Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease that severely challenges the yield of rice. Here, we report the identification of a “SAPK10-WRKY72-AOS1” module, through which Xoo infection stimulates the suppression of jasmonic acid (JA) biosynthesis to cause Xoo susceptibility. WRKY72 directly binds to the W-box in the promoter of JA biosynthesis gene AOS1 and represses its transcription by inducing DNA hypermethylation on the target site, which finally led to lower endogenous JA level and higher Xoo susceptibility. Abscisic acid (ABA)-inducible SnRK2-type kinase SAPK10 phosphorylates WRKY72 at Thr 129. The SAPK10-mediated phosphorylation impairs the DNA-binding ability of WRKY72 and releases its suppression on AOS1 and JA biosynthesis. Our work highlights a module of how pathogen stimuli lead to plant susceptibility, as well as a potential pathway for ABA-JA interplay with post-translational modification and epigenetic regulation mechanism involved.Bacterial blight caused by the infection of   •Xoo infection and JA synthesisWRKY72 negatively regulates rice resistance to •AOS1SAPK10 phosphorylates WRKY72 at Thr 129 to impair its DNA binding on •AOS1 transcription to attenuate JA synthesisWRKY72 directly represses •AOS1 promoterWRKY72 recruits hyper DNA methylation on  Biochemistry; Cell Biology; Genetics; Microbiology; Molecular Biology; Plant Biology The plant innate immune system is considered to contain two interconnected layers termed PTI (pathogen-associated molecular patterns-triggered immunity) and ETI (effector-triggered immunity) . Once plcis-regulatory elements, which are often found in many defense gene promoters. In addition to the W-box, it can bind other cis elements, such as sugar-responsive element (AA/TAA) in barley and pathogen response element (TACTGCGCTTAGT) in rice  and the fungal pathogen Magnaporthe grisea, which indicated that WRKY genes are extensively involved in plant defense to pathogens (M. grisea by up-regulating pathogenesis-related (PR) genes such as NPR1, ZB8, POX22.3, PR1b, PBZ1, and Sci2  genes , which was accompanied by a higher level of SA and JA. On the contrary, the variant WRKY45-2 was found to promote accumulation of JA, but not SA, and eventually play positive functions in response against Xoo and Xoc. The opposite roles of the two variants in rice-Xoo interaction are possibly attributed to their mediation of different defense signaling pathways.WRKY proteins have been reported to play broad and pivotal roles in plant-pathogen interactions and act in a complex signaling network as both positive and negative regulators of disease resistance . Till daathogens . Till daathogens . WRKY12 and Sci2 . Some of and Xoo . ReporteR) genes . Notablyng model . KnockouWRKYs have been achieved, it is believed that more WRKY members are involved in rice immunity, given the extensive involvement of WRKYs and complication of rice-pathogen interplays. The current study revealed that Xoo-inducible WRKY72 negatively regulates rice resistance against bacterial blight. WRKY72 directly binds to the promoter of a JA biosynthesis enzyme gene AOS1 and suppresses its transcription by recruiting DNA methylation on it. SAPK10-mediated phosphorylation on Thr129 of WRKY72 weakens its DNA-binding ability to AOS1, promotes the endogenous JA level, and finally enhances Xoo resistance.Despite the fact that tremendous progresses on WRKYs in a time line after Xoo inoculation and found that WRKY72 (LOC_Os11g29870) is highly induced. The 3,736-bp-long gene encodes a 243-amino acid protein. In its only intron, we identified a SINE (short interspersed nuclear elements)-type transposon  (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). WRKY72 displayed significantly elevated transcriptional level since 12 HAI (hours after inoculation) and reached highest level (over eight times up-regulation) at 72 HAI, when compared with the 0-HAI samples , as the lesion area on OxWRKY72-1 and OxWRKY72-7 were 37% and 39%, respectively, whereas 25% of the leaf area of WT displayed necrosis  (p < 0.05)  C. All thXoo infection, as its transcription was significantly suppressed by Xoo at the first 12 h after inoculation , which is out of, but very close to, the annotated WRKY domain (138–193 amino acid) (We screened over 1 million colonies from a cDNA library derived from rice young seedlings by using yeast two-hybrid method and detected an interactive kinase SAPK10 (LOC_Os03g41460). SAPK10 may be a negative regulator in response to culation A. We repculation B. The in protein C. Meanwh protein D. Thus, ed on it E. The phed on it E. Therefal sites , and finno acid) F.Figure OxWRKY72T129A lines, in which the only phosphosite Thr129 was mutated into an alanine (Ala) to block the phosphorylation on it. Two OxWRKY72T129A lines with comparable levels of WRKY72 expression as the OxWRKY72s were chosen for Xoo susceptibility assay , AOS1 , AOS2 , LOX1 , and LOX2  were significantly down-regulated in OxWRKY72, with the only exception of OPR7 , which showed an opposite tendency , which is in accordance with the observed higher susceptibility of OxWRKY72T129A. We also investigated the levels of PR protein genes PR1a, PR1b, PR5, and PR10. PR1b, and PR10 may contribute to the elevated susceptibility of OxWRKY72 and OxWRKY72T129A lines, as both genes were significantly down-regulated . In the WT, Xoo infection significantly stimulated the accumulation of JA to about 16 times higher level than that in the pre-infection condition. However, the Xoo infection appeared to have triggered the suppression function of WRKY72 on JA synthesis, as we found that the JA level was significantly reduced in OxWRKY72 at 72 HAI. Moreover, OxWRKY72T129A displayed even lower JA level than OxWRKY72 at 72 HAI, which is consistent with the observation that OxWRKY72T129A lines were more susceptible to Xoo than OxWRKY72s. The results above suggested that WRKY72 negatively regulates Xoo resistance by suppressing JA biosynthesis.Given the obvious down-regulation of the key enzyme genes for JA biosynthesis, we examined the endogenous JA level in , and WT C. BeforeOxWRKY72 lines intrigued us to speculate that WRKY72 directly suppresses the transcription of these genes. To test this hypothesis, we checked the in vitro binding of WRKY72 on AOC, AOS1, and LOX1 promoter regions by EMSA (electrophoretic mobility shift assay). The results showed that WRKY72 only bound to the promoter of AOS1, where a conserved W-box cis element exists , which is the only potential CpG island as predicted by MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi)  A. When cecreased . WRKY72TOxWRKY72 . The corAOS1 by impairing WRKY72 DNA-binding ability and lowering the DNA methylation level on AOS1 promoter, thus maintaining a normal endogenous JA level for growth. Under Xoo infection, the stimuli represses SAPK10 transcription and makes the WRKY72 in a non-phosphorylated status, which facilitates the binding of WRKY72 to the W-box cis element of AOS1 promoter to suppress AOS1 transcription by recruiting hyper DNA methylation on it, and eventually contributes to plant susceptibility by suppressing the endogenous JA biosynthesis  to 12,13-EOT -12,13-epoxy-9,11,15-octadecatrienoic), which is the first step toward JA biosynthesis (AOS genes in rice (AOS1-AOS4), AOS2 has been characterized as a pathogen-inducible gene, and its over-expression lines had higher levels of JA and stronger resistance to M. grisea  controlling juvenile-to-adult phase transition in rice. Pre exhibited long leaf with precociously acquired adult features in midrib formation, shoot meristem size and plastochron, and more importantly, lower endogenous JA level (AOS1 in disease resistance, it is rational to expect a higher susceptibility of aos1 to Xoo infection.WRKYs are very important transcription factors in plants. The majority of over 100 members in rice are found to be involved in plant defense response in either a negative or a positive manner. The negative regulator members include WRKY28, WRKY42, WRKY62, and WRKY76, but their regulatory mechanism may vary from each other. WRKY62 and WRKY76 cause pathogen susceptibility by regulating a list of defense-related genes or interacting with the intracellular kinase domain of Xa21 to affect its protein cleavage and nuclear localization . Recentlectively . In thisynthesis . Among t. grisea . LikewisJA level . AlthougArabidopsis, AtMPK3 and AtMPK6 directly phosphorylated AtWRKY33 to enhance the production of phytoalexin camalexin and phytohormone ethylene, whereas non-phosphorylated AtWRKY33 was not able to fully rescue wrky33 mutant, implying that MAPK-dependent phosphorylation activates AtWRKY33 function (M. oryzae compared with native WRKY53-over-expressing rice plants (Post-translational modifications, particularly protein phosphorylation, have been long recognized as a significant regulatory mechanism controlling transcription factor activity . In planfunction . Similare plants . It was e plants .AOS1 promoter, to thus release the inhibition on JA accumulation. In support of this finding, the OxWRKY72T129A lines, which had blocked SAPK10 target phosphosite, showed drastically reduced transcription of downstream JA biosynthesis genes, endogenous JA level, and resistance to Xoo infection than the native OxWRKY72 lines. Hence, the indication is that the SAPK10-dependent phosphorylation on WRKY72 turns down its function as a transcription repressor in plant defense, representing a diverse mechanism to the previously reported MAPK-WRKY module. SAPK10 is an ABA-inducible SnRK2-type kinase involved in ABA signaling (Xoo infection (In this study, we demonstrated that SAPK10 kinase cloud physically binds to and phosphorylates WRKY72 at Thr129. In contrast to the above-mentioned cases that phosphorylation activated WRKYs, the phosphorylation on WRKY72 weakened its DNA-binding ability to ignaling . So far,ignaling . Notablynfection . Our resWRKY45, whose two variants WRKY45-1 and WRKY45-2 play opposite roles in response to Xoo and Xoc infection (WRKY45-1 produces TE-siR815, which imposes DNA hypermethylation on ST1 via an RdDM mechanism to abolish WRKY45-mediated pathogen resistance (AOS1 promoter was negatively correlated with the AOS1 transcription level and endogenous JA level in WT, OxWRKY72, and OxWRKY72T129A lines, implying that the repressor role of WRKY72 may be achieved by inducing DNA methylations on the promoter region of its direct target AOS1. Nevertheless, the question that how was the DNA methyltransferase recruited to the target sites remain to be addressed. The identified SINE TE in WRKY72 intron might be a good clue that WRKY72 suppresses target genes through an RdDM mechanism, given the reported example of its homolog WRKY45 and many SINE-directed DNA methylation cases in mouse and human beings (DNA cytosine methylation is usually connected with transcriptional silence of the target genes in numerous biological processes, including plant defense . RdDM R. One of nfection . Only thsistance . In our n beings .Xoo infection suppresses JA biosynthesis to cause Xoo susceptibility. However, as we have discussed in the article, the final endogenous JA level in plant is determined by the counterbalance of both activation and suppression pathways. Therefore, figuring out the Xoo-activated JA biosynthesis regulatory pathways would be of great interest for us to elucidate the comprehensive reaction of rice in response to Xoo infection. In addition, although we provided clues that WRKY72 represses AOS1 transcription via RdDM, the detailed mechanism needs to be explored in future studies.In this study, we revealed a “SAPK10-WRKY72-AOS1” module, through which All methods can be found in the accompanying


Limited supply of nutrient normally causes cell growth arrest. Our recent study (Nat Cell Biol. (7):833-843) shows that fumarase (FH), a key enzyme responsible for the conversion between fumarate and malate in tricarboxylic acid cycle, is importantly involved in the cellular response to nutrient condition. To date, emerging evidence demonstrate that metabolites participate in a wide variety of cellular activities besides their direct role in metabolism. Previous studies have reported that metabolic activity of FH is critically involved in the precise regulation of histone 3 lysine 36 dimethylation (H3K36me2) upon DNA damage, which is based on the inhibitory effect of fumarate on α-ketoglutarate-(α-KG)-dependent histone demethylase KDM2A/B. Strikingly, through mass spectrum and immunoprecipitation analysis, here we first found that glucose deprivation largely promoted FH binding to ATF2, a canonical transcription factor responsive to stress signaling. Further ChIP analysis show that ATF2 recruits FH to the promoter regions of its targeted genes for cell cycle arrest. Accordingly, gene expression and functional analysis demonstrate that the ATF2-FH complex facilitates transcription and thus cell cycle arrest under glucose deficiency.in vitro O-GlcNAcylation assay indicate that FH is O-GlcNAcylated by OGT at Ser 75; and cancer cells exhibit high FH O-GlcNAcylation levels along with a strong OGT activity, which soundly impairs AMPK-mediated FH phosphorylation and the relevant cellular sensitivity to the low glucose availability.Subsequently, we performed a sequence of biochemical analyses and found that in normal pancreatic duct cells glucose deprivation readily triggered FH Ser75 phosphorylation by AMPK. This phosphorylation is required for FH interaction with ATF2 and the subsequent gene expression. In contrast, FH-ATF2 complex formation is weakly detected in pancreatic duct cancer cells under glucose deprivation, implying that a negative regulation for AMPK-FH-ATF2 signaling exists. After screening, we found protein O-GlcNAcylation, which is tightly related to glucose availability and is reversely catalysed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), implicated in this process. Intriguingly, mass spectrum and Figure 1).To understand the metabolic effects of FH on transcription, FH enzymatic dead mutant FH R233H and ATF2 binding defect mutant FH S75A were both utilized and the results indicate FH catalytic activity as well as its binding to ATF2 are both indispensable for ATF2-mediated transcription. Meanwhile, the importance of FH metabolic activity is validated by addition of exogenous fumarate which partially reverses the repressive effect of FH R233H and FH S75A on transcription and cell cycle arrest. Furthermore, the implication of locally generated fumarate is expanded to its epigenetic effect on H3K36me2 regulation; indirect evidence from sequential biochemical analyses exemplify that fumarate produced by promoter-associated FH, maintains H3K36me2 levels via inhibition of α-KG-dependent histone demethylase KDM2A directly at the promoter region, in which H3K36me2 level is positively related with the transcription of genes targeted by ATF-FH complex (in vivo. In consistence, clinical data show FH Ser75 phosphorylation is inversely correlated with OGT levels in human pancreatic tumour specimens, and patients with higher FH Ser75 phosphorylation display a longer median survival duration.At the physiological level, the FH S75A mutant significantly rescued the impaired xenograft tumor development resulted by OGT depletion, which indicates competition of OGT-mediated FH O-GlcNAcylation to FH Ser75 phosphorylation would be essential for cancer cell growth under condition of glucose deficiency The interplay between metabolism and epigenetic regulation are fundamentally involved in cellular response to microenvironmental nutrient status. Our study focuses on the subtle regulation of histone methylation-gene transcription by FH under glucose deficiency-induced signaling pathway. The flexible functional role of FH reflected here is attributed to the counteractive effect of OGT activity on AMPK signaling; this finding would valuably provide new molecular basis for developing effective cancer therapy in future.


Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of  Cyanobacteria are promising as platforms for biological production of fuels and other chemicals. Their fast growing photosynthetic nature makes them ideal from a sustainable production perspective. Within the vast and diverse cyanobacterial phylum, heterocyst forming cyanobacteria is a group of fascinating multicellular photosynthetic organisms that are able to differentiate a subset of their cells into specialized compartments dedicated to fixation of atmospheric nitrogen, called heterocysts. The interior of the heterocyst is kept micro-aerobic to let the oxygen sensitive nitrogenase, the enzyme-complex responsible for the nitrogen fixation, operate. The development of the nitrogen fixing cells is a complex process where environmental stimuli and a large number of signaling substances interact and give raise to the characteristic, semi-regular pattern of heterocysts along the filament. The process starts when no source of combined nitrogen is available and is coordinated by the global nitrogen transcription regulator NtcA . Anotheralr3808-promoter [hepA-promoter [Anabaena (Nostoc) sp. PCC 7120, and truncated versions of the uptake hydrogenase promoter from Nostoc punctiforme ATCC 29133 (N. punctiforme) [Heterocysts are interesting for production of biofuels and chemicals, as the unique cellular environment should be well suited for heterologous expression of oxygen sensitive enzymes, like hydrogenases –11. To spromoter ,14, the promoter  from Anatiforme) .The usage of native, often several hundred nucleotides long, promoter sequences in biotechnological applications is not optimal. Such native sequences are often heavily regulated and part of the complex and multi-layered internal metabolic regulatory system, which can give rise to unpredictable behaviors and unforeseen effects in the expression system. Naturally, long pieces of DNA are also less convenient when the expression constructs are being assembled, especially for large and more complicated constructs.nsiR1-promoter (PnsiR1) from Anabaena sp. PCC 7120 [nsiR1 (nitrogen stress inducible RNA1), a sRNA induced early in heterocyst development [PnsiR1 is well studied and has been used in a promoter-fluorescence reporter system in Anabaena sp. PCC 7120 for detection of proheterocysts before any morphological change specific to heterocyst are visible with microscopy [PnsriR1, as well as alr3808-promoter, belongs to the to the DIF+ class promoters [5’-TCCGGA-3’) located at the -35 position relative the transcription start site (TSS).The shortest promoter known to render heterocyst-specific expression of heterologous genes is the 70 nucleotides long native PCC 7120 . This prelopment . PnsiR1 croscopy . The Pnsromoters . This isromoters  . The alignment revealed no conserved patterns upstream -35 or between the -35 and -10 regions. We propose that the conserved heterocyst enhanced expression pattern from the DIF+ class promoters should be an effect of conserved regions in the promoter sequences. The lack of conserved sequence patterns upstream the DIF-motif suggests that a minimal promoter sequence could contain all genetic information needed for heterocyst-specific expression. Based on our observations, we designed a synthetic, 48 nucleotides long, ultra-compact promoter, PsynDIF, by combining the DIF-motif at position -35 with the further downstream consensus sequence from the alignment, making up an extended -10 region, spanning from -13 to TSS ..+ class PCC 7120 , we foun3 to TSS . To miniPCC 7120 ,17,18. U+ promoter had retained the heterocyst-specific expression pattern typical for the native DIF+ promoters, we designed a promoter fluorescence reporter construct for in vivo detection. The PsynDIF was put upstream of the gene eyfp, which encodes a yellow fluorescent protein (YFP), together with the cyanobacterial synthetic ribosomal binding site RBS* (“RBSstar”) [yfp   in the sctiforme . The twoN. punctiforme. After 24 h of nitrogen starvation, twenty heterocysts from three individual cultivations of the SynDIF strain were identified by morphology and reduced autofluorescence. In a second step, these heterocysts were investigated for YFP fluorescence. All of the investigated heterocysts, in total 60, were confirmed to have drastically higher fluorescence then the 5 closest cells on each side , distinct YFP fluorescence could be seen in both heterocysts and vegetative cells with no distinguishable difference in fluorescence level between the two cell types. It is thus clear that a DIF1-motif in the -35 position is not by itself enough to render heterocyst-specificity to a promoter sequence. Our result highlights the importance of the -10 region for the heterocyst-specific behavior of DIF+ promoters. The strain harboring the -35Ptrc-SynDIF-pSAW plasmid (SynDIF-35Ptrc strain) did not display any detectable YFP fluorescence in any cells, indicating that the combination of the -35 region from Ptrc and our consensus extended -10 region does not serve as a functional promoter in N. punctiforme. It is apparent that both the DIF1 palindrome and an appropriate -10 region are crucial for the heterocyst-specific expression pattern from our synthetic DIF+ promoter. Filaments bearing the promoterless control construct did not exhibit any detectable fluorescence. When N. punctiforme is grown in ammonium supplemented media, the formation of heterocysts is prevented, and under this condition the SynDIF strain showed only very low, irregular fluorescence from YFP. This is in agreement with what has been reported earlier for native DIF+ promoter-reporter constructs [After 12 h of combined nitrogen starvation, the expression patterns from the three promoter variants were investigated by fluorescence confocal microscopy. Representative filaments from each strain were imaged and can be seen in ach side , similarnstructs .nsiR1, that if the DIF1 palindrome of a DIF+ promoter is exchanged with a restriction site, the heterocyst-specificity is lost [trc. No detectable increase in fluorescence compared to the promotorless control was detected in the SynDIF-35Ptrc strain upon nitrogen depletion  on our synthetic minimal promoter, we chose a stretch of 35 base pairs upstream the DIF1 motif in the native UEsynDIF , was utiepletion , confirmepletion . The fluepletion . This re+ promoters have only been investigated in Anabaena sp. PCC 7120 and Nostoc punctiforme ATCC 29133, the SynDIF promoters are of potential interest for heterocyst-specific protein expression in cyanobacteria beyond these two model strains.In this work we use consensus sequences of core promoter elements to build a minimal synthetic promoter for heterocyst-specific expression. This promoter gives an approximate 10 time increase in expression 12 hours after the start of heterocyst differentiation. This well characterized promoter expands the potential for cyanobacterial biotechnology and is of special importance for expression of oxygen intolerant enzymes in a photosynthetic host organism. This work provides fundamental understanding of the promoter elements of a core promoter and adds to the knowledge concerning the importance of the cooperation of -35 and -10 regions, and the usefulness of upstream element to enhance the expression level. From recent work by Elhai and Khudyakovit , it can This work has expanded the available toolbox for heterocyst-based biotechnology. The developed promoters will be especially useful in bio-hydrogen production from heterocystous cyanobacteria, an area of importance in the search for sustainable energy-carriers for our society.Nostoc punctiforme and Escherichia coli was amplified from pSCR119 [ccdb  and inserted in pSAW by restriction/ligation using XhoI and XbaI, creating the plasmids SynDIF-pSAW and UESynDIF-pSAW. For the full sequence of the synthesized sequences, see supporting information  by standard procedure for conservation purposes and were transformed in Nostoc punctiforme ATCC 29133 by means of electroporation as described elsewhere [trc, SynDIF-10Ptrc and promoterless-YFP. These, were grown in 100 mL Erlenmeyer flasks under 40 μmol photons s-1 m-2 of light at 25°C with gentle agitation. BG11 supplemented with 2.5 mM NH4, 5 mM HEPES and 25 μg/mL kanamycin was used for growth under normal conditions. For nitrogen step-down experiments, the cells were harvested by centrifugation at 3500 rpm and resuspended in BG110 three times prior to a final resuspension in BG110 supplemented with 25 μg/mL kanamycin.The plasmids enlisted in the section “Promoters and plasmids construction” were transformed in lsewhere , obtainiN. punctiforme cultures where imaged using a Leica DM600 CS microscope and a HPX PL Fluotar 40.0x0.75 dry objective. Samples were excited using a 488nm laser and emission was detected between 530nm to 540nm (EYFP channel) and between 600nm and 700nm (autofluorescence channel). Prior to imaging, the different strains of N. punctiforme were fixed on solid media by spreading 20 μL of cell suspension onto 50 mL petri dish filled with BG110 0.8% agarose media with kanamycin 25 μg/mL. Z-stacks and 3D-projections were acquired using Lecia Application Suite Advanced Fluorescence (LAS AF) software, according to manufacturer’s instructions using recommended settings.The 4, 5 mM HEPES and 25 μg/mL kanamycin, at a light intensity of 40 μmol photons s-1 m-2 at 25°C to an OD750nm of 0.6. The cells were harvested at 0 and 12 hours and washed. RNA was isolated as previously described [TM qPCR machine (Bio-Rad). YFP and 16S were used as target and reference genes respectively. Primer efficiency value was considered to be reliable for further expression experiments if ranging between 95% and 110% [-ΔΔCt method [Triplicate cultures of the SynDIF strain and promotorless control strain were grown in 400 mL of BG11 medium, supplemented with 2.5 mM NHescribed . The RNAand 110% . Fold cht method . Three b+ promotors with the DIF1 palindromic sequence 5’-TCCGGA-3’  located close to the −35 were obtained from [+ promoters, were aligned and the consensus sequences were generated and visualized by WebLogo 3.0 [Sequences for 58 DIFned from . SequencLogo 3.0 .N. punctiforme strains SynDIF and UESynDIF were cultivated in BG110 medium. 5 mL of culture (at an OD750nm of approximately 1.0) was collected by centrifugation 12 hours after nitrogen step-down. Cells were resuspended in 2 mL of dH2O and sonicated at 40 W, for 1 min, and then cooled in ice-cold water. The samples were analyzed under microscope to ensure that the majority of filaments were ruptured into single cells. The samples were then centrifuged at 1,000 x g, for 3 min, at 4°C, to remove cell debris. Following centrifugation, cells were resuspended in 2 mL dH2O and incubated at RT.A minimum number of 10,000 cells were sampled on an image-based flow cytometer (Merck Amnis FlowSight) and analyzed using analysis software IDEAS. Single cells were identified based on the first gating with area scatter and aspect ratio scatter parameters. Thereafter, the cells were sub-gated with intensity scatter on YFP fluorescence channel, at 488nm channel  isolating all cells displaying fluorescence in the said channel. In order to isolate heterocysts, in case any other cells were displaying fluorescence in the YFP channel, a third step gating was performed. Based on the 488nm channel data, the cells were sub-gated with the intensity scatter on 745-800nm channel, indicating autofluorescence. The targeted single heterocyst cells were selected based on significantly decreased autofluorescence compared to vegetative cells.S1 Fignpt and origins of replication for Nostoc punctiforme (pDC1) and Escherichia coli (ColE1) from pSCR119 [ccdB gene for plasmid maintenance and selection in Escherichia coli [ccdB-operon. Terminators (brown) are named after their respective Parts Registry association number [Overview map of the plasmids pSAWccdb and pSAWyfp created in this work. Plasmid backbone contains kanamycin/neomycin resistance gene  pSCR119 . Toxin ehia coli  is expren number . Recomme(TIF)Click here for additional data file.S2 Fig3D-projection of a Z-stack containing 91 slices of a 30μm deep sample volume. Fluorescence images from YFP channel (530-540nm) and autofluorescence channel (600-700nm) are overlaid.(TIF)Click here for additional data file.S3 FigConfocal fluorescence images of a representative filament of the SynDIF strain at 6, 12, 24 and 48 h after nitrogen deprivation immobilized on agar plate. Autofluorescence (600-700nm) is shown in red, YFP fluorescence (530-540nm) in yellow. Rightmost column shows overlaid images. In the autofluorescence images, the arrows indicate a developing heterocyst.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 FileFull sequences of the promoter-RBS designs synthesized for this study.(DOCX)Click here for additional data file.S3 FileUsed for (XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.


Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice.Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging.Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.The online version of this article (10.1186/s13059-018-1489-y) contains supplementary material, which is available to authorized users. Old age is accompanied by increased frailty including a breakdown in functionality of the adaptive immune system mediated by B and T lymphocytes . This reThe advent of new technologies in functional genomics enables illumination of the changes in B cell development that occur during aging genome-wide. Recently, application of these technologies to aging human T cells has provided profound insight into widespread epigenetic changes that impair the function of closely related lymphocytes in the adaptive immune system. In particular, aging CD8+ T cells lose chromatin accessibility at promoters, which may compromise their metabolic state . CompariHere we present an integrated analysis of the transcriptome, epigenetic landscape, and higher-order chromatin structure in young and aged pro- and pre-B cells from mouse bone marrow. We show that aging affects the expression of a limited number of genes, in particular key components of the insulin-like growth factor (IGF) signaling pathway. These alterations in the transcriptome are accompanied by perturbations of multiple regulatory layers affecting transcriptional and post-transcriptional mechanisms, including microRNAs (miRNAs) and polycomb-mediated epigenetic regulation.Using multi-parameter flow cytometry analysis of the bone marrow B cell compartment, we found substantial changes in the B cell precursor pools in aged mice Fig. . Our anaRnf125, Dock9, Iigp1, Igj, and age-downregulated Irs1, Rftn2, Plxna2, and Igf2bp3). Overall, age-specific expression changes were correlated between pro- and pre-B cells, even when significance was reached in only one of the cell types , and genes coding for Major Histocompatibility Complex proteins, such as Cd74  as shown in Fig. pes Fig. . SeveralRftn2, Plxna2, Cdc42bpa, Plxdc2, and Pcdh9  and the PI3K-AKT pathway antagonist Inpp4b  level. We therefore examined IRS1 protein levels and found that these were decreased upon aging in both pro-B and pre-B cells  of DEGs detected in total RNA were also DEGs in the nuclear RNA-sequencing (RNA-seq) analysis, and overall the fold changes were correlated   and ethanol [2.5 × volumes]) overnight at − 20 °C, the DNA was spun down (centrifugation 3200 × g for 30 min at 4 °C). The pellets were resuspended in 400 μL TLE  and transferred to 1.5 mL Eppendorf tubes. After another phenol/chloroform (Sigma) extraction and DNA precipitation overnight at − 20 °C, the pellets were washed three times with 70% ethanol and the DNA concentration was determined using Quant-iT Pico Green (Life Technologies). For quality control, candidate 3C interactions were assayed (primers available upon request) by PCR and the efficiency of biotin incorporation was assayed by amplifying a 3C ligation product (primers available upon request), followed by digest with HindIII or NheI.Hi-C and PCHi-C libraries were generated as described  with modTo remove biotin from non-ligated fragment ends, 40 μg of Hi-C library DNA were incubated with T4 DNA polymerase (NEB) for 4 h at 20 °C, followed by phenol/chloroform purification and DNA precipitation overnight at − 20 °C. After a wash with 70% ethanol, sonication was carried out to generate DNA fragments with a size peak around 400-bp . After end repair  for 30 min at room temperature, the DNA was purified (Qiagen PCR purification kit). dATP was added with Klenow exo- (NEB) for 30 min at 37 °C, after which the enzyme was heat-inactivated (20 min at 65 °C). A double size selection using AMPure XP beads (Beckman Coulter) was performed: first, the ratio of AMPure XP beads solution volume to DNA sample volume was adjusted to 0.6:1. After incubation for 15 min at room temperature, the sample was transferred to a magnetic separator  and the supernatant was transferred to a new Eppendorf tube, while the beads were discarded. The ratio of AMPure XP beads solution volume to DNA sample volume was then adjusted to 0.9:1 final. After incubation for 15 min at room temperature, the sample was transferred to a magnet . Following two washes with 70% ethanol, the DNA was eluted in 100 μL of TLE . Biotinylated ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies) on a DynaMag-2 magnet (Life Technologies) in binding buffer  for 30 min at room temperature. After two washes in binding buffer and one wash in ligation buffer (NEB), PE adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 h at room temperature . After washing twice with wash buffer  and then once with binding buffer, the DNA-bound beads were resuspended in a final volume of 90 μL NEBuffer 2. Bead-bound Hi-C DNA was amplified with seven PCR amplification cycles  using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). After PCR amplification, the Hi-C libraries were purified with AMPure XP beads (Beckman Coulter). The concentration of the Hi-C libraries was determined by Bioanalyzer profiles (Agilent Technologies) and qPCR (Kapa Biosystems); the Hi-C libraries were paired-end sequenced  at the Babraham Institute Sequencing Facility.2O and hybridization blockers  were added to the Hi-C DNA. Hybridization buffers and the custom-made RNA capture bait system  were prepared according to the manufacturer’s instructions . The Hi-C library DNA was denatured for 5 min at 95 °C and then incubated with hybridization buffer and the RNA capture bait system at 65 °C for 24 h . After the hybridization incubation, 60 μL of MyOne Streptavidin T1 Dynabeads (Life Technologies) were washed three times with 200 μL binding buffer , before incubation with the Hi-C DNA/RNA capture bait mixture with 200 μL binding buffer for 30 min at room temperature, slowly rotating. Hi-C DNA bound to capture RNA was isolated using a DynaMag-2 magnet (Life Technologies). Washes  were performed according to the SureSelect Target enrichment protocol (Agilent Technologies). After the final wash, the beads were resuspended in 300 μL NEBuffer 2, isolated on a DynaMag-2 magnet, and then resuspended in a final volume of 30 μL NEBuffer 2. After a post-capture PCR , the PCHi-C libraries were purified with AMPure XP beads (Beckman Coulter). The concentration of the PCHi-C libraries was determined by Bioanalyzer profiles (Agilent Technologies) and qPCR (Kapa Biosystems); the PCHi-C libraries were paired-end sequenced  at the Babraham Institute Sequencing Facility.For PCHi-C, 500 ng of Hi-C library DNA was resuspended in 3.6 μL Hescribed , 66: 39,All next-generation sequencing reads were first trimmed using Trim Galore  and aligp value < 0.05 were labelled as DEGs; for the comparison of pre-B versus pro-B cells in young an additional filter requiring a log2 fold change > 1 was also applied. KEGG pathway analysis was performed using gProfiler [RNA-seq reads were aligned to the GRCm38 reference genome using HISAT2 . SeqmonkProfiler . To visuProfiler .p value threshold of 0.05. Complete-linkage hierarchical clustering of differentially expressed miRNAs was performed based on the Euclidean distance between the Pearson correlation coefficients between expression patterns for each replicate.MiRNA annotations from Ensembl and miRBase were merged and deduplicated. Reads were mapped to genome build GRCm38 using Bowtie 2  and straP values reflect multiple-test corrected (Benjamini–Hochberg) values.Target genes of differentially expressed miRNAs were identified using mouse mirTarBase v7 database, released in 2017 . Only fuTrimmed reads were aligned to the GRCm38 mouse reference genome using Bowtie 2 . Reads mp value threshold of 0.05. A lower stringency threshold  was used to identify a larger group of genes over which chromatin accessibility is altered, and the log2 fold gene expression changes were compared between these groups. Peaks were assigned to a gene if they overlapped with a window extending 1-kb up- and downstream of the gene. For comparison of ATAC-seq peaks with genes whose expression was significantly altered upon aging, assignment to genes was performed in the same way. Read counts for each peak were normalized with DESeq2 and the log2 fold change in the median counts (aged/young) was compared for peaks assigned to upregulated genes, downregulated genes or exclusively to genes whose expression was not significantly changed. For pro-B and pre-B cell ATAC-seq data, up- and downregulated genes were assigned from the total RNA-seq analyses from pro-B and pre-B cells, respectively. All upregulated genes predicted to originate from mature B cells were excluded.We used MACS2 , 79 withp value threshold < 0.05. To identify a larger set of genes for comparison of their log2 fold change in expression, lower stringency thresholds were used , with peaks assigned to genes as described for ATAC-seq. Comparison with genes whose expression was significantly up- or downregulated was performed as described for ATAC-seq.ChIP-seq data were mapped to the GRCm38 mouse reference genome using Bowtie 2. For all except H3K27me3, we called peaks using MACS2 with default parameters for narrow peaks. For H3K27me3, we instead used MACS V1.4 to call broad peaks. Similar to ATAC-seq analysis, peaks for aged and young were merged; those with low numbers of high-quality reads  or overlapping blacklisted regions were filtered out and differential enrichment analysis was performed using featureCounts and DESeq2, with adjusted p values from the DESeq2 differential enrichment analysis (see above), with the smallest (most significant) p value ranked highest. Each pairwise set of overlapping peaks was then filtered to retain only pairs of peaks for which at least one of the peaks was ranked in the top 10%, i.e. retaining those peaks with the greatest evidence for an age-related change in enrichment. Pearson correlation coefficients were then derived as above for these filtered sets of overlapping peaks.Overlapping peaks between all pairwise combinations of ChIP-seq and ATAC-seq data within a given cell type (pro-B or pre-B), and between pro-B and pre-B cells for a given chromatin feature, were first defined. These were filtered such that if a single peak in one dataset overlapped with multiple peaks in the second, one of the peaks from the second dataset was selected at random. Pearson correlation coefficients comparing the age-related log2 fold changes in enrichment for overlapping peaks from each pairwise combination were then calculated and displayed as heatmaps. To reduce the influence of stochastic variation in peaks that are unchanged upon aging, peaks from each dataset were then ranked based on their uncorrected For chromatin state analysis, we used chromHMM . We tookPromoter occupancy for each state (bp) was calculated over a 5-kb window centred on the TSS of each gene; the overall state of a given promoter was assigned to the state with the largest coverage, with the exception of the background state to which a promoter was assigned only if no other state was present. A genome-wide set of regulatory elements was defined by merging the MACS peaks called from ATAC-seq and ChIP-seq for H3K4me3, H3K27me3, H3K27ac, and CTCF in pre-B cells; the overall state of each was assigned in the same way as for promoters. States were assigned separately based on young and aged chromatin segmentation, allowing the proportions of promoters and regulatory elements in each state to be compared and to identify regions that changed state upon aging. To assess the overlap of regulatory elements in each state with transcription start sites, all mRNA isoforms (based on Ensembl annotations) were considered; any regulatory element within 1-kb of a transcription start site was considered overlapping. To extend the analysis of promoters, the absolute coverage of all states was considered for each promoter: genes displaying the greatest magnitudes of change in state coverage upon aging  were selected and changes in coverage were normalized by subtracting the mean change in bp coverage for a given state. Complete-linkage hierarchical clustering was performed based on the Euclidean distance between the Pearson correlation coefficients between state changes. Genes were segregated into nine clusters to maximize the distinction between state change patterns and the log2 fold change in pre-B cell total gene expression upon aging compared for each cluster. The fraction of the promoter occupied by each state was also calculated for selected age-up- and downregulated genes.We mapped and filtered the Hi-C reads using HiCUP  and analHindIII fragments with similar interaction distances. Each promoter and PIR was assigned to one of the six chromatin states for the chromHMM analysis as follows: active promoter = fragment overlaps with active promoter state and does not overlap with polycomb or bivalent states; polycomb = fragment overlaps with polycomb state and does not overlap with active promoter or bivalent state; bivalent = fragment overlaps with bivalent state or with both active promoter and polycomb states; active regulatory = fragment overlaps with active regulatory state and does not overlap with active promoter, bivalent or polycomb states; insulator = fragment overlaps with insulator state and does not overlap with any other state except background; background = fragment overlaps exclusively with background state. All active fragments were defined as those assigned to the active promoter or active regulatory state, or with overlapping ChIP-seq peaks of H3K4me3 or H3K27ac, excluding any fragments assigned to the polycomb or bivalent state or that overlapped with an H3K27me3 peak. To compare enrichment of ATAC-seq and ChIP-seq over PIRs, total read counts over the PIRs were normalized to the total library size and the log2 fold change in median read count compared for PIRs of up- and downregulated genes, excluding upregulated genes predicted to originate from mature cells. Genome browser interaction plots were generated using the WashU browser [HindIII fragment encompassing the Irs1 gene promoter. Read counts were quantified over merged HindIII fragments, such that each quantitation window comprised five adjacent HindIII fragments.Reads were mapped and filtered using HiCUP . BAM fil browser . VirtualPre- and pro-B cell pellets were sonicated in SDS sample buffer and proteins resolved by SDS-PAGE. Proteins were transferred onto PVDF membranes and immunoblotted with the indicated primary antibodies at 4 °C overnight, followed by 1 h at room temperature. Membranes were washed in TBST  and incubated with HRP-conjugated secondary antibodies. IRS1 antibody #2382 was from Cell Signaling Technology (CST)/New England Biolabs (NEB); anti-beta-COP antibody was a kind gift from Dr. Nick Ktistakis, Babraham Institute, UK; goat Anti-Rabbit and anti-Mouse IgG (H + L)-HRP conjugate was from BioRad. Membranes were washed and signal detected by enhanced chemiluminescence. Relative protein expression was quantified using Aida 2D Densitometry software (v3.27).Additional file 1:Supplementary Tables S1–S16. XLSX file describing datasets generated for this study. (XLSX 369 kb)Additional file 2:Supplementary Figures S1–S15. PDF of all supplementary figures S1–S15. (PDF 4237 kb)Additional file 3:Chromatin segmentation output for young pre-B cells. TXT file listing chromatin segmentation output for young pre-B cells. (TXT 4627 kb)Additional file 4:Chromatin segmentation output for aged pre-B cells. TXT file listing chromatin segmentation output for aged pre-B cells. (TXT 4190 kb)Additional file 5:Reviewer reports and authors’ response to reviewers. (DOCX 119 kb)


CDKN2B) and p21 (CDKN1A) are repressed by MYC at the transcriptional level. In contrast, MYC activates ARF, which contributes to the apoptosis induced by high MYC levels. At least in some cells types, MYC inhibits the transcription of the p27 gene (CDKN1B) but also enhances p27’s degradation through the upregulation of components of ubiquitin ligases complexes. The effect of MYC on cell-cycle brakes also opens the possibility of antitumoral therapies based on synthetic lethal interactions involving MYC and CDKs, for which a series of inhibitors are being developed and tested in clinical trials.Promotion of the cell cycle is a major oncogenic mechanism of the oncogene c-MYC (MYC). MYC promotes the cell cycle by not only activating or inducing cyclins and CDKs but also through the downregulation or the impairment of the activity of a set of proteins that act as cell-cycle brakes. This review is focused on the role of MYC as a cell-cycle brake releaser i.e., how MYC stimulates the cell cycle mainly through the functional inactivation of cell cycle inhibitors. MYC antagonizes the activities and/or the expression levels of p15, ARF, p21, and p27. The mechanism involved differs for each protein. p15 (encoded by  The oncogene c-MYC (referred to herein as MYC) was the first described gene that encoded for an oncogenic transcription factor with the ability to transform cells in culture. MYC is overexpressed by different mechanisms in 60–70% of human solid and hematopoietic tumors ,2,3,4,5.MYC is a transcription factor of the helix-loop-helix-leucine zipper (HLH-LZ) family that regulates the activation or repression of many target genes ,8. RegulThe number of MYC-binding sites revealed by genome-wide technologies ranks between 7000 and 15,000 in different models. Indeed, MYC is bound at one or more sites of the regulatory regions of 10–15% of human genes ,7,8,12. MYC protein contains several domains that play important roles in MYC functions, as well as many residues susceptible of being modified, modulating MYC’s activity and stability . MYC conActivation or repression of MYC-regulated genes is mediated by its interaction with a variety of partner proteins, many of them involved in chromatin structure regulation . TheCDKN1A (p21CIP1) [CDKN2B (p15INK4B) [Apart from transcriptional activation of gene expression, MYC also represses a great number of genes, many of them involved in processes such as the inhibition of cell-cycle progression and cell adhesion ,34. MYC p21CIP1) ,41, CDKN15INK4B) ,38,42.1-phase of the cell cycle, leading to the phosphorylation of the retinoblastoma protein (RB) and the release of the E2F transcription factors [1-phase further phosphorylate RB, allowing the expression of E2F target genes required for the transition to S-phase [2 phases. M-phase transition is regulated by CDK1 activated by B-type cyclins (B1 and B2) [INK4A, p15INK4B, p18INK4C, and p19INK4D) binds and inhibits CDK4 and CDK6 kinases, impairing their association with D-type cyclins. The CIP/KIP family  inhibits progression at every cell-cycle phase upon binding to several already formed Cyclin-CDK complexes [We will review here the role of MYC as cell-cycle brake releaser i.e., how MYC stimulates cell cycle mainly through the repression of cell-cycle inhibitors . Cell-cy factors ,46. Cycl S-phase . Later,  and B2) ,48. CDK omplexes . CDK inhomplexes  and someomplexes .MYC stimulates cell-cycle progression through the regulation of many genes related to cell-cycle control (recently reviewed in ) Figure. MYC indINK4A/ARF/INK4B gene locus is located on chromosome 9p21 in humans encoding three related proteins: p15INK4B (p15 herein after), p14ARF in humans or p19ARF in mice (ARF herein after) and p16INK4A (p16 herein after). p15 and p16 are characterized for their direct interaction with CDK4 and CDK6, blocking the formation of cyclin D-CDK4/6 complexes and provoking arrested proliferation through preventing phosphorylation of RB and S-phase entry [INK4A gene, while the first exon of each gene is totally different. They are transcribed from an alternative reading frame  within the same locus and thus, their amino acid sequences lack any similarity. ARF induces cell-cycle arrest in G1 and G2 phases [INK4A/ARF/INK4B locus is lost in a wide range of human tumors . This riewed in ). Disrupferation . In agreferation ,61 and Mferation ,63 throuferation . Here we1 phase by specifically inhibiting cyclin D-CDK4/6 complexes [INK4B upon TGFβ treatment. MYC-MAX heterodimers impair INK4B expression by interacting with MIZ-1 at the Inr element of its promoter, preventing p300 recruitment by MIZ-1 [INK4B induction by MIZ-1 through its interaction with SMAD proteins [INK4B expression independently of the Inr element. This mechanism involves the interaction of MYC with SP1 and SMAD proteins. MYC binds to activated SMAD, forming a repressor complex together with SP1, leading to the inactivation of INK4B expression upon TGFβ treatment [The cell-cycle inhibitor p15 arrests proliferation in Gomplexes . Moreoveomplexes . Treatmeomplexes . In factomplexes . The repomplexes ,68). Difomplexes ,70,71. Tby MIZ-1 . TGFβ inproteins . On the reatment .MYC transgenic mice [INK4A/ARF−/−-Eµ-MYC mice were more prone to develop lymphomas and displayed apoptotic defects despite the presence of wild-type p53, a phenotype similar to the one observed in p53-null lymphomas [Although MYC is always related to enhanced proliferation and cell growth, deregulated MYC expression paradoxically triggers apoptosis upon cellular stress conditions such as serum deprivation ,72. Thisnic mice , selectinic mice . In agrenic mice . Althougnic mice . Thus, lymphomas . Other symphomas ,81 showeymphomas , consistINK4A/ARF locus activating ARF expression. Thus, constitutive MYC signaling induces both nuclear FoxO levels and ARF expression [The mechanism of ARF expression induction by MYC remains largely unclear, although it seems to happen through an indirect mechanism involving the regulation of other factors that directly activate ARF expression. MYC induces FoxO transcription factors, which bind to and regulate the pression . On the pression , althougpression . As MYC pression . On the pression . This copression . Consistpression .1 arrest in a p53-independent manner [Apart from the p53-dependent ARF induction of apoptosis and arrested proliferation through MDM2 sequestration, ARF has been proposed to have p53- and MDM2-independent functions to suppress cell proliferation . Moreovet manner . An ARF t manner . In contt manner . This dit manner . Althougt manner . Notablyt manner . MYC-p14t manner . Chromatt manner . Thus, tt manner . Many tat manner ,95,96.Cip1/Waf1 (p21), encoded by the CDKN1A gene, plays key roles in controlling cellular processes such as proliferation, senescence, cell differentiation and apoptosis . Simiewed in ,100 in rA damage ,102. OneA damage . While aA damage , MYC-ER A damage . HoweverA damage . One of A damage . Thus, aA damage  constituThe better characterized and most studied mechanism by which MYC is known to counteract the antiproliferative activity of p21 occurs at the transcriptional level . In factCDKN1A transcriptional repression occurs in a HDAC-independent manner [CDKN1A upon MYC-ER activation, even in the absence of de novo protein synthesis. The inhibition of de novo protein synthesis diminishes the possibility that an intermediate protein could be responsible for this effect, meaning that MYC directly triggers p21 repression [CDKN1A promoter contains three non-canonical E-box sequences, two of them close to the transcription start site (TSS) (−5 to +1 bp and −20 to −15 bp) and another one around 150 bp upstream the TSS (−162 to −157 bp)  is enough for MYC to repress CDKN1A promoter’s activity [CDKN1A expression, being SP1/SP3 and MIZ-1 the main ones described so far [CDKN1A promoter fragments revealed that the TGFβ responsive element is not needed for MYC-mediated p21 repression. A luciferase construct containing from −62 to +16 bp of the CDKN1A promoter, was enough for MYC to mediate promoter repression and thus, MYC exerts its regulation independently of the rest of elements that act upstream that sequence, such as p53 or C/EBP [CDKN1A transcription start site that is enough for MYC to repress p21 expression, there are multiple SP1 binding sites and a potential Inr sequence. The initiator binding protein (TFII-I) induces gene transcription from the Inr of certain TSS and MYC is known to interact with the TFII-I impeding its activity in other models. However, that was not the case for CDKN1A, as depletion of the Inr sequence (+7 to +16 bp from the TSS) did not affect MYC repression of CDKN1A promoter in colorectal adenocarcinoma cells. Instead, MYC was found to interact with SP1 and SP3 transcription factors which play important roles in the induction of p21 expression [CDKN1A expression in CaCo cells [Several mechanisms have been reported as per which MYC is able to repress transcription , howt manner ,107. Bespression . The CDK−157 bp) a. Whetheactivity ,41,107. d so far ,41,108. or C/EBP . Within Co cells .CDKN1A promoter activity seems to be cell-type dependent. MYC also represses CDKN1A expression by interacting with the initiator-binding transcription factor MIZ-1. During hematopoietic differentiation, MIZ-1 levels increase and trigger CDKN1A expression, while ectopic MYC expression repressed basal or TPA-induced CDKN1A levels [CDKN1A promoter in this model was found to be between −49 and +16 bp from the transcription start site, a sequence already reported in other studies, as mentioned above. Nevertheless, opposite to previous reports [CDKN1A expression in other studies [CDKN1A repression. Instead, MYC was recruited to the DNA by interacting through its HLH domain with MIZ-1 [V394D mutant (HLH mutated domain), unable to interact efficiently with MIZ-1 although still capable of interacting with MAX, allowed p21 expression and cell differentiation, bringing to light that MYC-MIZ-1 interaction is essential for CDKN1A repression [The mechanism by which MYC represses A levels . The MYC reports , the Inr studies . Again, th MIZ-1 ,109. Thepression .CDKN1A core promoter resulting in p21 repression [CDKN1A expression by recruitment of HDAC1 and G9a [CDKN1A TSS, GFI-1 repression of CDKN1A expression happened through a mechanism that is independent of its DNA binding ability [CDKN1A core promoter. Knocking down MIZ-1 expression leads to a significant decrease in MYC and GFI-1 occupancy at the CDKN1A promoter region. Indeed, MIZ-1 binds GFI-1 through its C-terminal 1-12 ZFs, while the regions flaking the ZFs are required for MYC interaction [CDKN1A promoter region allowing p21 expression [More recently, MYC has been shown to form a ternary complex with MIZ-1 and GFI-1 able to bind the pression   and the histone demethylase KDM5B capable to bind and repress the core promoter of CDKN1A through the AP2-binding site located −111 to −103 bp from the TSS  is a direct MYC target gene that belongs to the bHLH-LZ family of transcription factors. Its basic DNA-binding domain is essential to mediate CDKN1A repression through recognition of the E-boxes located at the core promoter of this gene [CDKN1A expression [CDKN1A repression [Apart from the direct regulation of p21 transcription by MYC through its recruitment to the core promoter of the his gene ,130. AP4pression . AP4 is pression ,133. Nevpression , in agrepression ,107. Othpression ,135.miR-17-5p, miR-20a, and miR-106a, all of them belonging to the miR-17 family of miRNAs are induced by MYC and downregulate p21 expression [Finally, MYC has been shown to regulate p21 expression at the post-transcriptional level, by modulation of miRNA expression . p21 is pression . Thus, mKip1 (p27), encoded by the CDKN1B gene, is known to induce proliferation arrest in G1 by blocking the kinase activity of cyclin-CDK complexes, being cyclin E-CDK2 inhibition which exerts its main role in cell-cycle control. Besides, p27 behaves as a transcriptional regulator involved in a variety of cellular functions and in cancer  repression of p27 at the transcriptional level; (ii) induction of miR-221 and miR-222 that down-regulate p27 expression; (iii) induction of D-type cyclins and CDK4 and CDK6 that sequester p27 from cyclin E-CDK2 complexes; (iv) induction of CCNE expression directly or through E2F, leading to activation of cyclin E-CDK2 complexes that antagonize p27 function; v) induction of different components of the SCFSKP2 ubiquitin ligase complex  that targets p27 for proteasomal degradation . Since on rates . Moreoveon rates ,146. Theradation . Mechanin by MYC . These mCDKN1B core promoter, as already described for its related CDKN1A gene. CDKN1B mRNA expression levels inversely correlate with MYC expression in immune cells and other models. B cell receptor (BCR) engagement in immature B cells (upon anti-IgM treatment) lead to MYC downregulation, followed by p27 expression and induction of apoptosis [MYC and CDKN1B mRNA expression levels in this model upon IgM treatment. Luciferase assays showed that the CDKN1B promoter region containing from −2002 to +154 bp responded to anti-IgM treatment leading to an increase in CDKN1B promoter activity [CDKN1B promoter contains an Inr element at the TSS which, as already described for other MYC-repressed target genes, has been found to be crucial for CDKN1B downregulation and MYC has been reported to interact with it in different models [CDKN1B upregulation upon BCR engagement is abrogated by ectopic MYC expression [P115L mutant, in which the Phe at the position 155 is replaced by a Leu within the region of MYC needed for its transcriptional suppression function, enhances its repressor activity [CDKN1B promoter by direct interaction and inhibition of Foxo3a, a transcription factor known to upregulate CDKN1B expression [CDKN1A, MYC interacts with the CDKN1B Inr element through MAX, blocking CDKN1B expression [One of the mechanisms that accounts for the inhibition of p27 by MYC involves MYC-mediated transcriptional repression of the poptosis , an effepoptosis  and blocpoptosis ,149,150.activity . The CDKt models . Indeed,pression . MYC intactivity , consistactivity ,152,153.pression .CDKN1B at the post-transcriptional level by miRNAs has been recurrently reported during the last years. Indeed, aberrant up-regulation of miRNA clusters that regulate p27 expression have been linked with cancer development, progression and invasion [miR-221 family of miRNAs directly regulates the expression of p27 by targeting its 3′UTR sequence  [miR-221 and miR-222 are consistently overexpressed in liver tumors, showing an opposite correlation with low levels of p27 due to its aberrant pros-transcriptional regulation. Furthermore, miR-221 (but not miR-222) has been reported to enhance tumorigenesis not only in vitro, but also in vivo [Regulation of invasion ,157,158,sequence . miR-221e models . MYC plaand p57) . Besides in vivo .miR-34a, miR-15a/61, and miR-26a. MYC has been reported to induce the expression of D-type cyclins and CDK4/6 by repressing these miRNAs. Altogether, MYC induces the formation of cyclin D-CDK4/6 complexes promoting the switch of p27 from cyclin E-CDK2 to cyclin D-CDK4/6 complexes, thus inducing the G1-S phase transition as reviewed in [Inhibition of the cell cycle by p27 is controlled, in a great extent, by its recruitment to cyclin D-CDK4/6 complexes. The shift of p27 from cyclin E/CDK2 to cyclin D-CDK4/6 complexes relieves cyclin E-CDK2 from p27-mediated inhibition allowing progression through the cell cycle . Intriguiewed in .SKP2 ubiquitin ligase complex [SKP2 recognition [1 phase was first described in a Rat1-MYC-ER model [CCNE was later reported to be a direct MYC target gene and that MYC could also induce its expression via E2F1, another MYC target gene needed for the G1 to S-phase transition [MYC whereas E2F7 transactivate MYC [CCNE, such as miR-34a and miR-26a . Inognition ,180. Phoognition . Moreoveognition . In the ognition . In vivoER model  while thER model . Moreoveansition ,187. In vate MYC . On the iewed in . Thus, Miewed in ,190,191,iewed in . On the iewed in ,193, thuiewed in . Phosphoiewed in ,195. In iewed in ,197.SKP2 complex is composed by RBX1, CUL1, SKP1, and the F-box protein SKP2 [SKP2. The N-terminal portion of p27 packs with SKP2, a central Glu185 side chain inserts between SKP2 and CKS1 and the C-terminal portion containing the phosphorylated Thr187 binds to CKS1 [SKP2-CKS1 stimulating p27 ubiquitination [The SCFein SKP2 ,178. Celein SKP2 . Unlike  to CKS1 . Furthertination ,199,200.tination ,202.SKP2 complex. This is achieved as several components of this complex have been reported to be MYC-target genes: CUL1, CKS1, and SKP2 [MYC cells, which reduced p27 levels within the cells, and siRNA mediated depletion of p27, restored MYC’s wild type phenotype leading to normal proliferation rates. Thus, the SCFSKP2 complex is essential for MYC’s activity as a pro-proliferative transcription factor, by means of reducing p27 levels to allow cell-cycle progression. Altogether, it brings to light that MYC plays a critical role in the regulation of p27 degradation via proteasome through the combination of different regulatory mechanisms.MYC induces p27 proteasomal degradation through the upregulation of the SCFand SKP2 ,203,204.and SKP2 ,204. DepMYC would be a good target for therapy. First, MYC deregulation occurs frequently in human cancer. Second, MYC addiction has been shown in several models so that inactivation or depletion of MYC leads to tumor regression ,206,207.Third, whole-body inactivation of MYC in mouse models by expression of a dominant negative MYC form  only prSeveral molecules have been described to bind MYC and impair its function. Most of these molecules interrupt the MYC-MAX interaction b, as theA more promising approach is to target MYC as an indirect target via synthetic lethal approaches c. Severa2 phase, and some are being used in clinical trials [CDK1 is essential for mammalian cell division  and is tl trials ,223. Accl trials . Similarl trials . These rl trials . Therefol trials . It is wl trials . On the l trials ,228). MYl trials  and indul trials ,234,235.TP53 is frequently mutated in cancer and usually confers an adverse prognosis.Aurora kinases A and B (AURKA and AURKB) are serine/threonine kinases required for mitosis . MYC regOne of the effects of MYC overexpression is to induce DNA replicative stress , which iCDK9 is not a kinase involved in cell-cycle progression but in transcription initiation. However, it is worth noting this interaction, given its similarity with cell cycle CDKs. Inhibition or depletion of CDK9 (with shRNAs) in cells and mouse models of hepatocellular carcinoma, results in delay of growth and the extent of its effect correlates with MYC levels, suggesting a synthetic lethal inhibition .The importance in cancer research of the set of proteins acting as physiological brakes of the cell cycle has been well established. On the other hand, the impairment of CKI activities is a major mechanism for the tumorigenic effects of MYC. Therefore, the deciphering of the molecular clues of the mechanisms leading to MYC-mediated inhibition of p21, p27, and p15 functions or expression is critical for the design of therapeutic approaches of cancers with MYC deregulation.


Impact and Slc38a4 was lost in the offspring of female mice harboring deletions of the relevant murine-specific ERVs upstream of these genes. Our work reveals an evolutionary mechanism whereby maternally silenced genes arise from biallelically expressed progenitors.Imprinted genes are expressed from a single parental allele, with the other allele often silenced by DNA methylation (DNAme) established in the germline. While species-specific imprinted orthologues have been documented, the molecular mechanisms underlying the evolutionary switch from biallelic to imprinted expression are unknown. During mouse oogenesis, gametic differentially methylated regions (gDMRs) acquire DNAme in a transcription-guided manner. Here we show that oocyte transcription initiating in lineage-specific endogenous retroviruses (ERVs) is likely responsible for DNAme establishment at 4/6 mouse-specific and 17/110 human-specific imprinted gDMRs. The latter are divided into Catarrhini- or Hominoidea-specific gDMRs embedded within transcripts initiating in ERVs specific to these primate lineages. Strikingly, imprinting of the maternally methylated genes  Although many species-specific imprinted genes have been identified, how the evolutionary switch from biallelic to imprinted expression occurs is still unknown. Here authors find that lineage-specific ERVs active as oocyte promoters can induce de novo DNA methylation at gDMRs and imprinting. In both human and mouse female germline, the overall levels of CpG methylation increase from less than 5% in post-migratory primordial germ cells (PGCs) to ~40–50% in fully-grown, germinal vesicle oocytes (GVOs)9. In growing murine oocytes, 85–90% of this DNAme is deposited over H3K36me3-marked transcribed regions of the genome3. We recently demonstrated that SETD2-dependent deposition of H3K36me3 is required for de novo DNAme of transcribed gene bodies in mouse oocytes10. Such transcription-coupled de novo DNAme is also responsible for the establishment of regions of differential DNAme between the mature gametes , a subset of which are maintained throughout preimplantation development. This unique class of gDMRs, the imprinted gDMRs (igDMRs), can direct imprinted paternal allele-specific expression of the gene(s) under their regulation, the imprinted genes12. Importantly, transcription initiating within upstream oocyte-specific promoters has been reported to play a critical role in de novo DNAme at the maternal igDMRs of a number of mouse imprinted genes17, but the evolutionary origin of such promoters remains unexplored.During postnatal oocyte growth in mammals, transcribed genomic regions acquire two characteristic epigenetic marks: transcription-coupled trimethylation at lysine 36 of histone H3 (H3K36me3)18. Such LTRs function as oocyte-specific promoters for both novel protein-coding genes and non-coding transcripts, the latter of which are widely distributed in intergenic regions19. We recently identified numerous LTR-initiated transcription units (LITs) in mouse oocytes associated with H3K36me3-coupled de novo DNAme20. Notably, CpG islands (CGIs) embedded within such LITs also become hypermethylated by this mechanism in oocytes20 and the majority of maternal igDMRs associated with paternally expressed genes in both mice and humans overlap with CGIs21. Since ERVs are highly variable among mammalian species and de novo DNAme at maternal igDMRs is transcription-coupled, we hypothesized that active LTRs and their associated LITs may have played an important role in the genesis of lineage-specific maternal imprinting in mammals.Specific families of ERVs, also known as long terminal repeat (LTR) retrotransposons, are highly transcribed in mouse oocytesImpact and Slc38a4, both of which lead to loss of oocyte DNAme acquisition at the igDMR in mutant females and loss of imprinted expression in the offspring. Together, our data suggest a model in which species-specific imprinted genes emerge from biallelically expressed progenitors via the acquisition of novel LTR promoters active during oocyte growth.Here, we identify primate and rodent-specific igDMRs that appear to be de novo DNA methylated in oocytes as a consequence of transcription initiating within nearby LTR promoters. Our analysis of data from macaque and chimpanzee identifies Catarrhini- or Hominoidea-specific igDMRs embedded in oocyte transcripts emanating from species-specific LTRs. We further validate this phenomenon in two mouse mutants carrying deletions of upstream LTR promoters at 28 by interrogating published whole-genome bisulfite sequencing (WGBS) data from gametes, placenta and somatic tissues (detailed in Supplementary Data 29 to published human30 and mouse20 oocyte RNA-sequencing (RNA-seq) datasets and identified the transcript(s) and transcription start site (TSS) likely responsible for transcription-coupled deposition of DNAme over 20/21 mouse and 90/125 human igDMRs . Thus, the presence of a lineage-specific proximal LTR that initiates an oocyte transcript overlapping with a genic gDMR is associated exclusively with genes showing evidence of a species-specific igDMR.To investigate the contribution of LITs to maternal gametic imprinting in humans and mice, we first curated a list that includes well characterized and putative maternally methylated igDMRsretro-Coro1c , Cdh1527, Slc38a4 34 and Impact35, all initiate in rodent-specific ERVs 26 but are hypomethylated in adult tissues  are associated with LITs driven by LTR families that colonized the common ancestor of the Euarchontoglires , which may be due to a distinct non-LTR-initiated transcript -specific ERV families Fig. . Moreoveues Fig. . The remRHOBTB3/ Rhobtb3 locus for example, transcription in human oocytes initiates within an unmethylated primate-specific MSTA element located ~25 kb upstream of the promoter CGI/igDMR, forming a chimeric transcript that splices to the downstream genic exons of RHOBTB3  in adult tissues , the syntenic region in mice, including the Scin CGI promoter, is hypomethylated in each of these cell types and no upstream initiating transcript is observed in mouse oocytes  show no evidence of transcription initiating from the proximal LTR in this cell type. Rather, for all six genes  that show clear paternally-biased expression (>70%) in CT  families which colonized the common ancestor of the Hominoidea40 (LTR12C or LTR12E), while seven of them initiate within families that colonized the primate lineage, including the common ancestors of the Catarrhini. The remaining four initiate in more ancient elements derived from LTR families common to both primates and rodents (Euarchontoglires)  and rhesus macaques . All 16 relevant LTR insertions are present in the chimpanzee genome. Moreover, all four insertions from LTR families that colonized the Euarchontoglires common ancestor and 6/7 LTR insertions from families that colonized the common ancestor of the Catarrhini are also present in the orthologous loci in macaque. Similarly, the Hominoidea-specific LTR12C and LTR12E families, which include LTRs driving transcripts overlapping the ST8SIA1, SCIN, ZFP90, COL26A1, and SORD igDMRs in human oocytes, are absent from the macaque genome  Fig. . To charome Fig. .Fig. 2Co41 and genome-wide DNA methylome data42. As expected from the phylogeny of LTR12C elements, no LITs overlapping the region orthologous to the human igDMRs at the ST8SIA1, SCIN, or COL26A1 loci were detected in macaque oocytes, and their CGIs remain hypomethylated , DNAH7 (THE1C), HTR5A (LTR53B), AGBL3 (MER50), and ZC3H12C (MLT1A) putative igDMRs. In contrast, no LITs were detected at 4/11 loci despite the presence of a conserved LTR insertion in the orthologous macaque locus , which may impact transcription of the macaque LTR  genome , the MTC element upstream of Impact is absent.If the LTRs upstream of the mouse-specific igDMRs shown in Fig. ome Fig. , consistome Fig. 19. In co20. LITs emanating from the relevant LTRs were clearly detected at Cdh15 and Slc38a4 genes in rat oocytes, while in hamster, a LIT was detected only at the Slc38a4 locus  upstream start site in rat oocytes, but a gap in the reference genomic sequence precludes detailed analysis of the upstream transcript at this locus  embryonic stem cells using CRISPR-Cas9 and flanking guide RNAs  upon maternal transmission of the Slc38a4MT2AKO allele  highly expressed in the mouse placenta34, suggesting that variations in its overall expression levels might lead to placental and/or growth abnormalities, as described for other imprinted genes. However, we did not observe either phenotype in maternal heterozygotes . Further studies, such as those applied here in the mouse, will be required to determine whether alternative LTRs play a role in the imprinting of such loci in other species. Those species-specific maternal igDMRs not associated with a LIT are likely methylated as a consequence of transcription initiating in novel, single-copy, TSSs active in oocytes (as seen at the rat Impact locus).Our observation that 15.5% (17/110) of human-specific and 66.7% (4/6) of mouse-specific maternal igDMRs are potentially induced by ERV promoters highlights the importance of this mechanism and its impact on the evolution of species-specific imprinted genes in mammals. Furthermore, our analysis also revealed evidence for convergent evolution of imprinting, where a distinct LTR may be responsible for imprinting in different species  in oocytes, hypomethylated (<30% DNAme) in sperm, retain DNAme (>25%) in the blastocyst, and show 35–65% DNAme in placenta  or at least one adult somatic tissue72. We further validated and refined this list by only including gDMRs harboring either reported maternal, monoallelic or bimodal DNAme in the placenta and/or at least one somatic tissue. A bimodal DNAme pattern is a specific class of sequences methylated at ~50% for which individual DNA sequencing strands from WGBS datasets, although not overlapping a SNP with known parental origin, are either fully- (hyper) methylated or un- (hypo-) methylated. If the DNA sequence analyzed also encompasses a SNP, the bimodal pattern can be described as allelic when all hypo- or all hypermethylated strands contain the same variant at the SNP: only one of the parental alleles is methylated. Only when the parental origin of the sequence variants of this SNP is known in a given sample, can the allelic DMR be referred to as imprinted . In total, 125 human igDMRs  and 21 mouse igDMRs  are presented and analyzed in Supplementary Data http://genome.ucsc.edu/)73.To validate previously identified maternally methylated imprinted gDMRs (igDMRs)74. De novo transcriptome assemblies were produced using Stringtie v.1.3.5 32 or Cufflinks v.2.1.121 (macaque)29 with default parameters. All de novo transcripts overlapping or in proximity of putative imprinted gDMRs were identified, and their transcription initiation site was confirmed by visual inspection of splice sites. Transcripts initiating in transposable elements were identified using LIONS31. The 5′ ends of de novo assembled transcripts were classified based on overlaps with UCSC Repeat Masker annotation and our putative igDMR list, and transcripts with the Up or UpEdge classification (per LIONS raw output) were taken into consideration. For manual inspection of LITs over specific CGI promoters, LITs were either identified by manually validating transcripts with EInside transposable element contribution in the LIONS raw output or by intersecting de novo transcripts with the boundaries of hypermethylated domains.Oocyte RNA-seq libraries (see Supplementary Data http://timetreebeta.igem.temple.edu/). Integration of LTR families was imputed based on the presence or absence of the family in the species investigated, and colonization time is indicated at the root of those species in which it was found in common. For examining the evolutionary history of MT2A family, including the MT2A apparently responsible for de novo methylation of the Slc38a4 igDMR in mouse oocytes, we identified all annotated (UCSC RepeatMasker) MT2A elements >450 bp (the MT2A consensus sequence is 533 bp). Multiple sequence analysis and phylogenetic tree construction were carried out with MEGA X75 using MUSCLE UPMGA algorithm and by Neighbour-Joining method, respectively. Visualization and annotation of the phylogenetic tree were carried out with iTOL (https://itol.embl.de/)76.Phylogenetic tree of species shown was generated from TimeTree  of Kyoto University . Three rhesus macaque (Macaca mulatta) placentas and their parental blood DNA were collected and stored at −80 °C before use. Three chimpanzee (Pan troglodytes verus) placentas and their parental blood DNA were provided from Kumamoto Zoo and Kumamoto Sanctuary via Great Ape Information Network (GAIN).http://quma.cdb.riken.jp/top/index.html) to quantify CpG methylation levels of each CGI. From each sequence data (fastq files), the top 400 forward reads (R1) were extracted and mapped to each CGI with QUMA’s default conditions and uniquely mapped reads were used to calculate DNA methylation level per CpG dinucleotide site. Due to mappability issues, 20 mismatches were allowed for mapping the amplicons over the chimpanzee ZNF396 gDMR locus. When SNPs were available, reads perfectly matching each parental genome were extracted and aligned separately for allele-specific analyses.Regions orthologous to human placental igDMR were PCR-amplified with TaKaRa EX Taq HS (TaKaRa Bio) with primers specific for chimpanzee and rhesus macaque CGIs , diluted in native ChIP buffer  containing 1 mM PMSF (Sigma) and EDTA-free protease inhibitor cocktail (Roche), and incubated overnight at 4 °C with 0.1 μg of anti-RNA polymerase II monoclonal antibodies (Abcam ab817–8WG16-) and 5 μl of protein A: protein G 1:1 Dynabeads (Thermofisher). Antibody-bond chromatin was washed four times in low salt buffer  and eluted at 65 °C for 1 h in elution buffer . DNA was then extracted using phenol:chloroform and precipitated in 75% ethanol, followed by paired-end library construction. Libraries were sequenced (75 bp paired-end) on a NextSeq 500 according to manufacturer’s protocols.Germinal vesicle oocytes (GVOs) were isolated from 7–10-weeks-old C57BL/6J females. The zona pellucida was dissolved by passaging the oocytes through an acid Tyrode’s solution, followed by neutralization in M2 media. The oocytes were re-suspended in nuclear isolation buffer (Sigma), flash-frozen in liquid nitrogen and stored at −80 °C until usage. RNA polymerase II ChIP-seq libraries were prepared from ~200 GVOs using a modified version of ULI-NChIP-seqSlc38a4 and Impact were deleted in mouse ESCs using transient delivery of sgRNAs and Cas9 from an expression vector similar to pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene #42230)78. Plasmid phU6-gRNA-CBh-hCas9 was obtained from Rupesh Amin and Mark Groudine and first modified by insertion of the PGK-puro-pA selectable marker from pPGKpuro (Addgene #11349)79 to obtain pPuro2-hU6-gRNA-CBh-Cas9. Individual sgRNAs were designed from http://crispr.mit.edu/ and incorporated on a single forward oligonucleotide (IDT) including a 5′ SapI site and a 29-bp region of complementarity to a universal reverse primer containing an XbaI site. All primer sequences are shown in Supplementary Data 80 by lipofection . After 16 h, puromycin selection (4 μg/ml) was started and continued for 48 h, after which the cells were grown without selection for 5 days. Whole cell populations were collected and prepared for PCR by HotSHOT81. Genomic PCR was performed with primers flanking the sgRNA site  or Impact LTR KO (#3 and #5) ESCs were suspended in injection media (DMEM with HEPES + 25% KO ES medium without LIF/2i), and blastocysts were injected with 8–10 ES cells per embryo. After injection, 10–20 embryos were implanted into E2.5 pseudo pregnant mice via uterine transfer. In total, 28 and 7 chimeric pups were born for the Slc38a4MT2AKO and ImpactMTCKO mutant ESCs, respectively. From these, at least three chimeric males per line transmitted a deletion allele. Transgenic mice were bred and maintained in the Centre for Disease Modelling, Life Sciences Institute, University of British Columbia, under pathogen-free conditions. In all heterozygous genotypes, the maternally inherited allele is always presented first.Blastocysts were collected at E3.5 from female albino C57Bl/6J-TyrC2J mice following natural mating, and placed in a 200 ul drop of KSOM overlaid with embryo-tested mineral oil and incubated at 37 °C until ready for injection. Pooled clones of Impact MTCKO and placentae for the Slc38a4 MT2AKO crosses were used for RNA extraction using Trizol (ThermoFisher). RNA was DNase I-treated (Fisher) at 37 °C for 1 h, then heat inactivated at 65 °C for 15 min. cDNA was synthesized with MMLV-RT (ThermoFisher) using N15 oligonucleotides. RT-PCR was performed with primers described in Supplementary Data Slc38a4 or MluCI for Impact) and resolved on a 2% agarose gel. Uncut PCR products were sent for Sanger sequencing, and.ab1 files were analyzed using the PHRED software (http://www.phrap.org/phredphrapconsed.html).E13.5 embryos and placentae were dissected from reciprocal F1 crosses between heterozygous LTR KO animals on the C57BL/6 background and WT CAST mice. Embryonic heads for the 3 overnight at 50 °C, cleaned using the Wizard DNA cleanup kit (Promega) and desulfonated in 0.3 M NaOH at 37 °C for 30 min. Oocytes were harvested from females of the appropriate genotype at 21–28 days of age. GVOs were isolated by passing dissected ovaries through a 100 μm filter using a blunt tool, applying the filtrate to a 35 μm filter, then backwashing the trapped GVOs from the filter. GVOs were further purified by manual collection. >200 Oocytes were pooled from multiple females . Oocyte DNA was harvested by incubation in 0.1% SDS 1 μg/μl Proteinase K in the presence of 1 μg lambda DNA at 37 °C for 60 min followed by 98 °C for 15 min. DNA was converted using EZ DNA Methylation Gold Kit (Zymo) per manufacturer’s protocols. Three biological replicates for oocytes and three independent BS conversions for embryos were amplified using semi-nested primers (Supplementary Data 3) and touchdown-PCR conditions. Purified PCR products were TA-cloned into pGEM-T (Promega), and sequenced at the McGill/Genome Quebec Innovation Centre. Sequences were analyzed using BiQ Software . Informative SNPs were identified in final sequences and used to identify individual C57BL/6 or CAST strands.E13.5 embryos and placentae were dissected from reciprocal F1 crosses between heterozygous LTR KO animals on the C57BL/6 background and wild-type CAST/EiJ mice obtained from the Jackson Laboratory (stock number 000735). Bodies were minced and gDNA was extracted by proteinase K digestion followed by phenol:chloroform extraction. One hundred nanogram of gDNA was vortexed to shear the DNA, denatured in 0.2 M NaOH, bisulfite converted in 0.225 M NaOH; 0.0125% hydroquinone; 4 M NaHSOPpia. Primer sequences are available in Supplementary Data To measure total levels of expression, quantitative PCR (RT-qPCR) was performed on cDNA (described above) using Eva Green (Biotium) on a Step-One Plus Real time PCR system (Applied Biosystems). All reactions were run as follows: 2 min at 95 °C, then 40 cycles of 30 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and fluorescence was read at 80 °C. Ct values of six biological replicates from two litters for each stage were averaged and used to calculate relative amounts of transcripts, normalized to levels of the housekeeping gene Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary