The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. Figure 1). Microscopic visualization of DNA stains in ESC nuclei show diffuse staining indicative of a generally open chromatin state (Pluripotent embryonic stem cells (ESCs) from many organisms display strikingly different chromatin structure and overall nuclear architecture when compared with differentiated cells , while Lamin B1 and B2 are transcribed from distinct genes  of the NE . The lamThe B type Lamins (B1 and B2) are expressed in all cell types, while expression of Lamins A and C varies with cell type and developmental stage . ImmunofLamin A (LMNA) gene, which lead to an array of serious diseases called laminopathies, including cardiomyopathy, muscular dystrophy, lipodystropy, neuropathy and progeria , and support the notion that Lamins play functional roles in cell differentiation, as discussed further below.Further supporting separate roles for A and B type Lamins, studies of Lamin filaments in amphibian oocytes and HeLa cells indicate that Lamins A, B and C form discrete, but interconnected, lattice structures with differing physical properties . In agreprogeria . Togethelarge organized chromatin K domains” (LOCKS) grows from 17.5–24% in pluripotent human stem cells to 39.3–44.8% in differentiated cell lines (Lamin B1 Associated Chromatin Domains (LADS), exhibits a significant overlap between LOCKS and LADs, which supports a role for Lamin B1 in the peripheral localization of these heterochromatic domains . They further show this loss and the subsequent chromatin rearrangements coincide with terminal differentiation of the rod cells. Strikingly, the inversion phenotype was successfully recapitulated experimentally in additional cell types, such as the hair follicle (which does not express Lamin A/C), using LBR null mice, and examination of double null (Lbr-/- Lmna-/-) mouse pups indeed showed an inverted phenotype in all post-mitotic cell types studied. Conversely, artificially maintaining expression of LBR, but not Lamin C in these cells was enough to prevent chromatin inversion, suggesting that Lamin C does not bind chromatin directly but perhaps via other nuclear envelope associated proteins such as LEM domain proteins (discussed further below). Although it is not presently clear how the conventional versus the inverted heterochromatin architecture affects cell type specific gene expression, these results support the notion that Lamins B and A/C are needed to position heterochromatin in a cell type specific manner.In a series of elegant experiments the authors demonstrate that during cell differentiation, conventional chromatin architecture requires the sequential expression of first the NE transmembrane protein Lamin B receptor (LBR) and then its replacement by Lamin A/C, with some cell types expressing both proteins . The chrFigure 2A).Genome wide studies of LADs during neuronal differentiation in mice showed that while ESCs and terminally differentiated cells share a broad LAD structure, smaller sub regions of gene clusters undergo rearrangements corresponding to steps of the differentiation process . For exaDrosophila, the gene encoding a critical transcriptional factor hunchback was shown to move to the nuclear lamina during differentiation of neuroblast cells to neurons  repeats array, introduced into the genome of human culture cells, and the ability of the Lap2β-LacI to silence genes near its target site was similarly found to be dependent on HDAC activity.How do Lamins bind to heterochromatin or developmentally silenced genes? In addition to reports of a DNA binding domain in Lamin A  and in vhistones , there ahistones . The authistones . In thisOn a cautionary note, initial DamID studies of Lamin-chromatin binding sites required a population of cells, and thus the resulting LADs are reflections of both an average of many cells in a population as well as an amalgamation of binding events acquired over the time a DamID fusion protein is expressed. When these studies were repeated using the m6A-tracer technique which is able to label stochastic protein-Dam chromatin interactions in single, living cells, the authors found that at a given time only a subset of the initially described LADs was localized to the periphery while the rest were often located in the nuclear interior and further, this subset often changed following each cell division . Use of The nuclear envelope is a double lipid bilayer system made of the INM, directly adjacent and connected to the Lamin filaments, and the outer nuclear membrane (ONM), which is contiguous with the endoplasmic reticulum. The space between these membranes is called the perinuclear space (PNS) and is interrupted by NPCs which fenestrate the NM. Originally viewed as simply a protective barrier for the genome, the nuclear envelope along with its nuclear envelope transmembrane proteins (NETs) and associated soluble proteins are now known to participate in an array of cellular functions including genome organization, nuclear migration and positioning, cell cycle regulation, signaling, and cell differentiation . While tFigure 1). These tissue specific results were directly verified for several novel NETs by immunofluorescence staining and comparison with known tissue specific expression profiles . Secondly, these results suggest that NETs bind specific chromosomes in a reversible manner, linking chromosome positioning with differentiation . In this manner, tissue specific NETs may function to reposition entire chromosomes or large chromosomal regions to the nuclear periphery, which may further assist or stabilize the silencing of specific developmental genes by association with the nuclear lamina . Thirdly, multiple NETs can act to position the same chromosome, perhaps via cellular regulation of relative abundance of different NETs. Lastly, in addition to tissue specific expression levels, several of the NETs in this study appear to have tissue specific splice variants. Together these provide another layer of regulation to how a cell might fine-tune its gene expression profile during differentiation by utilizing NETs to position chromosomes at the NE in a tissue specificmanner.This study yields several important conceptual findings: firstly, it provides examples of tissue specific NET expression, giving rise to unique NE compositions correlating with cell type  experiments analyzing mobility of LBR truncation mutants revealed domains involved in interactions with histone H4, but not with Lamin B1 or B2 are required for formation of stable LBR microdomains in the NE. To investigate a role for LBR in heterochromatin formation, in vitro experiments using atomic force microscopy to measure chromatin compaction showed incubation of recombinant LBR with reconstituted chromatin resulted in highly aggregated chromatin fibers compared with controls. This study further demonstrated that LBR itself has the ability to repress transcription of a reporter plasmid.Lamin B receptor, discussed above, is an INM protein shown to interact directly with Lamin B and the chromodomain heterochromatic protein HP1 . Initialin vivo effects of LBR depletion or mutation  was first described .One can imagine a model where Nesprin isoforms are expressed in response to developmental signals and then themselves confer cytoskeletal changes as well as alter 3D organization of the genome to promote further tissue specific gene expression. They can do so via relaying these signals to INM proteins or through their own, yet undetermined, nucleoplasmic roles. Additionally, changes in nuclear size, shape and relative position within the cell can potentially influence the kinetics of nuclear processes and thus gene expression. Perhaps the most exciting implication of the LINC complex lies in its connection of the chromatin-associated INM proteins to cytoskeletal proteins via Nesprins, suggesting that cytoplasmic forces can directly move or alter nuclear chromatin positioning. This idea is supported by studies in both  bridges . The larThe NPCs are multi-component protein complexes that form selectively permeable channels through the NE. The primary function of the NPCs is to mediate nucleo-cytoplasmic transport of molecules and thus allow communication between the nucleus and the cytoplasm . They arVia its transport functions, the NPC plays an obvious role in gene regulation by controlling export of generated RNA and import of transcription and signaling factors. Yet, in addition to its canonical transport role, the NPC and individual Nups have been shown to play a role in genome organization and gene expression via direct binding to specific genomic locations . Multiplin situ RNA hybridization studies of the early Drosophila embryo revealed that Nups vary in their expression patterns relative to embryonic segments and developmental time points .Perhaps the most remarkable and well characterized example of a nuclear pore component playing a role in differentiation is that of Nup210. The transmembrane nucleoporin Nup210 is absent in mouse progenitor myoblasts and ESCs, but its expression is sharply upregulated during differentiation of these lineages into myotubes and neuroprogenitors, respectively . Nup210 Wnt signaling, a central developmental signaling pathway of multi-cellular organisms, has also been repeatedly linked to the nuclear pore . Wnt sigThese findings illustrate that Wnt pathway components are regulated by FG Nups both in terms of transport and function. Given the indispensable nature of Wnt signaling in stem cell maintenance, embryonic development and cell migration, these connections heavily implicate Nups in both normal development and oncogenic transformation. Intriguingly, the pluripotency state itself has been postulated to be regulated by the NPC via controlling levels of the pluripotency factors Oct4, Sox2 and Nanog in the nucleus . TogetheThe phenotypes of Nups in tissue specific differentiation, described above, can result from either the transport or the genome regulatory roles of Nups, or possibly, from the integration of both. Multiple examples of cell type specific transport have been reported, and proposed transport mechanisms of Nups in development have been reviewed recently . Here, wA functional relationship between nuclear pores and nuclear organization of chromatin was originally proposed based on EM close ups of mammalian nuclei that show frequent association of what appears to be decondensed chromatin with nuclear pores . Such liSaccharomyces cerevisiae demonstrated that some Nups, such as Mlp1, Nup2 and Nup60 often occupy regions of highly transcribed genes , such as tRNA and snoRNA genes, where they appear to be functionally required for correct RNA processing . In this manner, the NPC may represent a distinct nuclear environment that promotes a permissive chromatin state at the nuclear periphery, functionally opposed to the roles of the Lamins and NETs , but perhaps providing an accessible scaffold for switching between silenced and activated states during cell differentiation.Intriguingly and in line with their dynamic behavior,  nucleus . Both th nucleus . TogetheDrosophila Nup98 was found to associate with histone modifying complexes such as the histone methyl transferase Trithorax (Trx), the fly homolog of Mixed Lineage Leukemia (MLL), and the Non-Specific Lethal (NSL) Complex, which carries a conserved HAT males absent on the first . The earliest genome wide binding analysis of various Nups in yeast demonstrated that the stable yeast Nup84  binds to loci that are not enriched for transcriptional activity, and thus termed “neutral” chromatin , which bears direct relevance to the establishment of cell type specific gene expression programs. Whether the metazoan NPC primarily functions as a scaffold for expression of RNAP II and RNAP III genes, for establishment of chromatin boundaries or for additional regulation of silenced genes remains to be fully deciphered. It is possible that this large protein complex can accommodate interactions with all three types of loci via different Nups. Furthermore, the stable proteins of the NPC have been shown to be remarkably long-lived. Once assembled, the NPC core essentially does not turn over during the entire life span of post-mitotic cells, such as neurons  receptor signaling to maintain cyst stem cells and support differentiation of germ stem cells . Tissue specific expression of Lamin isotypes appears to be important for tethering heterochromatin to the nuclear periphery and for repositioning critical developmental genes to a silencing nuclear compartment . This role of nuclear lamina is closely linked to and likely executed through the functions of INM proteins, which have the ability to interact with chromatin bound regulators and histone modifying complexes. Expression of NETs has been shown to be highly cell type specific and likely drives the reorganization of chromosomes and large genomic regions needed for certain paths of differentiation . Finally, components of the NPC are functionally implicated in regulation of developmentally induced active genes and in setting up boundaries between chromatin domains . The composition of the NPC may vary depending on the cell type, with some Nups such as Nup210 being added to drive critical differentiation steps. Developmentally regulated genes and boundaries may thus be recruited to the NPC in a tissue specific manner, or recruit Nups to their intranuclear locations. An exciting new direction stemming from these models is how developmental signaling factors that enter the nucleus during differentiation of particular lineages may cofunction with NE proteins and influence their genomic binding.Accumulating evidence has demonstrated tissue specific presence and functions of various NE components. Much of that knowledge supports the model that many of these functions are carried out via cell type specific interactions between the NE and the genome, which contribute to the correct establishment of tissue specific gene expression (Together the presented data also illustrate the interconnected roles of nuclear compartments essential for cell fate determination, from the earliest steps of chromatin structure rearrangement to the last stages of morphological and other changes. Perhaps a more accurate view of the NE-genome interplay involves a myriad of overlapping mechanisms with increasing specificity during differentiation. The NE composition may be another “cellular code” for specifying tissue specific gene expression programs through its contacts with the underlying chromatin. Similarly to other highly complex regulatory networks, future applications of the systems biology view of this “code” may be particularly beneficial for fully understanding the role of the NE in genome function and cell fate.


Escherichia coli generates positive supercoiling in the DNA, which is relieved by the enzymatic activity of gyrase. Recently published experimental evidence suggests that transcription initiation and elongation are inhibited by the buildup of positive supercoiling. It has therefore been proposed that intermittent binding of gyrase plays a role in transcriptional bursting. Considering that transcription is one of the most fundamental cellular processes, it is desirable to be able to account for the buildup and release of positive supercoiling in models of transcription.Transcription in Here we present a detailed biophysical model of gene expression that incorporates the effects of supercoiling due to transcription. By directly linking the amount of positive supercoiling to the rate of transcription, the model predicts that highly transcribed genes’ mRNA distributions should substantially deviate from Poisson distributions, with enhanced density at low mRNA copy numbers. Additionally, the model predicts a high degree of correlation between expression levels of genes inside the same supercoiling domain.Our model, incorporating the supercoiling state of the gene, makes specific predictions that differ from previous models of gene expression. Genes in the same supercoiling domain influence the expression level of neighboring genes. Such structurally dependent regulation predicts correlations between genes in the same supercoiling domain. The topology of the chromosome therefore creates a higher level of gene regulation, which has broad implications for understanding the evolution and organization of bacterial genomes.The online version of this article (doi:10.1186/s13628-016-0027-0) contains supplementary material, which is available to authorized users. The dynamics of gene expression in single cells has been studied extensively in the last 15 years, yielding new insights into the processes of transcription and translation . PopulatOne repeated theme when studying single cell gene expression is the occurrence of bursts during the production of mRNA and/or proteins. In particular, the production of mRNA has been shown to deviate from a simple birth-death process, instead occurring in transcriptional bursts , 17. TraE. coli [E. coli there are two major factors when it comes to relieving supercoiling generated by transcription, topoisomerase I (Topo I) and gyrase. Topo I relieves negative supercoiling while gyrase relieves positive supercoiling. In E. coli Topo I has a higher activity than gyrase, as negative supercoiling can be very detrimental to the organism. This imbalance causes positive supercoiling to accumulate until gyrase binds and relieves the positive supercoiling [Recently, Chong et al. provided evidence supporting one contributing mechanism for transcriptional bursting in E. coli . As RNA E. coli . In the rcoiling . The resMany models of gene expression have been proposed and studied, e.g. see –25. HoweHere, we first develop a biophysical model to quantify the effect of supercoiling density on the transcription initiation rate. Then, using a simplified version of this model, we create a kinetic model of gene expression that directly accounts for the accumulation of supercoiling during transcription. When supercoiling is accounted for, our model predicts a change in the shape of mRNA distributions for genes with strong promoters, with an increase in low non-zero mRNA copy numbers. We then investigate the effect of having multiple genes in the same supercoiling domain and find a correlation in the expression of these genes. Having multiple genes in the same supercoiling domain also results in each gene’s expression influencing the expression of other genes in the same domain. These results not only provide insight as to how genes are expressed and regulated in bacteria, but also provides new directions for experimentally testing for the effects of domain coupled transcriptional bursting.E. coli RNAP. Their experiment monitored the production of mRNA from 160 individual molecules using an RNA-specific fluorescent dye. In the absence of gyrase, positive supercoiling accumulated, resulting in a decrease in the initiation rate of transcription. In their experiments, this manifested as a decrease in the fluorescence intensity with time as mRNA transcripts were being produced less frequently  and kf is the rate at which the RNAP transitions to the stable elongation complex. These two rates are considered to be irreversible because of the small reverse rates [kcat) is the rate limiting step [Vmax and kM differ.In order to study why the initiation rate decreases in this manner we use the following kinetic model: se rates , 29. Usiing step . In thosConsidering the substrate copy number, specific promoter DNA, is much lower than the RNAP in a cell, the production rate of mRNA can then be approximated as: koff rate. This assumption is supported by experimental evidence that the binding affinity of T7 RNAP for the single stranded promoter sequence is greater and it dissociates slower with a single stranded promoter sequence [The question of interest is how does supercoiling affect these different rates? It has been shown on linear pieces of DNA that the melting of the DNA in the promoter is a minor kinetic barrier . Howeversequence , 34.kcat. In the SI we repeat the below derivation for the kcat rate and show that one obtains the same final result Additional file E. coli for which positive supercoiling increases the transcription rate cast some doubt on such a simplifying assumption. However, additional experimental results would be needed to construct a more detailed model that differentiates how different RNAP states are influenced by supercoiling.We do not rule out the possibility that the stability of the DNA could also influence the other kinetic rates in the model and the rates involved could differ depending on the particular RNAP. For example, considering that supercoiling of the DNA influences the amount of energy needed to melt the DNA, it also seems plausible that the supercoiling could affect the rate nj junctions and a certain density of supercoiling, σ is: In the following, we make the assumption that the change in the free energy of the transition state for unbinding directly depends on the energy needed to melt the DNA of the promoter. In order to provide energetic insights as to how supercoiling would affect the DNA we utilized the statistical mechanical model of supercoiled DNA developed by Sen et al. , 36, whiΔS=.024 kcal/(K mol) is the conformational entropy due to melting a base pair. ε=7.9 kcal/mol and εo=2.5 kcal/mol are the base pairing and base stacking energies, respectively. The function g is the degeneracy factor with N total base pairs. α=23.4 depend upon the bending and the torsional stiffness, respectively [A=10.4 base pairs per rad2 for DNA in the relaxed state, see SI for derivation of supercoiling energy Gs. We use the same parameters as obtained in [Here, ectively , and A=1ained in .Starting with n melted base pairs in a circular DNA loop of N base pairs the probability of having k melted base pairs in the promoter, consisting of Np base pairs, would follow a binomial distribution with a probability of successes equal to n/N. This is only valid when a lower fraction of the DNA is melted n/N <0.06, otherwise more than one melted base pair will be inside the same melted junction . Here weσ is: Then the change in the free energy barrier of the transition to a melted promoter at a certain The main effect the supercoiling density has is to alter the probabilities of having a certain amount of DNA melted, which influences the probability of having a melted base pair inside of the promoter region and the amount of energy needed to melt the DNA in the promoter region. Taking the difference of the transition state relative to the transition state at no supercoiling we obtain: ΔΔG(σ), shown in Fig. ΔΔG(σ) according to: k(0) is the initial rate with no supercoiling density. Numerical values for k(σ) are shown in Fig. Using the parameters obtained in , with N k(σ) as a simple exponential, demonstrated by the fit to the data generated by the model in Fig. k(σ)=ewσ−. This is only true if ΔΔG(σ) is linear with σ, which has been shown in numerous papers dating back to 1975 [koff, where the negative in the exponent disappears because the open complex is trying to maintain the melted DNA, we obtain the following equation for the production rate of mRNA: In order to simplify further we approximate  to 1975 . Pluggink(o)off is the initial off rate with no supercoiling, w is a constant and k′=k(o)off/kcat. A fit to the experimental data from [where ata from  with thew. Though we do not explicitly use the energetic parameters in the remainder of the paper, the full form of Eq. By simplifying using an exponential we have combined the various energetic parameters that describe the biophysical characteristics of the system from the model into a single parameter σ<<1 for α>>1 [σ: Equation for α>>1 . We thenThis linear equation’s parameters have intuitive biological meaning: the y-intercept of the linear fit would then correspond to the production rate at zero supercoiling and the slope would determine how many times the loop of DNA could be transcribed before stalling.Figure The accumulation of supercoiling due to transcription is primarily based on the “twin-supercoiled-domain model” of transcription . PositivTo study the effect of supercoiling on mRNA and protein distributions, we combined our biophysical model, which describes the dependence of transcription initiation on the supercoiling state of the local DNA domain, with a kinetic model of gene expression. We based our kinetic model on a simple burst model of gene expression . Burst mRCoil and PCoil that track the amount of “regular”, the normal state, and positive supercoiling, respectively, inside the local domain. The sum of these two species is fixed and is denoted by max(RCoil). Production of a transcript converts one RCoil into a PCoil. Here, we assume an implicit fast relaxation of the corresponding negative supercoiling by Topo 1 [PCoil linearly decreases the transcription rate of the DNA supercoiling domain according to reaction (1) in Table max(RCoil) is therefore equal to the number of times the DNA domain can be transcribed before transcription stalls.Here we present a modified gene expression model such that the transcription rate is linearly dependent on the amount of positive supercoiling that has accumulated in the local DNA domain, in accordance with our simplified biophysical model of transcription initiation. We first define two additional species y Topo 1 . Though,Gyrase or bound with a gyrase molecule Gyrase′. We assume a constant pool of free gyrase such that binding is pseudo first order. Gyrase unbinding follows first order kinetics. When gyrase is bound PCoil is converted to Rcoil with a rate constant R that is fast relative to the other rates in the system, such that when gyrase is bound the local domain is effectively always in the “regular” state. The complete model is shown in Table To model the relaxation of the positive supercoiling we introduce a gyrase binding site in the local DNA domain as an additional species that can either be empty ao×max(RCoil)=0.0032 sec−1. The x-intercept corresponds to the number of transcription events that can take place before stalling, max(RCoil)=13. All other rates and species were set to zero. We ran 160 simulations with a single gene in the supercoiling domain. The results of the simulations show good agreement with the original data despite the numerous approximations made in the derivation, shown in Fig. To test our kinetic model, we simulated the in vitro experiment conducted by Chong et al.  by extraγ=50,bo=2,d=1,K1=10,K2=35,R=1000,max(RCoil)=4, except when specified in different specific situations.In order to take into consideration the stochastic nature of the reactions in Table γ, was chosen so that the lifetime of the molecule would be short, ∼2 min. The value of max(RCoil) was set to 4, as opposed to using the fit value from the single-molecule in vitro experimental data shown earlier for the T7 RNAP. In E. coli it has been previously suggested that 4 rounds of complete transcription in a supercoiling domain could result in inhibiting transcription, due to the environment of the cell [ao, and translation rate, bo, can take on a large range of values, we simply choose values that produced means that are physiologically relevant [The mRNA degradation rate, the cell . The ratThe range of rates for gyrase in the cell is a matter of debate and likely depends on many different factors. For instance, in vitro the dissociation constant has been measured to range from 0.2–0.5 nM for specific gyrase binding sites to 100 nM for weaker binding sites , 48, 49.ao= 100 and ao= 20, respectively. In the absence of supercoiling, these two promoter rates would result in Poisson distributions with a mean mRNA copy number of 8 for the strong and 0.4 for the weak promoter. To calculate the different distributions of mRNA generated by our supercoiling model, we simulated the equivalent of 4000 cells using the parameters specified above. Here, we ignore the protein part of the distribution, although we keep all rates relative to a protein degradation rate of 1.The probability distribution of the number of mRNA molecules per cell is an important quantity in gene regulation. Several previous studies have utilized smFISH to quantify the mRNA distributions of various genes , 18, 20.Figure ao×max(Rcoil), and γ can also be seen in Fig. Chong et al.  also devWhen the promoter strength of the gene is low, the distribution from our model shows only minor deviations from a Poisson distribution, with a slight enhancement at zero copy number Fig. b. The cThe degree to which a distribution corresponds to a Poissonian can be quantified by the Fano factor, the variance divided by the mean, which is always one for a Poisson distribution. To study the influence of promoter strength and gyrase binding rate on the deviation of our mRNA distributions from Poissonian, we performed simulations across a wide range of these two parameters. Figure E. coli genes could help to disentangle the various contributions.The increase in Fano factor with mean mRNA copy number is typically ascribed to extrinsic noise. However, extrinsic noise shifts the Fano factor of highly expressed genes’ mRNA by broadening the distribution around the mean whereas in our model the Fano factor increases due to the expansion of the distribution toward higher mRNA copy numbers, while maintaining a significant density at zero and low copy numbers. Thus, the Fano factor by itself cannot be used to distinguish between genes with high variability from extrinsic noise or supercoiling, full distributions are necessary. Measured smFISH distributions likely contain noise contributions from both of these and other sources of noise and obtaining distributions from a large variety of native γ>>1), as is typically the case in bacteria, the discrete negative binomial distribution can be approximated by the gamma distribution [Γ is the gamma function. The rates used in our model would correspond to the gamma distribution with a=ao·max(RCoil)/d and b=bo·γ/γ=bo.We next sought to compare the protein distributions produced by our supercoiling model against those produced by a typical model used for studying stochastic gene expression. The standard burst model of gene expression, shown schematically in Fig. ribution , 24: \doa, the burst frequency, and b, the burst size. We wanted to study how well these two parameters could be estimated using a gamma distribution if the underlying data were generated by our kinetic model, which accounts for supercoiling induced transcriptional bursting.Fitting protein abundance data to a gamma distribution gives an estimate for two key parameters from the model: ao=90, max(RCoil)=4, b=2, γ=50, K1=10, and K2=35. The stationary probability distribution for the protein is shown in Fig. E. coli protein distribution data. We then fit our simulation data to a gamma distribution. Although the distribution appears to be well-described by a gamma distribution, the a and b parameters from the fit no longer correspond to the model parameters. The estimated a value was 13.2 and the estimated b value was 15.1, which are each approximately an order of magnitude from the correct values.We generated simulation data from our supercoiling model using the parameters K1 is high and K2 is low, our model converges to the burst model. Likewise, when the initiation rate is comparable to the gyrase binding rate, the two models converge. To determine the parameter regions for which the two models give similar results, we performed a parameter scan of K1 and ao and compared the model parameters versus the gamma fit parameters. Figure a, which is to be expected as a is an effective burst frequency while a0 is the basal transcription initiation rate. We also saw a consistent overestimation of b.In a case where the gyrase binding site is predominantly occupied, i.e. when E. coli are thought to be loops roughly 10 kb in size [The supercoiling domains in  in size . The buimax(Rcoil) to be 20 for each to allow them to be fully transcribed on average four times before stalling. Each of the two domains has a single and independent gyrase binding site, based on evidence that there is roughly one gyrase molecule for each loop in E. coli and gyrase is thought to have relatively specific binding sites [To study any correlations introduced into gene expression by this coupling, we modified our model to include multiple genes in a supercoiling domain. When a series of genes are present in same topological domain we refer to this as a linked domain. Promoters for all of the genes in a linked domain share the same supercoiling state, RCoil. To analyze expression correlations within a linked domain and to control for background correlations between independent genes, we constructed a system containing two independent supercoiling domains, each with five genes. We maintain the assumption that each DNA loop can be transcribed a total of four times before stalling, as discussed above. Since there are now five genes in each linked domain, we set ng sites .ao values for the genes from 1 to 10 are 34, 20, 14, 10, 6, 2, 3, 2.4, 0.4, 1.0. We recorded the expression level of each gene over the course of the simulation. Figure We simulated a system in which the first linked domain contained genes with promoters ranging from strong to weak (genes 1−5) and the second linked domain contained genes with only weak promoters genes (6−10). The Next, we calculated the pairwise correlations between mRNA and proteins for all of the genes, ao, of a gene inside a linked domain would influence the expression level of other genes inside the domain. We performed simulations of a single linked domain containing five genes with decreasing promoter strength. The expression level of each gene in the linked domain was analyzed before and after the inhibition of gene 1, see Fig. Finally, we investigated how regulating the promoter strength, Our model linking supercoiling to transcription produces mRNA distributions that are noticeably different than those predicted by the burst and two-state models, as shown in Fig. E. coli genes contain contributions from both supercoiling inhibited transcription and extrinsic sources of noise.It has been shown that the Fano factor of the mRNA distributions is greater than one for highly transcribed genes , 14, 18.a and b values deviated greatly from the predicted values of the burst model even though the distribution could be fit by a gamma distribution. If the fit to a gamma distribution is justified, the a value will correspond to the number of mRNA produced in a proteins lifetime and the b value will correspond to the number of proteins produced per mRNA transcript. Our model shows that when supercoiling is introduced the a and b values determined from the fit do not correctly represent the underlying processes of the system. Therefore, studies that rely on a physical interpretation of a and b may need to be adjusted.When analyzing the protein distributions produced by our model, we found that the Our results do not necessarily mean it is impossible to extract the underlying biological parameters from the distributions of mRNA and protein. Though we consider it to be beyond the scope of this paper, if the gyrase binding constants could be varied in a controlled manner and the distribution of mRNA could be obtained at different gyrase binding rates, then the parameters in our model could potentially be found through fitting. Even though there is no unique analytical formula for the distribution produced by our model, the parameters could be found by fitting to numerical solutions. Additionally, other known sources of noise, such as extrinsic noise, would also have to be incorporated into the model , 52. ThiE. coli were shown to be correlated and dependent on supercoiling. The authors concluded that expression levels were directly linked to the distribution of gyrase on the chromosome. Our model predicts this effect; gyrase has differing binding affinities for different supercoiling domains, which effectively controls the overall expression level of each gene in the same supercoiling domain. Other authors have reported that expression of an inducible reporter gene represses downstream neighboring genes [Correlation in the transcription of genes in bacteria has been previously reported. In , the expng genes . Our modThough correlation in the overall expression level of genes has been observed, our model predicts an additional degree of correlation, namely correlation in the transcriptional bursts of all genes in a supercoiling domain. Our model gives rise to correlation of not only protein abundance but also mRNA abundance of neighboring genes in a supercoiling domain, such as shown in Fig. E. coli.The correlation of clustered genes has been probed experimentally and clustering was not found to have a significant impact on the expression levels . The autNegative feedback has been proposed to be an important factor in controlling the stochastic nature of the biochemical reactions that take place in gene networks , 60. GivHaving a built-in regulation network inside of the cell would help ensure the proper expression of certain genes without requiring extra energy for the production of transcription regulation factors specific for different genes. In this way, the expression level of genes in the same supercoiling domain would act as an intrinsic regulatory mechanism, providing yet another reason why the relative ordering of the chromosome is important. Such an effect would have implications in understanding the origin and widespread evolutionary conservation of operonal genome structure in Bacteria and Archaea.https://zenodo.org/record/45167, https://zenodo.org/record/45168, https://zenodo.org/record/45178, https://zenodo.org/record/45196, https://zenodo.org/record/45198.The data supporting the conclusions of this article are publicly available with zenodo, which can be accessed through the following:


Over the last 30 years, studies of aberrant DNA methylation in hematologic malignancies have been dominated by the primary focus of understanding promoter hypermethylation. These efforts not only resulted in a better understanding of the basis of epigenetic silencing of tumor suppressor genes but also resulted in approval of hypomethylating agents for the treatment of several malignancies, such as myelodysplastic syndrome and acute myeloid leukemia. Recent advances in global methylation profiling coupled with the use of mouse models suggest that aberrant promoter hypomethylation is also a frequent event in hematologic malignancies, particularly in chronic lymphocytic leukemia (CLL). Promoter hypomethylation affects gene expression and, therefore, may play an important role in disease pathogenesis. Here, we review recent findings and discuss the potential involvement of aberrant promoter hypomethylation in CLL. Cytosine methylation of DNA is an epigenetic modification affecting gene transcription and the integrity of the mammalian genome. The three catalytically active DNA methyltransferases in mammalian cells are DNMT1, DNMT3A, and DNMT3B. These enzymes are responsible for establishment and maintenance of DNA methylation during normal development and during mitotic cell division. Promoter methylation typically results in transcriptional repression of genes and plays a role in various normal physiologic processes, such as differentiation and hematopoiesis .One of the main observations that contributed to an interest in studying this phenomenon came from studies that discovered that in virtually all types of cancer aberrantly increased methylation in gene promoters was associated with transcriptional inhibition . As a reThe recent emergence of new high-resolution methylation profiling techniques, such as whole-genome bisulfite sequencing (WGBS), have revealed that the methylome of cancer cells frequently contains promoters that are hypomethylated relative to their normal cellular counterparts. Such aberrant hypomethylation is frequently accompanied by increased gene expression at differentially methylated loci. Whether or not such deregulated expression contributes to the initiation and progression of hematologic malignancies is currently unresolved and is actively under investigation.Here, we will first review data obtained in mouse studies focusing on DNA methyltransferase loss of function in various hematologic malignancies. We will then discuss recent findings that strongly support the idea that aberrant promoter hypomethylation accompanied by gene re-expression may contribute to the development of chronic lymphocytic leukemia (CLL) in a causative manner.de novo and maintenance. Early studies suggested that Dnmt3a and Dnmt3b are de novo enzymes capable of recognizing unmethylated DNA and catalyze the addition of a methyl group to cytosine  results unexpectedly in the development of a CLL-like disease after 1 year’s time. This finding was surprising in view of a lack of genetic alterations in the DNMT3A locus in B-cell malignancies. Since this time, other groups have reported that Dnmt3a loss in HSPCs results in myriad types of malignancies, such as myeloproliferative disorders, AML, T-cell acute lymphoblastic leukemia (T-ALL), and B-cell acute lymphoblastic leukemia (B-ALL)  , 10. DifD814V to induce B-ALL, T-ALL, and mastocytosis with myeloid blasts (G12DKras/+ to promote progression of juvenile and chronic myelomonocytic leukemia (CMML)  . Howeverhylation  and a mehylation . DespiteDnmt3aΔ/Δ mice and also promoted the development of T-cell lymphomas  almost exclusively (91%) develop T-cell lymphomas  is an indolent low-grade lymphoproliferation of mature B-cells  and is tAltogether, these data provide evidence for extensive promoter hypomethylation in human CLL that may be involved in the pathogenesis of the disease.Eμ-TCL1 is one of the first mouse models of CLL that was generated by using overexpression of the TCL1 gene in mouse B cells . Interestingly, these genes are among the top 25 most overexpressed genes in the Eμ-TCL1 mouse model of CLL (Eμ-TCL1 model has not yet been carefully evaluated, but these genes have been shown to be aberrantly hypomethylated in Dnmt3aΔ/Δ CLL. The functions of these genes in CLL development have not been evaluated to date. Therefore, we discuss here data for one protein coding gene – Zbtb32 – and one gene encoding a non-coding RNA – Pvt1– that support their potential role in CLL.A limited analysis of gene expression data revealed several genes that are upregulated in both l of CLL . MethylaZbtb32 was identified as a gene whose increased expression predicts whether healthy individuals without disease will develop CLL later in life (Zbtb32 is a Zinc Finger and BTB Domain Containing 32 gene that was originally identified as a transcriptional repressor protein that interacts with several proteins including the Fanconi anemia group C protein and PLZF . Recentl in life . This raPvt1 gene locus is a target of frequent tumorigenic translocations and retroviral insertions that result in overexpression of a long non-coding RNA that encodes several microRNAs that are overexpressed in hematological malignancies of B- and T-cell origin. For example, miR-1206 is overexpressed in human BLs, whereas miR-1204 is overexpressed in retrovirally induced T-cell lymphomas (The ymphomas –45. ThusGiven the large number of genes identified to be hypomethylated and overexpressed in mouse and human CLL, there is a clear potential that some of these genes are oncogenic drivers of CLL development. While convincing evidence for involvement of such genes in the pathogenesis of the disease is quite limited, a large body of data accumulated in recent years clearly points to such a possibility. A conceptual justification for such efforts is not limited to just an understanding of basic mechanisms governing cellular transformation but might also have direct implication for targeted therapies. Frequently, oncogenes that initiate tumor development are also important for maintenance of tumor phenotype. Therefore, their targeting may prove to be useful in anti-neoplastic therapy. Consequently, major efforts will likely be undertaken in a future to identify proto-oncogenes silenced in normal cells by promoter methylation that become de-methylated during tumor initiation/progression and functionally contributing to cellular transformation. Such efforts will likely not be limited to CLL only but will include other types of hematologic malignancies and potentially solid tumors.GU and RO discussed, designed, and conceptualized the review article. GU, RO, and SH wrote, edited, and revised the review article. Primary literature review was performed by GU and RO. SH and GU constructed Table 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. The reviewer BW and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Transcription elongation is a critical regulatory step in the gene expression cycle. One key regulator of the switch between transcription initiation and elongation is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) and several negative elongation factors to relieve the elongation block at paused promoters to facilitate productive elongation. Here, we highlight recent findings signifying the role of the PPM1G/PP2Cγ phosphatase in activating and maintaining the active transcription elongation state by regulating the availability of P-TEFb and blocking its assembly into the catalytic inactive 7SK small nuclear ribonucleoprotein (snRNP) complex. It has, more recently, became well-established in the field that transcription elongation control is vital for the regulation of several key biological processes including response to environmental cues, cell fate choice, differentiation, and development. Transcription elongation is precisely regulated by the action of two sets of elongation factors (negative and positive) that dictate transcriptional pausing and pause release, respectively. Sequence-specific transcription factors utilize co-regulators (co-activators and co-repressors), large protein complexes carrying enzymatic activities, to precisely modulate one or more steps in the transcription cycle. Great progress has been made in identifying several transcriptional co-regulators and in describing their critical importance for Pol II transcription such as acetyl transferases, deacetylases, kinases and phosphatases, among others . Here, we discuss the discovery of a novel transcriptional co-regulator, mechanistic insights into its role in promoting transcription elongation and future challenges.Studies over the past three decades have provided insights into the importance of transcription regulatory mechanisms in metazoan cells. RNA polymerase II (Pol II) activity can be regulated at several steps of the transcription cycle including pre-initiation, DNA melting, initiation, promoter clearance, and elongation –12. Notably, Pol II pausing is prevalent at genes involved in development and response to stimuli, suggesting that transcriptional pausing during early elongation plays important, variable, and regulated rate-limiting roles in the rapid and precise control of gene expression ,14.Promoter-proximal Pol II pausing is a key rate-limiting and highly regulated step in the transcription cycle. Genome-wide analysis in metazoan cells showed that the major form of Pol II is bound to promoter-proximal regions and poised for rapid entry into productive elongation . Interestingly, levels of DSIF and NELF at promoter-proximal regions directly correlate with total Pol II occupancy levels, suggesting that these factors associate with most early elongation complexes , and at most Pol II regulated genes thus implying a general role in controlling pausing , 10, 12.After transcription initiation and promoter clearance due to the action of the TFIIH kinase, Pol II pauses between the transcription start site (TSS) and the +1 nucleosome after transcribing a short (~20–60 nt) RNA chain by the action of negative elongation factors . There aSaccharomyces cerevisiae Spt5/Spt4 factors. DSIF was first identified by Wada et al. using HeLa cell nuclear extracts for factors that cause pausing of Pol II in conjunction with the transcription inhibitor DRB –18. In addition to its negative function in elongation, Wada et al. found that DSIF associated with Pol II and stimulated the rate of elongation by Pol II in vitro in a reaction containing limiting nucleotides , 19.DSIF is composed of two subunits (p160 and p14), which are the human homologues of et al. described the identification and purification from HeLa nuclear extracts of a factor required for DRB-sensitive transcription and they referred to it as NELF . NELF is composed of five polypeptides , the smallest (NELF-E) of which is identical to the RNA-binding protein RD, and whose RNA-binding activity is critical for NELF function as negative elongation factor . Upon NELF recruitment to Pol II at promoter-proximal regions, NELF inhibits Pol II activity by binding the nascent RNA chain and preventing further elongation ,20.Similarly, using an elegant biochemical approach, Yamaguchi –23 (. Both DSIF and NELF require a nascent RNA chain (>18 nt-long) to stably associate with the Pol II elongation machinery , in agreement with a lack of evidence for a role of DSIF/NELF in initiation and promoter clearance .While DSIF binds Pol II directly and stably without affecting its polymerase activity, NELF does not bind Pol II directly and is recruited to early elongation complexes through DSIF. Although the details of the interaction/recruitment mechanism are still unclear, one possibility is that the negative elongation function of DSIF/NELF could be related to their ability to bind the nascent RNA. Particularly, NELF-E and Spt5 are both known to interact with the short RNA transcript emerging from Pol II after transcription initiation and thus inhibit productive elongation –23 . Crossliet al. found that most genes marked by NELF in Drosophila also showed evidence of paused Pol II at promoter-proximal regions (+30 to +50 bp from the TSS) and proposed that NELF-mediated Pol II pausing might be an obligatory but sometimes transient checkpoint during the transcription cycle . Similarly, Rahl et al. has shown that NELF and DSIF co-occupy most Pol II regulated promoter-proximal regions of transcriptionally active but paused genes in mouse embryonic stem cells .Using permanganate genomic footprinting, Lee Drosophila (like Hsp70) are up-regulated by NELF genetic depletion, the majority show decreased expression levels, suggesting that the presence of paused Pol II might enhance gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region. Additionally, the loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in the active chromatin signatures surrounding the TSS . In fact, in 2010 Gilchrist et al. provided compelling evidence for a key role of paused Pol II in competing with nucleosomes for occupancy of highly regulated promoters, thereby preventing the formation of repressive chromatin architecture to facilitate further or future gene activation .Although a subset of NELF target genes in Drosophila and mammals have supported the model that NELF associates only with the 5′ end of genes, while DSIF travels with elongating Pol II since it is detected throughout the transcribing unit , 24, 25. For example, at the Drosophila hsp70 locus, following heat shock stimulation, DSIF and Pol II but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. Thus, both NELF and DSIF cause Pol II to pause in the promoter-proximal region, and the transcriptional activator heat shock factor (HSF) might cause NELF to dissociate from the elongation complex, and DSIF to travel with elongating Pol II.Several genomic approaches in S. cerevisiae. NELF orthologues are conserved from Drosophila to humans, but absent in budding yeast , thus suggesting a more complex regulatory network as species evolved. In fact, phylogenetic analyses of components of the elongation control machinery indicate that the number of mechanisms utilized to regulate P-TEFb function increase as organisms show more complex developmental patterns .Compared to the regulation in metazoan cells, the evidence for Pol II pausing is lacking in . There are several excellent reviews discussing the roles of GDOWN1 and other regulators in transcription elongation control. In this review article, we focus on the newly discovered PPM1G/PP2Cγ phosphatase ,30 and its functional interplay with the early elongation machinery in the control of Pol II pausing and pause release.Several additional factors are known to control Pol II pausing. For example, GDOWN1 (a substoichiometric Pol II subunit shown previously to render it responsive to Mediator) blocks the general transcription factor TFIIF and stabilizes paused Pol II at promoter-proximal regions of most human genes , 31, 32. Cdk9 exists in two functional isoforms (a major 42 kDa form and a minor 55 kDa form) both of which associate with a cyclin subunit . P-TEFb phosphorylates the C-terminal domain (CTD) of the largest subunit of Pol II and negative elongation factors (DSIF and NELF) to promote the escape into productive elongation  , 34, 35.Promoter-proximal Pol II pausing is relieved by the action of positive transcription elongation factors. One of these key factors is the positive transcription elongation factor b (P-TEFb), a heterodimer of a cyclin subunit and cyclin-dependent kinase 9 (Cdk9). The regulatory cyclin (Cyc) subunit associated with Cdk9 is usually CycT1 or CycT2 and in some cases CycK . With difference to the Pol II CTD residues phosphorylated by P-TEFb (Ser2 and Ser5), phosphorylation of Thr4 on the C-terminal repeats of Spt5 promote the elongation activity of DSIF. This phosphorylation event converts DSIF from a repressor to an activator and is thought to function in a manner analogous to the phosphorylated CTD  . Despite this remarkable discovery, it remains unclear how Spt5 phosphorylation contribute to the elongation process. It is likely that it may interact with different factors (protein and RNA) at promoter-proximal regions to relieve repressive associations with NELF or other negative factors in the early elongation complex.P-TEFb phosphorylates the Spt5 subunit of DSIF on C-terminal repeats (consensus = G-S-R/Q-T-P) to promote processive transcription elongation , in vivo studies showed that Ser2 phosphorylation is more strongly affected upon Cdk9 inhibition , 37. However, Cdk9 inhibition with DRB and flavopiridol can target several kinases, albeit with different affinities.In addition to DSIF and NELF, the primary target of P-TEFb is the Pol II CTD, which is an unusual polypeptide extension appended to the largest subunit (Rpb1). The CTD is inherently disordered and serves as a flexible binding scaffold for numerous protein interactions throughout the transcription cycle. In metazoans, the CTD comprises from 25 to 52 tandem copies of the consensus heptad repeat YSPTSPS. The repeats are post-translationally modified in almost every residue to orchestrate the association of different sets of factors with the transcript and influence the functional organization of the nucleus. Although recent in vitro studies have suggested that P-TEFb primarily targets Ser5 and Ser7 residues –41.Despite the critical role of Cdk9 in the control of transcriptional pause release, other kinases  have been implicated in CTD phosphorylation and the control of Pol II pause release and RNA processing. With the goal of keeping this review focused on the interplay between Cdk9 and the novel kinase regulator (PPM1G) here we include references to fundamental articles discussing roles of other CTD kinases in transcription control Taken together, mechanisms regulating Pol II pause release are vast and primarily rely on direct control of P-TEFb, which can be recruited to chromatin through interactions with different factors and probably in different catalytic states (active or primed) to phosphorylate its substrates at target genomic sites., 42, 43. However, the most unique and well-studied mechanism of P-TEFb regulation is through the reversible association with the 7SK small nuclear ribonucleoprotein (snRNP) complex. In rapidly growing cells, a large fraction of P-TEFb (up to 90%) exists in a catalytically inactive form assembled into the 7SK snRNP , which is composed of the 7SK RNA, P-TEFb, the kinase inhibitor Hexim 1 and/or 2 (here referred as to Hexim for simplicity), the 5′-RNA methyl capping enzyme MePCE, and the 3′-RNA stability protein Larp7 –51  , 53, 54 ., 56. The distal part of stems I and IV contain all necessary elements required for P-TEFb recruitment . Binding to 7SK RNA causes a conformational change in Hexim that allows it to interact with the CycT1 subunit allowing it to recruit P-TEFb, thus assembling the 7SK snRNP complex and inhibiting Cdk9 kinase activity , 58–61. Systematic 7SK RNA mutagenesis showed that alterations in Hexim binding abolishes P-TEFb’s recruitment thereby indicating its absolute requirement in formation of the 7SK snRNP . Moreover, 7SK snRNP assembly requires phosphorylation of the Cdk9 T-loop (at residue T186), which promotes interactions with Hexim and 7SK RNA in vitro .The distal portion of 7SK stem I contains two structurally similar motifs, which function in the binding of Hexim1/2 homo- or heterodimers in vitro and in vivo . The CCR2 motif mediates Hexim homodimerization resulting in the recruitment of P-TEFb through the CCR1 region , 59. Although the molecular details for the protein-RNA interactions are well established, the lack of a high-resolution structure so far impedes the better understanding of the assembly and kinase inactivation processes.In Hexim, two coiled-coiled regions [CCR1 (279–315) and CCR2 (319–352) in the C-terminus] and a basic RNA-binding motif (KKKHRRRP) that contacts 7SK RNA in a P-TEFb independent manner are involved in assembling the 7SK snRNP complex to inactivate the kinase , 62–64, probably to deliver primed kinase for ‘on site’ activation . These discoveries were initially seen to be controversial in the field because of the low crosslinking efficiency of 7SK snRNP components compared to factors directly bound to chromatin. Particularly because, biochemically, 7SK snRNP appears not to be stably associated with chromatin and can freely diffuse in the nucleus, as demonstrated by its extraction using mild conditions  , 66. However, several studies by our group and others have extended those initial discoveries by providing compelling evidence that components of the 7SK snRNP complex are recruited to genomic domains including promoters and cellular enhancers to control transcription elongation , 65, 67. Thus, the presence of P-TEFb as part of the 7SK snRNP complex at gene promoters correlates with the degree to which Pol II pauses probably indicating a direct role in positively controlling transcription elongation , 63–65. In this context, the 7SK snRNP complex appears not to simply be an inactive reservoir of P-TEFb but plays a functional role in delivering primed kinase to promoter-proximal regions containing paused Pol II.Although the majority of the 7SK snRNP complex exists in the soluble nucleoplasmic fraction, previous studies have shown that the snRNP is recruited to promoter-proximal regions , 69. The first Ser/Thr phosphatases to be functionally linked to P-TEFb activity control through the 7SK snRNP were PP2B and PP1α , members of the PPP phosphoprotein family of phosphatases . Both enzymes were proposed to cooperatively disrupt the 7SK snRNP complex to release P-TEFb for transcription in response to Ca2+ signaling .In order to accommodate the transcriptional needs of the cell, the P-TEFb kinase has to be released from the snRNP and then captured by promoter-associated factors to act locally on its substrates (Pol II and negative elongation factors). Just as Cdk9 T-loop phosphorylation promotes 7SK snRNP complex assembly, dephosphorylation of the T-loop by phosphatases (PPases) induces P-TEFb dislodgement from Hexim thereby causing 7SK snRNP disassembly and transcriptional pause release . While s. PPM1A overexpression in cells induces dephosphorylation of the Cdk9 T-loop . However, the biological relevance of this regulatory step in the control of P-TEFb activity and Pol II transcriptional pause release, as well as the target genes regulated remains to be elucidated.Another phosphatase is PPM1A/PP2Cα, which is a member of a large family of metal-dependent phosphatases (PPM/PP2C) that participates in diverse biological processes et al. identified another PPM/PP2C family member (PPM1G/PP2Cγ) that dephosphorylates the T-loop of Cdk9 to promote the disassembly of the 7SK snRNP complex in vitro   . In this context, protein interactions causing the induced fit of HIV Tat on P-TEFb exemplifies that transcription factors can promote conformational changes on P-TEFb and regulate its kinase activity , 74. These are two possible mechanisms that describe how P-TEFb (released from the 7SK snRNP complex) can undergo rapid activation through T-loop rephosphorylation  see . Interesrylation .Together, this underscores the importance of 7SK snRNP’s recruitment to chromatin and its interplay with cellular phosphatases (like PPM1G) to facilitate Pol II pause release of target genes in response to environmental stimulation.. In response to DNA damage signaling, PPM1G occupancy levels at NF-κB target gene promoters rises in parallel to the increase in P-TEFb/7SK snRNP. At these sites, PPM1G dephosphorylates the T-loop of Cdk9, which uncouples it from the kinase inhibitor Hexim, thereby causing disassembly of the 7SK snRNP complex, and inducing P-TEFb activation and Pol II pause release ,30 .While low levels of PPM1G are detected at promoter-proximal regions of inducible or primary responsive genes (such as those in the NF-κB network), its occupancy robustly and temporally increases in response to DNA damage stimulation as revealed by kinetic ChIP assays ease ,30 . Interes, to interact with the distal region of 7SK RNA stem I. PPM1G binding is in close-proximity to the Hexim-binding site, and blocks the CCR1 site on Hexim (the site of P-TEFb interaction), which is all consistent with formation of Hexim-PPM1G-7SK RNA complexes , 57, 75. Together, this suggests that assembly of the PPM1G-Hexim protein complex on 7SK RNA functions to block P-TEFb binding .PPM1G is composed of three discrete domains: N-terminal, C-terminal phosphatase domain containing a Lysine-rich region (Lys), and an intervening acidic-rich region. PPM1G uses the Lys-rich region, which bears similarity to the Hexim RNA-binding motif , 77. It would be interesting to determine whether these factors function in an analogous manner to PPM1G or whether they cooperate with PPM1G and Hexim in preventing P-TEFb association into the 7SK snRNP complex to sustain transcription elongation.Several heterogeneous nuclear ribonucleoproteins (hnRNP) such as hnRNP A1, A2/B1, R and Q have been previously shown to associate with 7SK RNA, but only after Hexim and P-TEFb have dissociated from the RNA . Consistent with the role of the ataxia telangiectasia mutated (ATM) kinase in regulating DNA damage signaling, we found that ATM regulates the interaction between PPM1G and the 7SK snRNP (by enhancing 7SK RNA interaction) through PPM1G site-specific phosphorylation. Due to the critical role PPM1G plays in transcription and activation of intrinsic and extrinsic pathways it would be interesting to determine if cancer-related mutations in the phosphatase could act as drivers of malignancy or remain passenger genetic alterations. Furthermore, if activation of key cellular programs is required to elicit critical responses to a DNA insult then it is possible that PPM1G would function as tumor suppressor. Honing our understanding about the roles PPM1G plays as transcriptional co-activator and in preventing the onset of malignancy become critical points as we move forward in defining the functional interplay between this enzyme and the transcription elongation machinery.The critical role that PPM1G plays in the transcriptional cycle and in the control of cell fate choice indicates that its malfunction can promote malignancy. Given that DNA damage activates the NF-κB signaling pathway and that PPM1G is a transcriptional co-activator of NF-κB, its function is essential in maintaining cellular homeostasis. In response to DNA damage, PPM1G regulates NF-κB mediated 7SK RNA dependent Pol II transcription ), PPM1G also emerges as a critical target for diseases where NF-κB is constitutively activated .Since PPM1G participates in the activation of NF-κB programs and NF-κB is a critical tumor suppressor or oncogene  as part of a larger 7SK snRNP complex? Furthermore, although the molecular and structural details of the interaction between PPM1G and 7SK snRNP provided important insights in the control of transcription elongation, several key questions remain for the future. For example: Do posttranslational modifications keep the Hexim-PPM1G complex bound to the snRNP? And if yes, which enzymes regulate this process? How does PPM1G dissociate from Hexim and 7SK RNA to allow re-association of P-TEFb and inactivate Pol II pause release?, in vitro studies defining the stoichiometry and molecular nature of the interactions would be beneficial to understand the assembly mechanism and to pave the way for future functional studies. Finally, due to the critical role of negative elongation factors (NELF and DSIF) in the control of Pol II pausing/pause release, whether PPM1G enzymatically targets these and other elongation factors while preventing escape into productive elongation remains to be investigated. In sum, understanding the intricate functional interplay between PPM1G, the 7SK snRNP, and other transcription co-factors are future challenges for the field.Since PPM1G interacts with other snRNP components such as MePCE and Larp7


Xist non-coding RNA and gradually silenced. This imprinted X-inactivation principally results from the apposition, during oocyte growth, of an imprint on the X-inactivation master control region: the X-inactivation center (Xic). This maternal imprint of yet unknown nature is thought to prevent Xist upregulation from the maternal X (XM) during early female development. In order to provide further insight into the XM imprinting mechanism, we applied single-cell approaches to oocytes and pre-implantation embryos at different stages of development to analyze the expression of candidate genes within the Xic. We show that, unlike the situation pertaining in most other cellular contexts, in early-growing oocytes, Xist and Tsix sense and antisense transcription occur simultaneously from the same chromosome. Additionally, during early development, Xist appears to be transiently transcribed from the XM in some blastomeres of late 2-cell embryos concomitant with the general activation of the genome indicating that XM imprinting does not completely suppress maternal Xist transcription during embryo cleavage stages. These unexpected transcriptional regulations of the Xist locus call for a re-evaluation of the early functioning of the maternal imprint on the X-chromosome and suggest that Xist/Tsix antagonist transcriptional activities may participate in imprinting the maternal locus as described at other loci subject to parental imprinting.During the first divisions of the female mouse embryo, the paternal X-chromosome is coated by  P), which characterizes extra-embryonic tissues, as opposed to embryonic and adult tissues, which display a random inactivation of the XP or of the maternal X (XM), leading to tissues that are mosaic for the expression of X-linked genes. Imprinted X-chromosome inactivation (I-XCI) is first established at the 4-cell stage and manifests itself as overexpression of the Xist gene from the XP.Cis-coating of the XP by Xist ncRNA, recruitment of chromatin remodelers, and heterochromatinization of the chromosome subsequently maintain the silent state of the inactive X in extra-embryonic derivatives of both the placenta and the yolk sacP is reactivated in the embryonic lineage to allow the establishment of random X-inactivation in the epiblast of the late blastocyst.The paternal and maternal genomes are not fully equivalent. In mammals, differences include the presence of parent-of-origin specific marks or “imprints,” which lead to monoallelic expression of either the maternally-inherited or paternally-inherited alleles of imprinted genes in embryonic and/or adult tissues. In female mice, an extreme example of imprinting is represented by the inactivation of the paternal X chromosome  or XMXMXP and XMXMY embryos, which show a delay in the establishment of XCI during pre-implantation development in the first case,Xic) and includes—among others—the Xist gene,cis-repressor, the Tsix antisense ncRNA,Ftx and Jpx,Xist expressionThe nature of the imprint(s) on the XXist and Tsix result in abnormal placental development associated with aberrant, parent-of-origin specific I-XCI profiles, which are consistent with the repression of Xist by Tsix on the XM of extra-embryonic tissues.Xist allele during pre-implantation development is likely independent of Tsix, since this antisense transcript is not transcribed during embryo cleavage stages.Xist is the ubiquitin ligase RLIM (encoded by the X-linked Rnf12/Rlim gene), as suggested by the lack of Xist coating in female embryos lacking RLIM.M imprint is established in the female germline and protects the maternal Xist allele from subsequent activation by RLIM at the time of zygotic gene activation.Mutations of both During early post-implantation development (E7.25), female germ cells originating from the epiblast proliferate and migrate to the genital ridge. Around E10.5, the primordial germ cells colonizing the gonads are subject to global DNA demethylation and extensive histone modifications associated with global genome resettingM imprinting to the oocyte growth and final maturation stages.Xic up until now. Additionally, mutant embryos derived from oocytes that have been depleted either for the maintenance DNA methyltransferase DNMT1 or for the de novo DNA methyltransferases DNMT3A and DNMT3B during the oocyte growth develop normally at least until the blastocyst stage without any noticeable defects in the establishment of I-XCI.M-imprint is unlikely to involve DNA methylation during cleavage stages. An alternative appealing hypothesis proposes that transcription itself participates in the XM-imprinting mechanism through the creation of a specific chromatin environment, which could allow imprint apposition. Such a mechanism has been described at the Gnas or at the PWS/AS (Prader-Willi/Angelman syndromes) imprinted loci, where transcription through DMRs during oogenesis is thought to promote the formation of an open chromatin structure allowing the imprinting complex access to DMRs.Experiments exploiting nuclear transfers of GV from early oocytes into enucleated fully-grown oocytes have restricted the time window of XXic genes and of X-linked genes in oocytes at different stages of folliculogenesis. Unexpectedly, all the Xic genes that we tested—including the Xist gene— appear heavily transcribed in early-growing oocytes. Strikingly, the activation of Xist takes place simultaneously with Tsix transcription, indicating that the antagonist relationship that exists between these 2 Xic actors in other cellular contexts cannot be operating in the growing oocyte. Maternal Xist, but not Tsix transcription, is then re-initiated, transiently, in late 2-cell embryos just before paternal Xist upregulation and XP coating. This furtive maternal transcription may represent a “leakage” of the maternal Xist promoter that is selectively suppressed at later developmental stages. Alternatively and more interestingly, this permissive maternal expression may represent a backup system that is activated in abnormal developmental contexts, for example in parthenogenote and gynogenote embryos, to restore X-inactivation from the early morula stage onwards.Following on the hypothesis of the involvement of transcription in the establishment of the maternal imprint on the X-chromosome, we have analyzed, using single-cell RT-qPCR and RNA-FISH approaches, the expression of candidate Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture to perform experiments on live mice , in appliance of the French and European regulations on care and protection of the Laboratory Animals .Protocols were approved by the veterinary staff of the Institut Pasteur animal facility and were performed in compliance with the NIH Animal Welfare Insurance #A5476-01 issued on 02/07/2007.in vitro.Early-growing and late-growing GV-intact oocytes were collected without any hormonal stimulation, from freshly isolated ovaries obtained from 12-day old 129.Tgn.GFPX4,Figure S1A and S1B. Pre-amplification was followed by exonuclease I treatment (NEB) and qPCR using nested allele-specific primers (when required) was performed on the BioMark thermocycler (Fluidigm). Raw efficiencies of each PCR assay were measured on control DNA or cDNA within each experiment. The allele-specificity of allelic primer pairs was controlled as previously described.Figure S1C. Transcript levels were extrapolated using the raw PCR efficiencies allowing the direct comparison of different genes. Cells that fail to show any amplification, that are inherent to single-cell RT-qPCR analysesGapdh, Rplp0, and Hist2h2a). Only cells showing a significant expression for 2 of these control genes are shown in the manuscript figures. Detailed analyses, statistical tests, and hierarchical clustering have been performed using the Qlucore Omics Explorer 2.3 (QLUCORE Company). As recommended for single cell gene expression analyses that are designed to reveal the variability of expression levels from cell to cell, no standardization with reporter genes has been applied. The variability of RNA levels from one cell to the other reflects the gene expression heterogeneity within the cell population.Single-cell gene expression analyses were performed as described in Guo et al.Figure S5A. Compared to the standard protocol of double-strand priming, a step of DNase I treatment was added and performed as recommended by the manufacturer . Forward or reverse primers (50 nM) each were used to prime the reverse transcription reaction.For single-cell strand-specific RT-qPCR, the general protocol and controls are described in Table S1 for primer sequences).Embryos sexing was performed using PCR assays specifically detecting repeated sequences on the Y chromosome . Allele-specific qPCR primers were controlled for specificity and efficiency as described in Xist/Tsix ; Tsix double-stranded probe (17E)Rlim ; Linx ; Kdm5c (RP24-148H21); Xic  labeled by nick translation ; Xist specific fluorescent oligonucleotides ; Tsix specific fluorescent oligos  together with mouse Cot-1 DNA (Invitrogen) and sonicated salmon sperm DNA (Invitrogen). Slides were washed 2× in 50% Formamide/2XSSC (pH7.4) at 37°C for 5 min, 1× in 2XSSC at 37°C for 5min and mounted in Vectashield + DAPI (Vector Laboratories). The efficiency and specificity of the Xist and Tsix fluorescent oligos were measured through hybridization of differentiated ES cells, in which the Xist nuclear domain was easily detected by the single-stranded probe. We noted, however, that the Xist signal appeared weaker than when using a larger, double-stranded probe. This may induce a slight underestimation of the number of Xist transcribing loci. For sequential DNA-FISH procedure, slides were treated with RNaseH (10 U/ml NEB) for 30 min at 37°C, washed in 2XSSC, denatured in 70% Formamide/2XSSC (pH 7.4) for 1 min 30 sec at 75°C, dipped in ice-cold 2XSSC and hybridized overnight at 42°C with a probe mixture denatured for 10 min at 75°C and containing, approximately, 100 ng of each spectrum-labeled fosmid probe together with 3 μg of mouse Cot-1 DNA and 10 μg of sonicated salmon sperm DNA per 22×22 mm coverslip. Washing and mounting steps were as described for the RNA-FISH procedure. Z-stacks were captured (step = 0.2 μm) on a Zeiss Axioplan2 microscope equipped with a Hamamatsu orca-ER CCD camera and controlled by the Perkin Elmer acquisition software Volocity.The procedure was adapted from.Drosophila larvae carrier RNA and poly(A)+ RNAs were separated from poly(A)- RNAs on oligo(dT) Dynabeads (Invitrogen). Following reverse transcription, qPCRs for Taf11  for Xist and for Rlim (see primer sequences in Table S1) were performed on poly(A)+ and poly(A)- RNA fractions in parallel. RNAs from female embryonic stem cells (LF2 cell line) differentiated for 4 days served as positive control for the presence of Xist poly(A)+ RNAs.Approximately 1000 mature MII oocytes were collected from 3-week old hormone stimulated 129Sv mice. Total RNA from MII oocytes was mixed with Xic and with X-linked genes during oogenesis, we collected early-growing MI-oocytes (n = 7), late-growing MI oocytes (n = 22), and mature MII oocytes (n = 20) from 129Sv mice of various ages . We analyzed the transcription levels at the Xist, Tsix, Jpx, Ftx, Linx, and Rlim loci, at a selection of X-linked genes and, for reference, within the body of the lncRNAs Airn, Kcnq1ot1, and H19 genes involved in the imprinted expression of the autosomal gene clusters Igf2r, Kcnq1, and H19-Igf2, respectively .In order to analyze the transcriptional activities associated with the Xic in most early-growing oocytes compared to late-growing MI oocytes or to MII oocytes (Table S2 for raw quantification results). Two out of 7 oocytes classified as early-growing, however, showed low levels of primary transcripts overall and clustered with SN oocytes , suggesting that these oocytes were in fact more advanced in their growth. About half of NSN-oocytes showed significant—yet reduced compared to earlier oocytes—transcription. In agreement with previous reports indicating that transcription activity is restricted to the growth phase of oogenesis, transcription could barely be detected in SN MI- or in MII-oocytes.Linx locus appeared more heterogeneous than those of other loci. While most Linx noncoding transcription occurs in early-growing oocytes, significant levels of transcripts were observed in some transcriptionally silent NSN and SN oocytes. The significance of this result is unclear, since the characteristics of the Linx non-coding transcription and, notably, the respective contributions of transcriptional activity, of primary transcripts, and of alternative spliced isoforms to global transcription activity at this locus have yet to be fully established.We observed much higher transcriptional activity for genes within the Xic, like most of the X-linked and autosomal loci we tested, undergo transcriptional activation in early-growing oocytes.Taken overall, these observations indicate that the component loci of the Xist and Tsix, two sense and antisense transcriptions known to be mutually exclusive in most cellular contexts,Xist gene is almost completely embedded within an intron of Tsix, the intronic PCR assay for Xist (Xist IN) theoretically also detects ongoing Tsix transcription  surrounding Xist IN and compared the levels of Xist spliced transcripts measured with Xist Trans-EX, to RNA levels quantified with Xist IN and to the intensity of Tsix transcription outside of the Xist gene (Tsix assay) in oocytes at different stages . We noted however that the level of Xist mature transcripts appeared highly heterogeneous among MII oocytes. The reason for this is not clear but may be linked to variable fertilization competency characterizing MII oocytes.Intriguingly, Xist/Tsix locus in growing oocytes, we designed additional PCR assays along the locus that were used to analyze a replication set of 15 freshly collected early-growing oocytes . All three Tsix-specific assays detected significant levels of Tsix molecules indicating that, most probably, Tsix transcription extends over the entire locus. Similarly, no significant difference was observed between Xist trans-exonic or between Xist intronic assays, suggesting that Xist is fully transcribed and spliced in growing oocytes . Using strand-specific RT-qPCR on another batch of 21 early-growing oocytes , we could detect, using primers within Xist intron 1, transcription in the Xist orientation in the majority of oocytes. This Xist transcription was accompanied by a significant transcription in the Tsix orientation in ∼¼ of oocytes . Surprisingly, at this position, Tsix RNA levels appeared very reduced compared to Xist levels or to Tsix levels at a downstream position within Tsix exon 4 . This suggests that, as previously characterized in ES cells, Tsix transcription is less abundant within Xist span and that Tsix spliced forms are more stable than Tsix primary transcripts.In order to further characterize the transcription at the Xist and Tsix are co-expressed in early-growing oocytes. Interestingly, similar sense and antisense co-transcriptions were observed at 2 other autosomal imprinted loci: Airn/Igf2r  and Kcnq1ot1/Kcnq1 (see Table S2).These results indicate that Xist/Tsix locus by RT-qPCR quantification led to 2 different questions: (1) Is the accumulation of mature Xist transcripts in MI oocytes associated with the formation of a Xist domain on the X chromosome, which could directly participate in the imprinting process? (2) Are Xist and Tsix transcripts produced from the same locus or from distinct X chromosomes and/or sister chromatids present in the early-growing oocyte? In order to address these questions we analyzed, by RNA-FISH, the distribution of Xic transcripts in the nucleus of oocytes at different stages.The detection of major levels of transcription at the Xist/Tsix, Rlim, and Linx transcripts formed 3 to 4 pinpoint foci located in the same nuclear vicinity . In later growing MI oocytes, while Linx was still transcribed in ∼40% of nuclei, Rlim transcription and transcription from the Xist/Tsix locus were detected in only 20 and 26% of the GVs in NSN oocytes, respectively  tail compared to mature Xist RNAs found in adult cells, which may affect the ability of Xist RNAs to coat the X-chromosome in the context of the oocyte .In most early-growing oocytes, Xist/Tsix locus originated from the same chromosome/chromatid, we performed double RNA-FISH on early-growing oocytes using a strand-specific oligonucleotide probe that specifically detects Xist transcripts and with a double-stranded probe lying 3′ to Xist within the Tsix 5′ end . Since strand-specific single-cell RT-qPCR reflects the quantities of a given RNA molecule in a given cell at a given time and, therefore, depends on transcript stability while RNA-FISH detection only reflects, under our experimental conditions, ongoing transcription, it is possible that the above results indicate differences in the stability of Tsix ncRNAs compared to Xist nascent transcripts in this cellular context. For comparison, we performed the same RNA-FISH analysis on ES cells, which constitute the only known somatic cell type in which Xist and Tsix are co-transcribed, although Xist steady-state levels, in these cells, are dramatically reduced compared to Tsix levels.Xist loci showed simultaneous sense and antisense transcription  indicating that Xist and Tsix co-transcription is more frequent in early-growing oocytes than in somatic cells.In order to address whether sense and antisense transcription at the Xist spliced forms that do not coat the X-chromosomes in fully-grown MI and MII oocytes, suggesting either that these Xist RNA molecules are unable to associate with the X, potentially due to a short poly(A) tail, or that the X-chromosome structure is not accessible or, alternatively that some factors involved in the recruitment of Xist RNAs to the X-chromosome are not present in oocytes. Since Xist transcription in early-growing oocytes frequently occurs simultaneously with and from the same chromatid as antisense Tsix transcription, this strongly suggests that the repression of Xist by Tsix that is known to take place in other cellular contexts is less—or not—effective in early-growing oocytes.To summarize, both our RNA-FISH and RT-qPCR results identify an accumulation of Xist expression from the XM in early-growing oocytes opens up the possibility that maternal Xist transcription also occurs during the first embryo cleavage stages. To test for this, we first performed allelic RT-qPCR quantifications of the Xist locus on whole 129Sv/Pwk heterozygous embryos at different pre-implantation stages . As expected from previous reports, no Tsix expression could be detected during pre-implantation development either from the XM or from the XP. In contrast, paternal transcription at the Xist locus appeared induced from the late 2-cell stage onwards, with Xist spliced transcripts progressively accumulating in the 4-cell female embryos. This kinetics is in agreement with previous reports of an initiation of XP inactivation occurring around the 2 to 4-cell stage.Xist expression from the XM in the late 2-cell embryos of both sexes, which appeared to be turned off by the 4-cell stage . We noted however that maternal Xist induction from an X chromosome of Pwk origin appeared reduced compared to transcription from a 129Sv XM despite the fact that Pwk oocytes showed higher Xist levels than 129Sv oocytes  indicating that the genetic background may influence the level of maternal Xist expression.Xist/Tsix transcription to establish the actual number and proportion of blastomeres showing this maternal expression of Xist. While in individual cells of the early 2-cell female embryos, Xist expression was absent from the vast majority of cells, at the late 2-cell stage, we were able to detect an induction of maternal Xist in ∼½ embryos in which either one or both blastomeres appeared to produce both Xist primary and spliced transcripts . At the 4-cell stage, this maternal expression decreased drastically and Xist expression then became progressively restricted to the high levels of paternal transcription seen in cells of the 8-cell embryos (Xist at the late 2-cell stage in 2 out of 4 embryos (3 out of 8 blastomeres) .We next performed single cell RT-qPCR analysis of Xist locus in the nucleus and determine the sex of each embryo, we co-hybridized the Xist probe with 2 other probes detecting the expression of the nearby Rlim gene and of Kdm5c, a gene known to escape from XCI  of late 2-cell embryos . No significant difference was observed between crosses. In agreement with our RT-qPCR analysis, no Tsix transcription could be detected at any stage. In contrast, a Xist transcription from the XM was observed in 13% of the male nuclei and a biallelic Xist transcription was detected in 20% of female nuclei at the late 2-cell stage , biallelic Xist expression characterized by a Xist pinpoint located at the Xic at the vicinity of the GFP marked XP and a second Xist pinpoint at the Xic located away from the GFP locus , pinpoint signals scattered in the nuclear space in 27.3% of the late 2-cell embryos we analyzed .Our observation that Xist is significantly induced in early-growing oocytes and that this Xist transcription frequently occurs simultaneously with and from the same chromatid as antisense Tsix transcription. Interestingly, simultaneous antagonist transcriptions also characterize sense/antisense counterparts involved in the regulation of imprinting at other autosomal loci. In the latter cases, this unusual transcription feature is associated with differential histone modification states and with allele-specific DNA methylation.M-imprinting either via direct recruitment of chromatin remodelers or, indirectly, via the triggering the formation of endo-siRNAsXist/Tsix locus. In this regard, a recent study reported a significant derepression of maternal Xist allele upon expression of the H3K9me3 demethylase KDM4B in early parthenogenote embryos, suggesting that this histone mark participates in XM imprinting both by preventing the binding of RLIM on the maternal Xist promoter and by precluding maternal Xist activation.Xist promoter during oogenesis concomitantly with Xist/Tsix co-transcription. From a mechanistic point of view, concomitant sense/antisense transcription should trigger RNA polymerase II (RNAP II) collision, followed by RNAP II stopping, which, in turn, may lead to primary transcripts remaining associated with the locus for longer periods of time than in the case of a fully processive enzyme event.A major finding of our study is that Tsix or Xist transcription during oocyte growth using classical cre-inducible deletions appeared to be impossible due to allele incompatibilities. Notably, we were unable to create the mother that would produce ΔXist oocytes because of an incompatibility between the ZP3-cre inducer allele and the Xist floxed allele during spermatogenesis (data not shown). Functional analyses addressing the role of antagonist Xist and Tsix transcriptions will therefore require developing alternative approaches such as in vitro models of oogenesis allowing for ex vivo mutagenesis.Abrogating either