elegans.We constructed metaplots for each of the selected organisms using Coolpup.py27 with—reference Point TSS (upstream loop anchor), and -a and -b parameters were selected as half of the mean loop size for each Hi-C dataset. In the case of enhancer-promoter interaction analysis, we set -a and -b parameters to 2 Mb.To analyze the enrichment of proteins involved in looping, we applied deepTools222 (GSM1872886). Following the methods of the original paper, we removed from the consideration CTCF-PET clusters that had less than four interactions. Then we binned the resulting CTCF-PET cluster interaction map using a 10 kb window. The resulting interaction coordinates of 10 kb windows, we considered as CTCF-mediated loops. The data for CTCF-HiChIP in .hic format was obtained from Mumbach et al.26 (GSE115524). In accordance with the original paper, we annotated CTCF-mediated interactions using HiCCUPS with the following parameters: -m 500 -r 5000, 10,000 -f 0.1,0.1 -p 4,2 -i 7,5 -d 20,000, 20,000.A table containing the contacts of the CTCF-PET clusters was obtained from Tang et al.https://genome.ucsc.edu/cgi-bin/hgLiftOver). The base of the loop was considered to contain a peak if it contained at least one peak from the corresponding mark.Data for GM12878 cells were obtained from the ENCODE . Coordinates of ATAC-seq peaks in hg38 have been translated to hg19 using the LiftOver utility  to 1 kb upstream of that TSS. We intersected these promoters and significant contacts and left only those contacts for which one of the anchors fell inside the promoter region; another anchor, therefore, was assigned as an enhancer.Supplementary Information.


Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication–transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution. Developing organisms rely on cell division (mitotic cells) to distribute their genetic material equally and ideally errorless to subsequent daughter cells. During differentiation most cells do not continue to divide (postmitotic cells) and transition into senescence while the whole organism is ageing. For a long time, this course into cellular expiration by telomere shortening was thought to counteract cellular division progress into malignant overgrowth . HoweverEvery cell is exposed to up to thousands of single lesions from exogenous and endogenous sources to mostly challenge the integrity of the genetic material within one generation when affecting somatic cells , 5. HoweOne of the major drivers of endogenous DNA damage is replication, i.e. the accurate copying of the genetic material into two identical copies, each kept available for the spreading to two daughter cells during cell division. Hindered and slowed replicative processes in interphase can lead to stretches of under-replicated DNA where doubling of the genetic material is not finished on time before the onset of mitosis . FurtherTraditionally, fragile regions have been subdivided into ‘common fragile sites’ (CFSs) and ‘rare fragile sites’ considering the frequency of appearance of these sites in a normal population . During The above mentioned understanding of fragility is not any more based on the attempt to define parallel mechanisms to explain every breakage event, but rather use the term ‘fragile site’ as a converging definition of multifactorial aspects resulting in the same cytogenetic manifestation. What we see is most likely the consequence of earlier events and not the cause of further downstream events, such as breakage and genomic rearrangements. The defined mitotic instability of FSs raises questions about events in preceding interphases leading to a later uncondensed chromatin structure. While some genetic regions are known to replicate early in S-phase of the cell cycle, others are usually late replicating. This temporal difference is also reflected in the spatial organization of the genome during interphase. Whereas the nuclear centre usually harbours early replicating sites, the nuclear periphery is mostly composed of late replicating areas of the genome . Since eAre FSs genomic regions that purely represent the existence of large and difficult-to-replicate units with a low number of activatable origins leaving behind large stretches of DNA that cannot be replicated timely within one cell cycle , 29–33? The before-mentioned replication challenges seem to cause delayed chromosomal condensation in later cell division cycle stages. Transcription may further delay condensation of late replicating regions for mitotic chromosomal separation. The coincidence of replication and transcription causes those two acting multiprotein machineries to encounter in the same genomic region thereby suppressing replication initiation  or even The so-far mainly cited reasons for chromosomal fragility summarize in a set of large genes and replication units that are late replicating and concomitantly transcribed in interphase . HoweverThus, genomic regions later manifested as FSs should be located in the nuclear periphery during interphase concerning their replication profile, but closer to the nuclear interior concerning their transcriptional profile. A peripheral location favours the idea of possible nuclear membrane or cytoskeletal anchor points that prevent certain genomic areas from early replication, hamper them from being transcribed that may leave behind uncondensed chromatin in mitosis when they continue to do anyway. A low transcription level is sufficient to cause later fragility , 53 indiDespite it is object of recent discussion, the possible link between lamina anchorage in interphase and chromosomal breakage in mitosis has not been addressed in detail yet , 60. ThiThe nuclear material has a cell type-specific organization, i.e. the relation of chromosomes to each other and to the nuclear periphery is specific to each cell type , 55. ThiPartially it has been described that FSs are located in the nuclear periphery—visualized by the localization of FANCD2  what indIn embryonic cells of fruit flies it was shown that lamina interacting regions are characterized by a certain degree of AT-richness, transcriptional repression with according histone modifications, late replication and long intergenic distance . SimilarAnother example for chromatin-lamina-interaction is the lamin B receptor which is the direct link between lamins and chromatin . It was Fhit, an ortholog to the human FRA3B, is largely associated with the NM in naïve B lymphocytes where FRA3B is a highly expressed CFS. The same region is only partially anchored to the NM in primary hepatocytes and postmitotic neurons which are not expressing this CFS. Furthermore, global analyses of structural DNA-NM interactions in naïve B lymphocytes displayed that most of the genome has no tight interaction, emphasizing that the fragility of Fra14A2 might be related to the association with the NM [Recently published data suggests that DNA regions tightly connected to chromatin loop anchors or to the nuclear matrix (NM) are involved in chromosomal fragility in a cell type-specific manner , 77. Theh the NM . This lah the NM .versus repressed chromatin and DNA damage on a cellular versus evolutionary level.The question remains what establishes first during development: inactive genomic regions marked by specific repressive chromatin marks will be read out to be tethered to the nuclear periphery thereby creating two nuclear regions leaving space in the centre for protein accumulation and transcription , and allThe cytogenetic appearance of FSs as gaps and breaks has mainly been assigned to under-replicated DNA stretches that might be resolved by diverse repair pathways before the onset of mitosis to prevent chromosomal instability. Specifically in cancer cells, chromosome integrity and recurrent copy number alterations are discussed with involvement of FSs , 36. It The time of manifestation and visibility of chromosomal fragility is not necessarily the time of occurrence of an actual DNA damage. By focusing on the factors contributing to imbalances at the time of their action, but not at the time of visible consequences, it seems that even primarily assessed detrimental lesions can have beneficial functions on a cellular and evolutionary level. Therefore, in addition to consider FSs as harmful chromosomal lesions, they also have a certain function for chromosomal stability as sites of uncondensed chromatin, similarly to constrictions, such as centromeres or nucleolar organizing regions at the short arms of acrocentric human chromosomes.By further contemplating the conservation of FSs not only within the mammalian lineage –87 but a signalling points late and/or long-lasting replication as a sensor for cell cycle delay preventing genomic instability in subsequent cell divisions,(II)transcriptional activity in a surrounding of delayed replication for clashes leading to DNA instability as sensors for DNA repair,(III)transcriptional activity in a surrounding of delayed replication to antedate replication timing to prevent genome instability,(IV)transcriptional activity accompanied by nucleosome exclusion as a marker of euchromatic regions associated with a decondensed chromatin structure and(V)anchoring to the nuclear lamina as a sensor for nuclear envelope disassembly and reassembly before and after mitosis on a cellular level.We, thus, propose FSs asnts Fig. a combini functions recognition sites for the establishment of cell type-specific transcriptional profiles,(II)balancing gene expression in a surrounding of tightly regulated expression profiles and areas with a low threshold towards changing expression—specifically in the brain,(III)recognition sites for the determination and maintenance of the nuclear architecture after cell division,(IV)platforms for non-genetic chromatin marks as reusable evolutionary hot spots in subsequent generations and(V)hot spots for structural variants as a source of diversity on an organismal as well as evolutionary level.Additionally, FSs merge theons Fig. b of:(I)rTherefore, future studies should address in more detail functional and beneficial consequences of FS expression with the focus on their caretaker functions. It should be asked whether every site that is physically anchored to the periphery can indeed result in a mitotic FS and whether FSs per se are located at the nuclear periphery in the course of cellular differentiation.


The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications. Saccharomyces cerevisiae (ScTOR1 and ScTOR2) and the fission yeast, Schizosaccharomyces pombe (SpTor1 and SpTor2). Signal transduction pathways act to communicate extracellular and intracellular signals to effector proteins that control a range of fundamental cellular processes. The discovery of the TOR (target of rapamycin) serine/threonine protein kinases, first in yeast cells and later in human cells, revealed TOR-dependent signaling pathways that control metabolism, proliferation, and survival in response to nutrients, energy status, growth factors, and hormones . TOR is an atypical serine/threonine protein kinase that belongs to the family of phosphatidylinositol-kinase-related kinases (PIKKs). Other members of the family include ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) which, like TOR, are also conserved from yeast to human. ATM and ATR have traditionally been considered nuclear proteins that function in response to genotoxic stress, but more recent studies established that they also have cytoplasmic functions ,9. InterS. cerevisiae or S. pombe cells two TOR genes are present. The basic composition of the TORC1 and TORC2 complexes is highly conserved throughout evolution. The presence of the Raptor protein (Kog1 in S. cerevisiae or Mip1 in S. pombe) defines TORC1, while the presence of the Rictor (Avo3 in S. cerevisiae or Ste20 in S. pombe) and Sin1 (Avo1 in S. cerevisiae and Sin1 in S. pombe) proteins defines TORC2. In S. cerevisiae, Tor1 or Tor2 can act as the catalytic subunit of TORC1, while only Tor2 forms TORC2 . Much alysosome ,59. mTORlysosome  and alsolysosome ,61,62). revisiae ,63,64,65S. pombe . The presence of active TOR proteins in the nucleus was first controversial. This was due to the use of different cell types, the limitations of biochemical fractionation and indirect immunofluorescence techniques, as well as the lack of direct nuclear substrates ,55,67. AScTORC1 and mTOR in association with gene promoters, including promoters of Pol I- and III transcribed genes . D. D82]. DHMO1 which encodes a high mobility group box (HMGB) protein . . ScTORC1hibition . ScTORC1pression A 113]. . ScTORC1nditions . Thus, SScTORC1 signaling also regulates global histone H3 lysine 56 acetylation (H3K56ac) which depends on the histone chaperone Asf1 and the acetyltransferase Rtt109 . . ScTORC1Wnt target genes to repress their transcription. This pathway controls the development of mature adipocytes and formation of white adipose tissue. S6k1-/- mice have reduced global, and Wnt gene specific, H3K27 methylation which prevents adipocyte development and makes these animals resistant to high-fat diet induced obesity [In mammals, H3 lysine 27 (H3K27) methylated by the Polycomb methyltransferase Ezh2 represses gene expression. In pre-adipocytes, mTORC1 signaling activates S6K1 which phosphorylates histone H2B at serine 36 (H2BS36ph) . This H2 obesity . Recentl obesity . TherefoScTORC1 regulated genes [ScTORC1 inhibition alters Rsc9 genome-wide binding [ScTORC1 to integrate nutrient signaling with chromatin remodeling. Indeed, distinct RSC subunits were shown to either activate or repress ScTORC1 regulated genes, including RP genes and the rDNA [ScTORC1 controls the transcription initiation of a large number of growth-promoting genes via RSC-dependent NFR maintenance. In this scenario, nutrient stress would suppress ScTORC1 signaling and impair RSC function. Blocking RSC activity then could allow nucleosome encroachment into the NFR, promoter occlusion, and inhibition of transcription initiation. The yeast RSC (Remodeling the Structure of Chromatin) complex is an essential and abundant ATP-dependent chromatin remodeler that either activates or represses transcription through nucleosome repositioning . RSC hased genes . A previ binding , thus suthe rDNA ,137. HowScTORC1-dependent transcriptional regulation [ScTORC1 pathway, and that it contributes to ScTORC1-dependent metabolic gene regulation. Histone H3 lysine 18 acetylation (H3K18ac) is a histone post-translational modification dependent on active ScTORC1 signaling [ScTORC1-activated genes. Intriguingly, loss of the Ino80 catalytic subunit reduces H3K18ac at these genes [ScTORC1-dependent epigenetic modifications. Cells lacking functional INO80 exhibit sustained ScTORC1 signaling after rapamycin treatment and are more rapamycin-resistant relative to wild-type cells [ScTORC1, how it attenuates ScTORC1 signaling, and the role this process has during nutrient stress responses, has yet to be determined  mutation was the only mutation on either H3 or H4 that caused cell death after ScTORC1 stress. This cell death consisted of both apoptosis and necrosis, and it was found to be independent of the H3K37 post-translational modification state. Instead, the H3K37A mutant reduced chromatin binding by specific HMGB proteins which resulted in their migration to the cytoplasm where they deregulate ScTORC1 signaling through an unknown mechanism [ScTORC1 signaling is a well-established mechanism for extending chronological longevity and, consistent with this aging role, the elevated ScTORC1 activity in H3K37A cells substantially reduced their longevity [ScTORC1 signaling resembles that which occurs in set2Δ and INO80 mutants [ScTORC1. Deregulated ScTORC1 activity would make cells more sensitive to environmental stress by decreasing their ability to repress growth responses and prevent the initiation of key stress-coping mechanisms. HMGBs also contribute critical functions to ScTORC1 and mTORC1-dependent transcription of RP and/or rRNA genes, including Hmo1 in yeast and UBF in mammals [Rapamycin-based chemical genomic screening of a histone H3/H4 mutant yeast library identified additional epigenetic pathways functionally connected to the fication . These rechanism . Reducedongevity . The inc mutants ,139, thu mammals ,101,142.ScTORC2 in the regulation of gene expression. ScTORC2-Ypk1 controls ATG8 expression by repressing the stress-response transcription factors Msn2 and Msn4, thus positively regulating autophagy [ScTORC2. Of the three kinases that are targeted by ScTORC2, only Pkc1, but not Ypk1 or Ypk2, has been shown to shuttle between the nucleus and the cytoplasm [ScTORC2 in these Pkc1-dependent transcription regulation. Less is known about the roles of TORC2 in gene expression compared with TORC1. In particular, relatively little is known about the roles of utophagy ; howeverytoplasm . Pkc1 reytoplasm . FurtherS. pombe cells , a developmental program that is enhanced by increased ribosome biogenesis and is associated with wound healing and tumor metastasis . InteresS. pombe, TORC2 has been implicated in the regulation of nutrient-dependent and stress-induced gene transcription, including the regulation of amino acids permeases [SpTORC2, or its target kinase Gad8, resulted in a decrease in the binding efficiency of the MBF complex to its cognate promoters and loss of MBF-dependent gene induction in response to DNA replication stress [In ermeases , nitrogeermeases  and DNA-ermeases . The MBFermeases . Loss ofn stress . The inan stress . Co-immun stress . As mentn stress ,85,86, sn stress . In partn stress . S. pombe suggest that Gad8 is regulated via de-phosphorylation by PP2A-B55 [SpTORC2 in Gad8 [SpTORC1 positively regulates PP2A-B55; this in turn inhibits SpTORC2-Gad8 signaling either at the level of Gad8, or downstream of Gad8 signaling [Other studies in PP2A-B55 , a phospPP2A-B55 . Gad8 im in Gad8 . Under nignaling ,151,155.SpTORC2 [SpTORC2-Gad8 mutant cells to induce the sexual development genes ste11+ and mei2+ and partially suppressed the sterility of gad8 mutant cells [S. pombe FOXO transcription factor, Fkh2 has also been implicated in regulating ste11+ induction and sexual development [SpTORC2-Gad8 phosphorylates Fkh2 in vitro [SpTORC2-Gad8 phosphorylation awaits further study. Yet another physical interaction between Gad8 and a nuclear protein is its interaction with Taf12, a component of the SAGA (Spt-Ada-Gcn5 Acetyltransferase) transcriptional co-activator complex and the general transcription factor TFIID . Gad8 inSpTORC2 . Consistnt cells . The S. elopment . SpTORC2in vitro , howeverin vitro ,86. TherSnail-dependent gene expression [Metabolic signals play critical roles in determining chromatin structure . Since mpression . In addipression . The mTOpression . mTORC2 pression , demonstSIRT6, which as mentioned above affects FoxO1 acetylation , has alsS. pombe implicated TORC2 in another aspect of epigenetic regulation, one that controls heterochromatin spreading and gene silencing. Genome-wide transcription analyses of SpTORC2 or gad8 disruption mutant cells indicated an extensive defect in gene expression, specifically resulting in increased non-coding RNA and subtelomeric gene transcription. Transcription profiles of SpTORC2 or gad8 mutant cells resembled the transcriptomes of cells lacking Clr3 or Clr6 which are histone deacetylase class I and II enzymes, respectively, or the transcriptome of a SWI/SNF chromatin remodeling complex RSC mutant [SpTORC2 is required for chromatin-mediated gene silencing. Further studies implicated SpTORC2-Gad8 in chromatin-mediated gene expression and in the regulation of epigenetic states and their stability [SpTORC2-Gad8 mutant cells, as its expression stochastically alternated between repressed and expressed configurations [SpTORC2 function is conserved in higher eukaryotes. Studies in C mutant ,164. TheSpTORC2-Gad8 mutant cells is accompanied by shrinkage of the H3K9me2 spreading, as demonstrated by genome-wide ChIP analysis [SpTORC2 may be involved in regulating the response to adverse conditions via chromatin-mediated gene expression of subtelomeric genes. The PAF complex is required in S. pombe to antagonize subtelomeric heterochromatin spreading [SpTORC2-Gad8 mutant cells is fully suppressed by the loss of Leo1 or Paf1, two subunits of the PAF complex [SpTORC2 and PAF [SpTORC2 activity, while restoration of heterochromatin at this region following two weeks in nitrogen starvation (late-quiescence) correlated with re-activation of SpTORC2. Thus, the finding that the loss of heterochromatic structures in early quiescence is antagonized by Leo1 inactivation suggests that PAF may act downstream of TORC2 to regulate chromatin-mediated gene expression in response to changing environmental conditions [Heterochromatin is characterized by histone H3 lysine-9-dimethylation (H3K9me2) . The subanalysis . The subanalysis . Thus, Spreading ,166,167. complex . Moreove and PAF . Reductinditions . SpTORC2-associated defects by disrupting positive transcription regulators, such as PAF or Gnc5, as well as the similarities between transcription profiles of SpTORC2-Gad8 mutant cells and histone deacetylases, suggest that SpTORC2-Gad8 acts to restrain transcriptional permissiveness. This is in a sharp contrast with the role of ScTORC1 in stimulating histone acetylation and transcriptional permissiveness (see above). Whether SpTORC1 acts like ScTORC1 to maintain transcriptional permissiveness via histone modifications is still unknown. Such a scenario will be particularly interesting in view of the opposite effects of SpTORC1 and SpTORC2 on many different cellular functions [In conclusion, it is interesting to note that the suppression of unctions ,155,168.Our current understanding of the nutrient and/or growth factor/mitogen-specific mechanisms activating TOR kinases is restricted solely to those functioning outside of the nucleus, specifically pathways that function on the cytosolic face of organelles such as vacuoles/lysosomes. How nuclear TOR kinases respond to these upstream signals remains completely unknown, so addressing this question should be a priority area for future investigation. One distinct possibility is that TOR kinases are activated in the cytoplasm, and then they shuttle to the nucleus to mediate their epigenetic and/or transcriptional effects. This possibility is supported by the observation that the TOR kinase shuttles between the cytoplasm and nucleus . AnotherNuclear TOR kinases are detectable across species, yet whether these kinases phosphorylate substrates in the nucleus remains unclear. Structural studies of mTORC1 reveal that it forms dimers, and that the kinase is held in a constitutively active state . CatalytFinally, nuclear TOR kinases, and the associated subunits, may provide non-catalytic functions critical for gene regulation. TOR kinases are very large, as are their Raptor and Rictor associated subunits. Their interactions with transcription factors or the transcriptional machinery on DNA may create novel surfaces which recruit additional coregulators to control gene expression. These unknowns remain critical deficiencies in our understanding TOR signaling, which are essential to address for understanding how aberrant TOR signaling causes disease.


Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1’s downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to  SIX1 cause Branchio-Oto-Renal (BOR) or Branchio-Oto (BO) syndrome (The transcription factor (TF) Six1 belongs to the sine oculis (So/Six) homeobox protein family that plays important roles in the development of multiple organs, including ear, urinary system and kidney . Overexpsyndrome . Approxisyndrome ,8. The msyndrome . Failurecis-regulatory elements (CREs) of genes. In order to regenerate hair cells after damage by altering differentiation programs that control cellular states in the sensory epithelium, we must understand the causal relationship between TF activities and cellular phenotypes. The TF Six1 is critical for neurosensory cell development and −/−Six1 mice lack neurosensory structures of the inner ear  was dissolved in corn oil  and administrated (1.5 mg/10 g body weight) by oral gavage.For Six1 ChIP, 50 (E13.5) to 40 (E16.5) cochleae were used, while 10 cochleae were sued for H3K29ac or H3K27me3 ChIP. Cochleae were dissected from E13.5, E15.5 or E16.5 wild-type embryos and cochlear epithelia were dissected from cochleae, which also contained surrounding mesenchyme tissues, like we did previously for cochlear explant culture . The dist-test was used to determine the significance of enrichment changes for the ChIP-qPCR experiments. Error bars indicates SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test. The primers used for ChIP-qPCR are listed in For quantitative real-time ChIP-qPCR, chromatin derived from cochleae was precipitated with IgG, anti-Six1 , -Rfx1  or -Rfx3 respectively. The ChIPed DNAs were subjected to quantitative real-time PCR (qPCR) amplification with StepOnePlus PCR system and SYBR green PCR Master Mix kit . This experiment was repeated three times and each qPCR was performed in triplicate. The enrichment fold of IP over mock IP (IgG) was calculated using the comparative Ct (threshold cycle) method. IPs and mock IPs were normalized to inputs and the enrichment of mock IP was considered 1-fold. The Student's For ChIP-qPCR using chromatin prepared from 293 cells, 293 cells were cotransfected with each reporter transgene driven by CRE or CRE carrying mutated SIX-motifs or SIX:RFX motifs in combination with empty pcDNA3.0 vector, His-Six1/pcDNA3.0 or Flag-Rfx3 expression plasmid alone or in combination. Cell fixation, chromatin preparation and ChIP assay were performed as described in the ChIP protocol above. Transfection was repeated three times and each qPCR was performed in triplicate.http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Raw sequencing reads were then aligned to the mouse mm10 genome using default settings of Bowtie (v2.2.0)   ((v2.2.0) . Only un(v2.2.0)  with var(v4.8.3) . The pea(v4.8.3)  and Pant(v4.8.3) .The Hsp68 minimal promoter was inserted into pWhere vector (Invivogen) to drive LacZ or eGFP expression flanked by the H19 insulators and individual enhancer element was inserted upstream of the Hsp68 minimal promoter. Pronuclear injection was performed at our Mouse Genetics and Gene Targeting facility. Transgene expression was analyzed in G0 embryos at different stages.Site-directed mutagenesis of Six1/2-binding sites or Six1/2:RFX motifs in combination in the enhancer sequences was performed to generate mutant reporters. The primers for site mutagenesis were listed in supplementary materials .Two hundred ninety-three cells were cultured and used for transfection as described previously . ReporteExpression plasmid: His-Six1 pcDNA3.0(20) and pRRLHA-Atoh1 was constructed in our lab. Flag-Rfx3 , Flag-Gfi1 , Flag-Pou4f3 , Flag-Gata3  or Flag-Pbx1 .in situ hybridization were performed as described previously , pre-cleared with protein A/G beads . After removal of the beads, the lysates were incubated with ∼1 μg primary antibodies overnight at 4°C and the protein–antibody complex were pulled down by adding 20 μl beads pre-blocked with BSA. The IPed protein complex was washed by IP buffer plus 0.2% NP40 for four times and analyzed, separated in SDS–PAGE and detected with differentiation primary antibodies and HRP-conjugated secondary using the enhanced chemiluminescence (ECL) method .Cochleae of E14.5, E15.5 or E17.5 or 293 cells transfected with His-Six1 and HA-Atoh1, Flag-Pou4f3, -Gfi1, -Rfx3 or -Pbx1 expression plasmids were lysed in homogenized and lysed in 10 mM HEPES, pH 7.9, 1.5 mM MgClPrimary antibodies: anti-Six1 , -Rfx1 , -Rfx3 , -Flag , -CTCF , -Gfi1 , -Pou4f3 , -Atoh1 , -Pbx1. The secondary antibody: anti-Rabbit IgG light chain (HRP)  and mouse IgG light chain binding protein m-IgGκ BP-HRP .t test was used for statistical analysis. Primers used are listed in Cochlear epithelia were dissected from P0 inner ears and used for total RNA extraction using Trizol Reagents (Invitrogen). Total RNAs were treated with RNase-free DNase Set (QIAGEN) and then used for reverse transcription and PCR were performed as described previously using the Applied Biosystems StepOnePlus Real-Time PCR Systems . ExpressWe performed ChIP-seq to investigate the global occupancy of Six1-binding from undifferentiated prosensory progenitors in the auditory sensory epithelium at E13.5 to differentiation at E16.5 Figure . To bettWe also observed stage-specific differences in the genomic distribution of Six1 peaks. The majority (∼96%) of E13.5 peaks were intronic and intergenic Figure  and 84% GREAT and Gene Ontology analyses revealed overrepresentation of genes related to inner-ear/cochlea development in Six1 targets Figure . Notably+ cells are capable of differentiating into hair cells in response to Wnt signaling  and one precursor-transient intronic region ∼+94-kb  , BMP , and Fgf   and involved in the Wnt, Notch, Shh, and Fgf signaling pathways that are crucial for prosensory primordium specification and both hair cell and supporting cell fate selection. The Wnt signaling mediator Lgr5 is expressed in prosensory progenitors and maintained in a subset of supporting cells during differentiation . Recent ignaling . We idenb Figure , which wb Figure . Similar) Figure , Shh   pathways1 Figure .In summary, our Six1 ChIP-seq demonstrates the time course of binding dynamics of this key TF in both hair and supporting cell fate selection and subsequent differentiation, thus uncovering a broad role for Six1 in auditory sensory epithelium development.Atoh1 as a target of Six1 based on the dual criteria of changes in Atoh1 expression in response to Six1 loss- or gain-of-function experiments and Six1-binding to the 1.4-kb 3′-Atoh1 autoregulatory enhancer , suggesting that this precursor Six1-occupancy may ‘prime’ Atoh1 by limiting binding to other TFs. In contrast, a 500-bp of Atoh1+70 000 drove hair-cell-restricted expression in all inner-ear sensory organs in all five transgenic lines  and outer-hair-cell-specific Slc26a5 (Prestin)  and Slc1a3 . Thus, Six1 may engage target sites in chromatin for later activation.Intriguingly, we discovered that Six1 pre-occupies CREs of hair-cell-subtype-specific genes at the precursor stage, including inner-hair-cell-specific Prestin) . Six1-ocP = 10−3138 or P = 10−2311) matched to the Six1/2-binding motifs , IRF and NF1/CTF are among the top five most enriched motifs Figure . CTCF/BORfx1 and Rfx3  compared to control littermates  , whereas SIXmt2 disrupted Six1-binding in 293 cells  Figure . ChIP-qPs Figure  and weaks Figure  and abolDusp6 is a downstream effector of Fgfr signaling and inactivation of Dusp6 causes hearing loss . However, a 4-bp mutation of SIX alone (SIXmt2), which completely disrupted Six1-binding . On the apical surface, F-actin and anti-acetylated tubulin staining and scanning electron microscopy (SEM) revealed V-shaped stereocilia packed with actin filaments and a kinocilium centered next to the tallest stereocilia on each hair cell, which are uniformly aligned along the medial-lateral axis across the entire sensory epithelium (referred as PCP)  Figure , B. The ) Figure . The apin Figure -K.The primary hair-bundle defects include flat bundle Figure , G, multThe bundle orientation as a readout of hair cell PCP was also significantly disrupted in both inner hair cells and outer hair cells with outer hair cells more affected than inner hair cells Figure –K. The aVangl1/Vangl2/Celsr1 and Ptk7  with various motifs for TF binding and are the DNA platform for the recruitment of TFs and transcription regulatory machineries. Genome-wide characterization of enhancers and developmental programs that drive the generation of distinct cell types in the auditory sensory epithelium is a prerequisite to finding ways to repair it when damaged. Due to technical challenges in material collection and others, genome-wide identification of inner ear-specific enhancers has not yet been performed and no ChIP-seq data for any TFs that use inner ear sensory organs are publicly available. Previous studies on brain development discovered very few enhancers, including the 1.4-kb 3′enhancer  and Sox2er  and Pendrin (Slc26a4)  , whose mSlc26a4) ,65, our The ChIP-seq data reported in this paper were deposited to the Gene Expression Omnibus (GEO) (GSE108130 and GSE119545).https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE108130.We have two GEO associated with this project. GSE108130, token is ehmxuaqqrbwphuv: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119545.GSE119545, token is ytmxskuwrbwzncb: gkaa012_Supplemental_FilesClick here for additional data file.


Although many signalling pathways have been discovered to be essential in mesenchymal stem/stromal (MSC) differentiation, it has become increasingly clear in recent years that epigenetic regulation of gene transcription is a vital component of lineage determination, encompassing diet, lifestyle and parental influences on bone, fat and cartilage development.This review discusses how specific enzymes that modify histone methylation and acetylation or DNA methylation orchestrate the differentiation programs in lineage determination of MSC and the epigenetic changes that facilitate development of bone related diseases such as osteoporosis. The review also describes how environmental factors such as mechanical loading influence the epigenetic signatures of MSC, and how the use of chemical agents or small peptides can regulate epigenetic drift in MSC populations during ageing and disease.Epigenetic regulation of MSC lineage commitment is controlled through changes in enzyme activity, which modifies DNA and histone residues leading to alterations in chromatin structure. The co-ordinated epigenetic regulation of transcriptional activation and repression act to mediate skeletal tissue homeostasis, where deregulation of this process can lead to bone loss during ageing or osteoporosis. The stromal component within bone marrow is a hierarchical continuum of mature functional stromal populations and committed progenitor cells. This is sustained by a minor population of long-lived, self-renewing and multipotential skeletal stem cells or bone marrow-derived mesenchymal stem/stromal cells (BMSC) . PurifieEpigenetics is the cellular modification of reversible and heritable changes in gene expression that occur without changes in the DNA code . EpigeneThe histone 3 lysine 27 (H3K27) methyltransferase, EZH2, is essential for embryogenesis and postnatal tissue development –12. We hMethylation of histone 3 on lysine 4 (H3K4me3) is associated with transcriptional activation and pivotal for differentiation , 23. AbsAnother important epigenetic modification is methylation of histone 3 lysine 9 (H3K9me), a marker for gene repression. The H3K9me demethylase, KDM4A, was found to promote adipogenesis and conversely inhibit osteogenesis in primary cultures of BMSC . SubsequSome epigenetic enzymes exhibit different functional roles targeting more than one epigenetic mark such as NO66, a H3K4 and H3K36 demethylase, which has been shown to bind to the Osterix promoter and inhibit transcription . SimilarHistone acetylation is an epigenetic modification associated with transcriptional activation, where histone deacetylation causes chromatin to compact, leading to transcriptional repression , 40. MemInvestigations into mechanical bone stimulation demonstrated enhanced Notch signalling and downregulated HDAC1, in human osteoporotic BMSC ••. HDAC1To date, the gene promoter of the osteogenic master regulatory factor, Runx2, has been reported to undergo specific changes in epigenetic modifications including, enrichment of histone 3 acetylation (H3Ac), H3K4me3 and reduced H3K4me1, H3K27me3 and H4R3me2 . ChromatCollectively, these studies demonstrate that the interplay between different epigenetic factors targeting histones is delicately balanced in order to orchestrate correct developmental pathways by targeting lineage-specific gene sets that mediate BMSC cell fate determination.There is overwhelming evidence implicating DNA methylation as an inhibitor of BMSC osteogenic and adipogenic differentiation in vitro , 54. DNAIn more recent years, the hydroxylases, Ten-eleven-translocases  have emerged as key regulators of stem cell renewal. They catalyse the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) where it opposes the function of 5mC and leads to DNA demethylation –62. In hThe complexity of BMSC cell fate determination is dependent on interacting epigenetic modifications that control key lineage-specific transcription factors. Osterix is one of the most important lineage determinants for bone formation and is expressed specifically by pre-osteoblasts and chondrocytes , 66. ExaIdentifying genome-wide epigenetic signature changes and chromatin remodelling regions during BMSC multi-differentiation enables the identification of essential genes, promoters and enhancers that function in lineage determination. Studies of histone modifications have been examined using C3H10T1/2 cells and BMSC following osteogenic differentiation in vitro . InteresWhole-genome histone modifications and DNA methylation have been mapped during chondrogenic differentiation of human BMSC . The traGenome-wide epigenetic map analysis during adipogenesis, using human adipose-derived MSC and embryonic derived 3T3L1 cells, found increased H3K4me3 levels along with gene promoters during adipogenesis, while H3K4me1 and H3K27Ac marks were found mainly in promoters, introns and intergenic regions . H3K36meGene ontology analyses investigating changes in epigenetic profiles of murine BMSC during osteogenic differentiation revealed genes associated with cell cycle, DNA replication and non-bone lineage transcription factors were downregulated, whereas bone differentiation genes were upregulated . Highly The use of EZH2 inhibitor GSKJ126 reduces H3K27me3 around transcription start sites, enhancing expression of osteogenic initiating factors, PTH, BMPs, Wnt genes and extracellular matrix genes in murine MC3T3 cells ••, resulAn osteoporotic model based on injecting dexamethasone into the tibia of mice showed an increase in adipogenesis and expression of adipogenic genes, PPARγ2, C/EBPα and GLUT4, and a reduction in osteogenic genes Runx2, alkaline phosphatase, bone sialoprotein and Osteocalcin . IncreasThe H3K36me3 demethylase, SETD2, has been shown to be downregulated during adipogenesis, whereas SETD2 levels increase during osteogenesis . AnalysiThe H3K4 methylation mark is associated with gene activation, where the H3K4 demethylase, KDM5A, was found to be specifically upregulated in both human and mouse BMSC derived from osteoporotic bone samples . FunctioCollectively, these preclinical studies highlight the reversible nature of the epigenetic landscape under pathological settings, which is amenable to drug targeting Fig. . HoweverEpigenetic regulation of MSC differentiation is an area that has progressed rapidly in the last decade with the use of technologies such as ATAC-seq, ChIP seq, bisulphite sequencing and 3C-chromatin capture. With more than 20 known histone modifications and two key DNA modifications, the potential for unique epigenetic combinations is staggering, illustrating the field is still in its infancy. The literature up to now shows that during differentiation, activating histone modifications consisting of combinations of H3K4me1/2/3, H3K36me3, H3R17, H3K9Ac and H3K27Ac prevail along with lineage-specific genes related to osteogenesis, adipogenesis and chondrogenesis, depending on the differentiation path. The most tightly associated mark with lineage transcription is H3K36me3/H3K4me3 along with gene bodies and transcription start sites together with H3K4me1 and H3K27Ac that mark enhancers. The enhancer modifications closely correlate with active lineage-associated transcription and are sites of active chromatin remodelling. Commonly seen is the appearance of repressive marks  on lineages not activated to ensure their repression. Although DNA methylation on promoters is not entirely consistent with BMSC differentiation, DNA methylation along with gene bodies and enhancers is closely linked with transcription as removal of DNA methylation marks is associated with activation. Epigenetic deregulation in osteoporotic BMSC implies loss of cell identity with representation of non-bone markers, compensatory mechanisms cause an increase in early osteogenic gene expression to synthesise more bone and changes in the active and repressive histone marks and DNA methylation on lineage-associated genes lead to decreased osteogenesis and enhanced adipogenesis. Further investigations into the identification of novel epigenetic factors and target genes regulating MSC differentiation will help progress the development of epigenetic drug-specific targeting of skeletal tissues for a range of orthopaedic-related indications, by coupling with new drug screening and drug delivery approaches.


MBTPS2 gene on the X-chromosome encodes the membrane-bound transcription factor protease, site-2 (MBTPS2) or site-2 protease (S2P) which cleaves and activates several signaling and regulatory proteins from the membrane. The MBTPS2 is critical for a myriad of cellular processes, ranging from the regulation of cholesterol homeostasis to unfolded protein responses. While its functional role has become much clearer in the recent years, how mutations in the MBTPS2 gene lead to several human disorders with different phenotypes including Ichthyosis Follicularis, Atrichia and Photophobia syndrome (IFAP) with or without BRESHECK syndrome, Keratosis Follicularis Spinulosa Decalvans (KFSD), Olmsted syndrome, and Osteogenesis Imperfecta type XIX remains obscure. This review presents the biological role of MBTPS2 in development, summarizes its mutations and implicated disorders, and discusses outstanding unanswered questions.The  MBTPS2) on the X-chromosome encodes site-2 protease (S2P) which is an integral membrane protein that plays vital roles in regulating membrane-tethered transcriptional factors. It is ubiquitously expressed within the Golgi membrane where it functions sequentially with site-1 protease (S1P), encoded by the membrane-bound transcription factor protease, site-1 (MBTPS1), to proteolytically activate membrane-tethered latent transcription factors. This mechanism is known as regulated intramembrane proteolysis (RIP).The Membrane-Bound Transcription factor Protease, Site-2 gene , not only for the transcription factors that it processes but also for its involvement in unexpected pathways that are critical for diverse biological process, such as ER stress, unfolded protein response, and gluconeogenesis [The second intramembrane cut, that releases the membrane bound fragments into the cytosol is carried out by one of the four distinct and evolutionarily conserved proteases named intramembrane cleaving proteases (I-CLiPs): the aspartyl protease-like, the zinc metalloproteinase S2P, the serine protease family of rhomboids, and the glutamatergic intramembrane proteases . Among togenesis . AccordiMBTPS2 mutations. We highlight the role of MBTPS2 in skin disorders and X-linked recessive form of osteogenesis imperfecta (X-OI), as these pathological states provide good examples of the diversified functions and their dysfunction in diseases.In this review, we focus on MBTPS2 and provide an update overview of the current understanding of how MBTPS2 contributes to a range of functions in maintaining cellular integrity and examine diseases caused by The landmark discovery of the human zinc metalloproteinase S2P in 1997 as a critical component of the processing machinery in cleaving TM precursor proteins has transformed our conceptual molecular understanding of the regulation of fundamental cellular processes carried out via RIP. Somewhat serendipitous, S2P was discovered through a series of independent studies that deciphered the SREBP pathway to understand the global regulation of cholesterol homeostasis in mammalian cells , 10. In 2)-terminal DNA-binding domain is a basic-helix-loop-helix-leucine zipper (bHLHL-Zip) family of transcription factors. The middle domain is a helical hairpin membrane anchor, with two TM helices separated by a short loop that projects into the lumen of the ER and nuclear envelope. The carboxy (COOH)-terminal, which also projects in the cytosol, functions as the regulatory domain [2-terminal intermediate domain at site 2, a site within the transmembrane helix, to release the bHLHL-Zip domain from the membrane. The bHLHL-Zip domain is then translocated to the nucleus to initiate the transcription of genes encoding enzymes involved in the biosynthesis and uptake of cholesterol, fatty acids, and triglyceride  that is auxotrophic for cholesterol due to a defect in carrying out the site-2 cleavage to release the bHLHL-Zip domain of SREBPs from the membranes, and the HfT1M19(c) CHO that is a revertant cell line of the M19 CHO, as a recipient for complementation cloning, the S2P was isolated and characterized [The characterization and appreciation of the vital role that these cleavage machineries serve in the feedback regulatory system of cholesterol synthesis and uptake warranted studies to identify these proteases. Grasping this opportunity was the Rawson lab who conducted pioneering work in isolating the cterized , 17. Thecterized . Furthercterized .k factor regulates gene expression in the mother cell after engulfment of the forespore. Cleavage of pro-σk and liberation of transcription factor require the membrane protein SpoIVFB, which this protein contains the HExxH motif [The proteolytic function of S2P is further supported from the discovery of a family of related proteins in bacteria. These proteins are vital for the proteolysis of membrane-bound transcription factor needed for sporulation. For instance, the σxH motif . In anotxH motif . These s2-terminal cytosolic domain, and a stress-sensing domain in the ER lumen [ATF6 activates the transcription of genes in response to the accumulation of unfolded or misfolded proteins in the ER . As a tyER lumen . Under rER lumen . Upon acER lumen . The cleER lumen  Fig. 1)2-terminaER lumen –27.2-terminal cytosolic domain resembling a transcription factor of the bZIP family and undergo proteolytic cleavage by S1P and S2P. They have been reported to have major roles in development, however, other roles have been identified such as metabolism, secretion of signaling proteins, cell survival, differentiation and division, and tumorigenesis.The CREB3 family of transcription factors consist of five members—CREB3, CREB3L1, CREB3L2, CREB3L3, and CREB3L4. Like SREBPs and ATF6, all members of this family harbor a single TM helix with an NH2-terminal fragment relocates to the nucleus to activate target transcription genes and proteins such as cAMP response element (CRE), ER stress response element II, and unfolded protein response element [CREB3 or Luman was first within its family to be identified as an ER-bound transcription factor as a counterpart of the herpes simplex virus transcriptional activator VP16 that binds to the host cell factor regulator , 29. It  element , 37. The element  2-terminal fragment enters the nucleus to initiate the transcription of COL1A1 by binding to a CRE-like sequence in its promoter region. Its prominent role in bone formation was further supported by its contribution as a genetic cause of OI [CREB3L1 downregulated the expressions of genes encoding components of the COPII coat, Sec23A and Sec24, protein components responsible for the transport and secretion of cartilage matrix proteins from the rough ER to the Golgi apparatus [CREB3L1, originally known as OASIS, plays a critical role in bone development. In response to bone morphogenetic protein-2 (BMP-2), a cytokine required for bone formation and osteoblast differentiation, CREB3L1 is cleaved sequentially by S1P and S2P , 39. These of OI –42 2-terminand SEC24 .2-terminal nuclear form activates the transcription of C-reactive protein and other acute phase proteins in the liver to provide an early defense against microbial infection [NFATC1), a prominent transcription factor for osteoclast differentiation, to induce osteoclast. Inhibition of ER stress, however, reduced the expression of osteoclast-related genes and CREB3L3 activation, and thereby prevented RANKL-induced bone destruction, suggesting the inactivation of ER stress CREB3L3-induced signaling pathways may be of therapeutic use for the treatment of osteoporosis [CREB3L3, also named CREB-H, was originally isolated as a hepatocyte-specific bZIP transcription factor and has an important role in innate immunity , 46. Folnfection , 47 , is highly abundant in the prostate where it is involved in the chromatin organization during spermiogenesis –54. Likehe Golgi . In a mohe Golgi . The acthe Golgi  or the phe Golgi .The primary function of MBTPS2, as aforementioned above, is to cleave its substrates to enable them to perform vital cellular functions. To accomplish this, MBTPS2 relies on both of its conserved catalytic site motifs, HExxH and LDG, that are not only hallmarks of MBTPS2 but also core to its activity. Illuminating this was the structural studies of MBTPS2, via sequence-based topology predictions or X-ray crystallography methods, which has provided a substantial amount of information regarding the fundamental structural requirements of MBTPS2 and how they conform to its function as a membrane-embedded protease.MBTPS2 gene spans greater than 4 kilobase pairs on human chromosome X and has 11 exons  is an extremely rare congenital disorder with only about 60 cases reported worldwide since it was first identified by McLeod in 1909 . The syn08205 is MBTPS2 2- or COOal delay . Additioal delay . Yet, anInitially, BRESEK or BRESHECK was considered as a distinct entity that was termed ‘BRESEK or BRESHECK syndrome’ by Reish et al. (1997); however, as majority of its symptoms are often presented along side the IFAP triad, particularily Hirschsprung disease in severe cases of IFAP, IFAP, and BRESHECK have been classified within the same entity as ‘IFAP with or without BRESHECK (MIM #308205) , 85, 86.Keratosis follicularis spinulosa decalvans  is a rare hereditary disorder of keratinization recognized by widespread hyperkeratotic follicular papules . First dMBTPS2 have been identified as one of the causative genes of KFSD. Alike mutations leading to the IFAP triad, mutations underlying KFSD lie within the TM domains of MBTPS2. In three unrelated families, a missense mutation in MBTPS2 (p.N508S) was identified in affected males displaying mild phenotypes [MBTPS2 mutation was speculated  [Olmsted syndrome  is an extremely rare keratinization genetic disorder classically marked by the combination of bilateral mutilating palmoplantar keratoderma, and periorificial hyperkeratotic plaques . The dis (TRPV3) , 99–104.MBTPS2. First of the three is a missense mutation (p.F464S) harbored by two males in an Iranian pedigree [MBTPS2 mutations to not only consist of mutations on TM domains but also ones at the exon–intron boundaries of MBTPS2. The third mutation is another recurrent mutation (p.F475S) that was reported in two Lebanese brothers that displayed clinical features for both OS and IFAP [TRPV3 mutations which affected individuals only display classical OS phenotypes without any IFAP features. Whether the nomenclature of OS should be incorporated with IFAP in a similar manner that the BRESHECK syndrome was grouped as ‘IFAP with or without BRESHECK’ requires further considerations.Among the 106 cases of OS, three of these are to date reported to be caused by mutations in pedigree , 105. Thpedigree . Interespedigree . By an ipedigree , 78. Thiand IFAP  Fig. 4)MBTPS2. Fand IFAP ; howeverand IFAP . These rand IFAP , 106. ThOsteogenesis Imperfecta (OI) or brittle bone disease is a congenital heterogenous skeletal deformity disorder that affects approximately 1 in 10,000–20,000 births worldwide . IndividCOL1A1 or COL1A2, causing an alteration in the structure or function of collagen type 1, the most abundant protein of bone extracellular matrix [CRTAP, PPIB, LEPRE1/P3H1) [SERPINH1, FKBP10) [SEC24D) [BMP1) [MBTPS2 [MBTPS2 missense mutations. In both pedigrees, the resulting substitutions (p.N459S and p.L505F) were found to occur within or adjacent to the zinc ion coordinating site required for enzymatic activity  –115, fol FKBP10) , 116, in[SEC24D) , and ext) [BMP1) , 119 hav [MBTPS2 , 121  and/or animal models . Any insMBTPS2 mutations in which several studies have revealed a prominent concordance in the molecular and biochemical properties of the mutations to the catalytic sites. What is also rather striking is the very distinctive disorders that result from the variants of MBTPS2, which may be suggestive of MBTPS2 having pleiotropic properties. Additionally, the disorders that so far have been reported arise from mutations that all lead to loss of function of MBTPS2, an area that has not been delved into are effects or disorders of variants that lead to the hyperactivity of MBTPS2. Nonetheless, the challenge lies in deciphering the complex pathomechanism underlying the associated distinctive disorders. Investing efforts into these important, albeit challenging, technical areas will most definitely accelerate our understanding of MBTPS2 in pathophysiological conditions even further. Looking into the future we hope that many of the fundamental remaining questions about MBTPS2 will be addressed and look forward to the exciting advances in both the mechanistic features of MBTPS2 functions and to utilize it as a therapeutic target.Since the serendipitous discovery of MBTPS2 in 1997, tremendous strides have inarguably been made in understanding the role of this once heretical protease. It has transition S2P from just a component in the cleavage machinery of SREBPs to now a protease that is widely regarded as a critical regulator of serval cellular processes in health and disease. The biochemical and structural biology studies have offered spectacular insights into the mechanisms and functions of S2P, disposing the longstanding doubt of whether its proteolytic action via hydrolysis of peptide bonds can occur in the membrane, provided a conceptual platform in appreciating the broader functions of S2P and revealed a plethora of substrates that are entirely dependent on S2P for their downstream target pathways. Another notable insight gained is the identification of both of the catalytic active sites of S2P that is not only crucial to its proteolysis, but also seemingly an essential aspect in the pathogenesis of the disorders resulting from


Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in  Adh is isoforms . These tn adults . It has promoter . Howeverpromoter . TherefotubGAL4 line was obtained from the Bloomington Drosophila Stock Center (ID 5138). All fruit flies in the stock were heterozygous for tubGAL4 and the balancer TM3, Sb1Ser1, as the tubGAL4 chromosome is homozygous-lethal. For experiments requiring adult fruit flies, a mixture of males and females was used. The AdhDIST∆ line was homozygous for the deletion allele. In experiments requiring induction of AdhDIST* in larvae, we crossed homozygous AdhUAS males to virgin female tubGAL4/TM3, Sb1Ser1 or Oregon-R control fruit flies in collection cages with molasses plates spread with live yeast. After 8 hr, plates were removed, and embryos were allowed to age for 72 hr at 25°. The population consisted of predominantly first and second instar larvae. To collect the samples, larvae were washed off the plates using PBS and then washed three times in PBS. In between washes, larvae were left undisturbed to allow settling by gravity. ∼2 mL of larvae were aliquotted, flash-frozen in liquid nitrogen, and stored at -80° for later processing.Fruit flies were raised on standard molasses medium at 25°. Oregon-R was used as wild type (a generous gift from Don Rio). The AdhDIST∆ (pUB1041) expressed sgRNAs 5′-AGTGGGCTTGGTCGCTGTTG-3′ and 5′-TAATATAGAAAAAGCTTTGC-3′. The sgRNA plasmid for generating AdhUAS (pUB1038) expressed sgRNAs 5′- CATAACTCGTCCCTGTTAAT-3′ and 5′-ACACATTTGTTAAAAGCATA-3′. The repair templates were cloned into the pGEX-2TK cloning vector . To generate the repair template for the AdhDIST∆ allele (pUB1094), two 1-kb homology arms were amplified from Oregon-R genomic DNA, with the Adh distal promoter region removed. When used as a repair template donor, this results in the removal of the region spanning -387 to -1376 bp upstream of the proximal isoform transcriptional start site. A similar allele was described previously (AdhUAS (pUB1091) contained two 1-kb homology arms amplified from Oregon-R genomic DNA, flanking a 10xUAS-hsp70(core promoter) construct amplified from pVALIUM20 (10xUAS-hsp70(core promoter) construct at the -1 position relative to the distal transcriptional start site.We cloned sgRNAs into pCFD4 (eviously . The rep1 w M{nos-Cas9.P}ZH-2Ay embryos (Bloomington 54591), which express maternal Cas9, by BestGene Inc. . The resulting mosaic fruit flies were outcrossed to 1118w, and the F1 progeny were individually crossed to CyO or CyO, twi > GFP balancer lines prior to being genotyped. Introduction of the desired allele in the genotyped parent was tested by PCR and sequencing. F2 progeny carrying the desired allele and balancer were then crossed inter se to generate homozygous animals.sgRNA plasmids and their corresponding repair templates were injected into Adh isoforms were quantified using primers specific to AdhDIST and AdhPROX  according to a previously described protocol (Bogart and Andrews 2006). 450 ng of isolated RNA was treated with DNase  and reverse transcribed into cDNA  according to the manufacturer’s instructions. The RNA levels of specific Adh isoforms, the total RNA of each sample was treated with RNaseH prior to RNA blot analysis. A total of 15 µg Trizol-extracted RNA was added to 1x RNaseH buffer . Next, a site-specific DNA oligo  was used for full-length, RNA ligase-mediated rapid amplification of 5′ cDNA ends according to manufacturer’s instructions. 2 μg of total RNA was isolated, as described above, from L1/L2 larvae and adults. The gene-specific primer used is listed in Chromatin immunoprecipitation in larval samples was performed as previously described  and 50% sucrose buffer  were used to generate a sucrose gradient of 10–40% in a Beckman Coulter 9/16x3.5 PA tube (Cat #331372) SW-41 ultracentrifugation tube. The gradient tube was stoppered and the setting “long sur 10-40%” was used to make the gradient. Gradients were centrifuged at 35,000 g using a SW-41 rotor for 3 hr at 4° and fractionated on a Brandel flow cell (Model #621140007) at 0.75 mL/min with the sensitivity setting at 0.5 Abs. A volume of 750 μL was collected for each fraction. The samples were then pooled as indicated in Figure S1. 5 ng rcc1(xl)-polyA spike RNA was added to each pooled fraction prior to RNA extraction. RNA was extracted from the fractions using standard acid phenol:chloroform extraction as described in Whole fruit flies or larvae harvested in 1X PBS were transferred to a microcentrifuge tube on liquid nitrogen. Samples were homogenized on ice in 200 μL cold lysis buffer in the presence of cycloheximide. The lysis buffer for cycloheximide samples is as follows: 500 mM KCl, 15 mM Tris-HCl pH 7.5, 15 mM MgClhttps://doi.org/10.25387/g3.10565984.All the reagents generated in this study are available upon request. Supplemental material available at figshare: Adh proximal promoter produces a transcript of 1001 nucleotides in length (hereon referred to as AdhPROX), whereas the Adh distal promoter activates a transcription start site (TSS) located 715 base pairs (bp) upstream of the proximal TSS. The resulting transcript from the distal promoter, hereon referred to as AdhDIST, has a unique 5′ leader located in exon 1  using isoform-specific primers  and normalized AdhDIST and AdhPROX transcript measurements to αTUB84B, a ubiquitously expressed transcript. This analysis revealed that the AdhPROX transcript level was ∼20 fold higher in larvae compared to in adults, whereas the AdhDIST transcript had the reciprocal pattern with more than 8000-fold enrichment in adults compared to its expression level in larvae . Interestingly, in the adults, AdhDIST enrichment in the high polysome fraction was more than fourfold higher relative to αTUB84B enrichment . We conclude that both AdhPROX and AdhDIST are well translated. Furthermore, AdhDIST appears to be noticeably more enriched in the high polysome fractions than αTUB84B, indicating enhanced translational efficiency.To determine the translational status of the two PROX expression at the endogenous locus, we used CRISPR/Cas9-based editing    . The Adh4 driver . Even inPCR data  and 3C. Adh locus, we performed chromatin immunoprecipitation (ChIP) against H3K36me3 and H3K4me3 in larvae collected from wild type and homozygous AdhUAS lines, where both Adh alleles express AdhDIST*. H3K36me3 is a co-transcriptionally established modification that occurs in regions downstream of active promoters  in the heterozygous GAL4AdhUAS line appears to be inconsistent with a cis-mediated transcriptional interference mechanism at a first glance. We attribute this unexpectedly high reduction of AdhPROX level in the heterozygous lines to transvection, a common phenomenon in Drosophila in which interallelic promoters are co-regulated due to somatic pairing of homologous chromosomes. It has been shown that the GAL4-UAS system is subject to transvection , thus excluding repressive uORF translation. The difference between the apparent regulation in these two cases is important: poor translation in the case of the 5′ extended LUTINDC80 isoform and superior translation in the case of AdhDIST. It is interesting to note that an earlier study, which examined the consequences of a natural transposon insertion at the Adh locus in the fruit fly (copia retrotransposon between the Adh adult enhancer and the Adh distal promoter leads to an unusually low level of the Adh protein and enzyme activity. The reduction was found to occur as a result of a decrease in the level of the AdhDIST transcript. Surprisingly though, in this case, the AdhPROX transcript levels were proportionally increased in adults (PROX might not be as efficiently translated as AdhDIST, which is consistent with our polysome analysis. One possibility is that tissue-specific, trans-acting factors could differentially modulate the translation of the two Adh mRNA isoforms. Such spatial effects are likely to be missed by the whole organism polysome fractionation approach that was used in this study.A key difference between examples of ruit fly , along wruit fly , showed n adults . Given tAdh example is likely to be one of many cases where developmentally controlled transcriptional interference from ORF-distal promoters can alter genome decoding and cellular function in a manner that has not been anticipated previously.More broadly, the switch from one mRNA isoform to another may alter not just the translational efficiency of the transcript, but also localization, stability, or alternative splicing as well. In this regard, transcript toggling driven by developmental switches in promoter usage and the subsequent transcriptional interference from distal gene promoters may serve to alter gene expression in respects other than gene silencing. We posit that the


Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment. Osteoblast lineage commitment is regulated by a coordinated set of extra cellular stimuli and developmentally-regulated signaling pathways, including those mediated by bone morphogenic proteins (BMPs), Wnt-ligands, steroid hormones, and growth factors, among others . FollowiWe discuss research demonstrating the role of epigenetic mechanisms in controlling gene transcription during mesenchymal cell commitment to the osteogenic lineage. We first overview key components of epigenetic mechanisms that control gene expression in mammals and then describe how these epigenetic processes contribute to regulate transcription of the Runx2 and Sp7 genes, two critical osteogenic master regulators.Nuclear DNA is organized as chromatin. Nucleosomes are the fundamental units of this chromatin and are composed of an approximately 147 bp DNA segment wrapped around an octamer of histone proteins  . A portiMolecular components that regulate chromatin organization and transcription are critical players during cell differentiation. Among them the large group of “histone post-translational modifications” (HPTMs), which are considered a principal “epigenetic” mechanism . HPTMs mGenetic studies allowed the identification of the Polycomb Group (PcG) and the Trithorax Group (TrxG) complexes, which mediate inhibition and activation of transcription, respectively, during embryogenesis . One sigSeveral mammalian TrxG complexes, including COMPASS (Complex of Proteins Associated with Set1a/b) and the Mixed Lineage Leukemia (MLL1–5)-containing COMPASS-like complexes have been reported . TrxG-meSet1-COMPASS mediates global genomic deposition of H3K4me3 in most mammalian cells and therefore its function is tightly associated with transcriptional activation of a large number of genes. MLL2-COMPASS-like has been shown responsible for H3K4me3 deposition at promoters in embryonic stem cells (ESCs), contributing to transcriptional upregulation of homeobox genes . InteresChromatin domains with decreased enrichment of H3K4me3 or H3K27me3 can also be maintained through the activity of lysine demethylases . In partMethylation at H3K9  is strongly associated with the formation of highly compacted and transcriptionally repressed chromatin (heterochromatin) . The metcis or by transferring histone octamers in an ATP-dependent manner. This changes the exposure of regulatory motifs thereby facilitating or preventing recognition by cognate factors  activities (Genomic DNA can be methylated at cytosines that are followed by guanosines (CpG dinucleotides). The DNA methyl-transferases that mediate this modification belong to a well-conserved family of proteins that include both maintenance (Dnmt1) and tivities . Most retivities . DNA demtivities . Importativities .Enrichment of H3K9me3/2 at transcriptionally-inactive chromatin can be associated with the presence of 5mCpG. This is due to the ability of the proteins that “write” and “read” these two epigenetic marks to form complexes  thereby Differentiation of mesenchymal stem cells (MSCs) to the osteogenic lineage requires the expression and function of two master transcription factors: Runx2 (Runt Related Transcription Factor 2) and Sp7 . These factors bind to and control the expression of numerous downstream target genes that code for proteins that are necessary for establishing the osteoblast phenotype . Runx2 aActive transcription of the Runx2 and Sp7 genes is accompanied by an epigenetic landscape that includes increased H3K4me3 enrichment at both promoter regions . Hence, It has been determined that an enrichment of H3K4me1 accompanies transcriptional silence at the Runx2 and Sp7 genes in non-osteoblastic and mesenchymal uncommitted cells ,b. As MLMaintenance of chromatin with low H3K4me3 enrichment (and hence elevated H3K4me1) at the Runx2 promoter can be also achieved by binding and activity of the H3K4 demethylase Jarid1b/Kdm5b , which cFindings from several teams revealed that regulating the repressive mark H3K27me3 represents an important step during the differentiation of MSCs toward osteoblasts. It was demonstrated that the activity of the H3K27 demethylases Utx/Kdm6a and Jmjd3/Kdm6b is critical for erasing this mark from the promoter of the Runx2 and Sp7 genes  and hencThe repressive mark H3K9me3 plays an important role during differentiation of MSCs to the osteoblastic lineage ,b. H3K9mAlthough most studies support the role of the H3K27me3 and H3K9me3 marks during repression of both master osteogenic genes, recent analyses indicate that for certain cells, this role may have to be re-evaluated. Thus, analysis of uncommitted human umbilical cord-derived Wharton Jelly MSCs (WJ-MSCs) showed that the Runx2-P1 promoter region does not exhibit significant enrichment of H3K9me3 and H3K27me3 . InteresHistone lysine acetylation also plays a relevant role during bone formation as it supports increased expression of critical genes for osteoblast differentiation . ChromatDNA methylation (5mCpG) can regulate osteogenic differentiation of MSCs. Addition of the DNA methyl-transferase inhibitor 5-Azacytidine (5-Aza) to human and murine mesenchymal cells enhances their ability to engage osteogenesis by reducing 5mCpG enrichment at regulatory regions of osseous genes . In partex vivo. Recent results demonstrate that the substrate stiffness at which hMSCs are grown in culture can induce rapid and stable changes in chromatin organization and the associated epigenetic landscapes that permanently up- or down-regulate genes (Controlling lineage commitment in MSCs represents a critical challenge to overcome skeletal deficiencies in patients. Extensive research during the last 20 years has shed light into potential targeting of specific epigenetic mechanisms that control bone-related gene expression. Future studies will need to consider the influence of the mechanical environments at which MSCs are maintained and differentiated te genes , hence mMM analyzed the data and wrote most parts of the manuscript. MC wrote parts of the text and prepared the figures. GN analyzed the data, wrote parts of the text and helped preparing the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers.Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477  SIM, which is required for EBNA1 to exert inhibitory effects on a SUMO2-modified complex (SC2) including STUB1, KAP1 and USP7. Disruption of EBNA1SIM leads to loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxia-mediated reactivation of viral lytic replication induces the EBNA1 dissociation from STUB1 in the SC2 complex. This discovery not only opens a new insight on the interplay between host and virus, but it also provides a therapeutic target specific against EBV-associated cancers.The Small Ubiquitin-related modifier (SUMO) modification of proteins is a reversible post-translational regulation involved in control of gene transcription, among other functions. Epstein-Barr virus (EBV) infects most people worldwide and contributes to the development of several types of cancers due to its ability to induce cell proliferation and survival. EBNA1 is expressed in all forms of EBV-associated tumors. In this study, we found that EBNA1 contains a SUMO-interacting motif (SIM) named EBNA1 Epstein-Barr virus (EBV) was the first human tumor virus to be discovered. It was identified from Burkitt’s lymphoma (BL) in 1964, and accounts for approximately 2% of all cancer deaths in the world to date ,4. The EOriP) has been demonstrated to be critical for driving the latent cycle of viral episome DNA replication and segregation, and to maintain its stability in proliferating cells  with high molecular weight (>170kDa) to some extent, although we found a moderately higher association of EBNA1 with (SUMO1)n-sb than with (SUMO2)n-sb  purified from the E.coli expression system ,49. InteTo determine whether the SIM2 or SIM3 motif impairs the EBNA1 dimerization, we performed a co-IP assay to detect the interaction of FLAG-tagged EBNA1 with myc-tagged wild type (WT) EBNA1 or its SIM-deleted mutants in HEK293 cells. As shown in 441-619) and its K477R or SIM3-deleted mutants using an E.coli expression system, and then carried out in vitro SUMOylation assays by incubation with His-tagged SUMOylation E1 and E2 enzymes in the presence of SUMO1 or SUMO2. Strikingly, the results showed that the K477R mutation could efficiently reduce both the SUMO1 and SUMO2 modification of EBNA1 in vitro  within the DNA-binding domain of EBNA1 is a putative SUMOylated site with an associated highly probability (> 0.90) based on the prediction analysis . To deteTo further confirm that both the K477 residue and the SIM3 motif are required for SUMOylation of EBNA1, we investigated the subcellular localization of both EBNA1 and SUMO1 (or SUMO2) by co-expressing cherry-labeled SUMO1/2 with the WT EBNA1 and its K477R or SIM3-deleted versions in HEK293 cells. Intriguingly, in the absence of exogenous SUMO1/2 molecules, the results showed that both WT EBNA1 and its SIM3-deleted mutant are diffusely distributed within the nuclear compartment, while the K477R mutation resulted in the clear co-localization of EBNA1 with DNA as punctate dots . In contcis-acting OriP and trans-acting viral protein EBNA1  was expressed as fold changes relative to the reporter construct alone. Assays were performed in triplicate.OriP-GFP-Hygromycin plasmid at 48 h post-transfection, were cross-linked with 1.42% (v/v) formaldehyde. The following protocols were performed as described previously  is highlighted.(TIF)Click here for additional data file.S2 FigHEK293T cells were co-transfected with expression plasmids as indicated. Whole cell lysates (WCL) were harvested at 48 h post-transfection, and subjected to co-IP and IB as indicated. The relative density (RD) of EBNA1-binding Ubc9 and Ubc9-SUMO is quantified and shown on the right panel.(TIF)Click here for additional data file.S3 FigRelated to (TIF)Click here for additional data file.S4 FigRelated to (TIF)Click here for additional data file.S5 FigLCL1 cells were subjected to hypoxia (0.2% oxygen) treatment for overnight. Endogenous EBNA1, SUMO1, and SUMO2 were individually stained by EBNA1 (green) and SUMO1/2 (red) antibodies. The profile of EBNA1 and SUMO1/2 immunofluorescence were quantified and shown on the right panels. SUMO2 co-localization with EBNA1 was highlighted by the arrows and enlarged at the bottom panels.(TIF)Click here for additional data file.S6 FigA) or LCL (B) cells with KAP1, STUB1 or USP7 knockdown were individually subjected to immunoblotting (IB) with the indicated antibodies. The luciferase knockdown (shCtrl) was used as control.Whole cell lysate of 293T ((TIF)Click here for additional data file.